UNIVERSITI PUTRA MALAYSIA

IDENTIFICATION AND CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES IN GANODERMA BONINENSE-INFECTED OIL

PALMS (ELAEIS GUINEENSIS)

ZETTY NORHANA BINTI BALIA YUSOF

FS 2007 50

IDENTIFICATION AND CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES IN

GANODERMA BONINENSE-INFECTED OIL PALMS (ELAEIS GUINEENSIS)

ZETTY NORHANA BINTI BALIA YUSOF

MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA

2007

IDENTIFICATION AND CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES IN GANODERMA BONINENSE-INFECTED OIL

PALMS (ELAEIS GUINEENSIS)

By

ZETTY NORHANA BINTI BALIA YUSOF

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Master of

Science

August 2007

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science

IDENTIFICATION AND CHARACTERIZATION OF DIFFERENTIALLY EXPRESSED GENES IN GANODERMA

BONINENSE-INFECTED OIL PALMS (ELAEIS GUINEENSIS)

By

ZETTY NORHANA BT. BALIA YUSOF

August 2007

Chairman: Associate Professor Faridah Abdullah, PhD Faculty : Science Basal stem rot (BSR) caused by Ganoderma boninense is the most serious

disease of oil palms in Malaysia. Thus, changes in the gene expression patterns

of infected palms have gained interests among researchers as a tool to

understand the disease. An indication that there are differences in susceptibility

to BSR between germplasm materials from different genetic origins have

provided hope in generating oil palm varieties with reduced levels of

susceptibility by using existing genetic materials. The study of differentially

expressed genes may also pave the way towards developing diagnostic tools for

early disease detection in oil palms. A novel method combining elements of

suppression subtractive hybridization (SSH) permits the efficient and rapid

cloning of rarely transcribed differentially expressed genes. The experimental

strategy virtually excludes the possibility of isolating false positive clones. This

study used SSH and isolated 1,038 differentially expressed cDNAs from the G.

boninense-inoculated oil palm seedlings (T1) when subtracted from its

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uninoculated counterpart (T2), using the basal stem and spear leaf tissues.

Sequence data indicated that of the 1,038 clones obtained, 86% showed

sequence similarity to proteins already registered in public databases, 10%

showed similarity to putative protein sequences and 4% were unknown proteins

with no records in public databases. Seven clones harboring genes encoding for

defense mechanisms against fungal and insect pathogens in plants were

identified. They were pathogenesis-related (PR)-genes and defense-related

genes depending on their direct or indirect roles in plant defense against

pathogens. Reverse northern analysis of these 7 clones demonstrated that 4 were

differentially expressed in T1 but northern analysis showed that only 3 were

differentially expressed. Further analysis via reverse transcription-PCR (RT-

PCR) confirmed these 3 genes to be differentially expressed in G. boninense

infected oil palms. They were MAG 43 that codes for serine

palmitoyltransferase, MAG 59 for chitinase and MAG 225 for endochitinase.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PENGENALPASTIAN DAN PENCIRIAN GEN YANG DIEKSPRESKAN SECARA BERBEZA DI DALAM SAWIT (ELAEIS GUINEENSIS) YANG

DIJANGKITI OLEH GANODERMA BONINENSE

Oleh

ZETTY NORHANA BT. BALIA YUSOF

Ogos 2007

Pengerusi: Profesor Madya Faridah Abdullah, PhD Fakulti : Sains Penyakit reput pangkal batang (RPB) yang disebabkan oleh Ganoderma

boninense merupakan penyakit sawit yang paling serius di Malaysia. Justeru itu,

pelbagai kajian biologi molekul terhadap sawit yang dijangkiti oleh G.

boninense semakin giat dilakukan bagi mengetahui perubahan pengekspresan

gen akibat jangkitan. Terdapat juga petunjuk tentang perbezaan kadar jangkitan

penyakit RPB yang bergantung kepada kandungan sel daripada keturunan

genetik berbeza. Petunjuk ini telah memberi harapan untuk menghasilkan

variasi sawit yang mempunyai kadar jangkitan yang rendah dengan

mengeksploitasikan unsur-unsur genetik yang sedia ada. Selain itu, kajian

terhadap gen yang diekspreskan secara berbeza juga mungkin merupakan satu

proses permulaan bagi menghasilkan alat diagnostik untuk mengesan kehadiran

patogen di dalam sawit. Kaedah baru yang menggabungkan elemen penindasan,

penolakan dan penghibridan membolehkan pengklonan gen yang diekspreskan

secara berbeza. Strategi ujikaji sebegini dapat menyingkirkan secara mutlak

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kebarangkalian penghasilan klon positif palsu. Potensi kaedah yang digunakan

telah dibuktikan dengan pengasingan 1,038 cDNA yang diekspreskan secara

berbeza di dalam anak sawit yang telah diinokulasi dengan G. boninense (T1)

jika dibandingkan dengan anak sawit yang tidak diinokulasi dengan G.

boninense (T2). Dua jenis tisu yang digunakan di dalam kajian ini ialah tisu

pangkal batang dan pucuk sawit. Data jujukan yang diperolehi menunjukkan

daripada 1,038 klon yang diperolehi, 86% daripadanya menepati dengan protein

yang telah didaftarkan, 10% menunjukkan homologi dengan jujukan protein

putatif dan 4% tidak menepati sebarang jujukan di dalam GenBank/EMBL

Database. Sebanyak 7 klon yang menepati jujukan gen yang berfungsi di dalam

sistem pertahanan daripada serangan kulat dan serangga di dalam tumbuh-

tumbuhan telah dikenalpasti. Kesemua 7 klon tersebut mengandungi jujukan

protein yang berkaitan dengan patogenesis dan gen-gen yang terlibat di dalam

sistem pertahanan tumbuh-tumbuhan. Analisa penghibridan northern berbalik

terhadap ketujuh-tujuh gen tersebut menunjukkan hanya 4 daripadanya

diekspreskan di dalam sampel T1. Seterusnya, analisa northern membuktikan

hanya 3 gen diekspreskan di dalam T1. Kaedah PCR transkripsi berbalik (RT-

PCR) dijalankan bagi mengesahkan keputusan yang diperolehi daripada analisa

northern ke atas ketiga-tiga gen tersebut. Keputusan RT-PCR mengesahkan

bahawa ketiga-tiga gen tersebut adalah diekspreskan secara berbeza di dalam

anak sawit yang telah dijangkiti G. boninense. Ianya adalah MAG 43 yang

membawa jujukan gen serine palmitoyltransferase, MAG 59 yang membawa

jujukan gen chitinase dan MAG 225 yang membawa jujukan gen endochitinase.

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ACKNOWLEDGEMENTS

In the name of Allah, the Most Beneficent, Most Gracious, Most Merciful

First and foremost, I would like to thank Allah S.W.T for all His blessings that

enable me to complete this thesis successfully. My heartiest appreciation goes to

my first supervisor, Associate Professor Dr. Faridah Abdullah for her precious

advice and guidance.

Special recognition and appreciation goes to Dr. Mohamad Arif Abd Manaf, my

immediate supervisor, and also my mentor throughout my 2 years in MPOB.

The quality of his supervision is the best. His support, understanding,

encouragement and above all his trust in me are invaluable. Thank you very

much, Dr. I would also like to express my sincere appreciation to Professor Tan

Soon Guan for his support and guidance in the completion of this thesis.

My deep appreciation also goes to all Gene Expression Lab members, especially

Puan Anita, Mr. Mohd Shafiq, Puan Aminah and Puan Zaini. I am extremely

grateful to all of them for their invaluable help and suggestions based on their

experience.

I wish to dedicate my thesis to my parents Dato’ Balia Yusof Haji Wahi and

Datin Faridah Dato’ Amiruddin who have always been proud of me, believed in

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me and supported me all the way. Your love, belief, strong support and most

important, your blessings have made it possible for me to complete this thesis.

My most profound gratitude goes to my family, Mohd Rafael, Izyan Darlina and

Mohd Arief who supported me throughout the process of completing my thesis.

Finally, a special heartfelt appreciation goes to Mr. Mohd Hefni Rusli for

always being there for me through thick and thin. He gave me the emotional and

spiritual strength to succeed in this dream of mine. Your love, help, endless

motivation and continuous encouragement during the process of completing this

thesis has made it a success. Thank you for everything.

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I certify that an Examination Committee has met on 13th August 2007 to conduct the final examination of Zetty Norhana Binti Balia Yusof on her Master of Science thesis entitled “Identification and Characterization of Differentially Expressed Genes in Ganoderma boninense-infected Oil Palms (Elaeis guineensis)” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the degree of Master of Science. Members of the Examination Committee were as follows: Umi Kalsom Yusuf, PhD Associate Professor Department of Biology Faculty of Science Universiti Putra Malaysia (Chairman) Suhaimi Napis, PhD Associate Professor Department of Cell and Molecular Biology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Faridah Qamaruzzaman, PhD Lecturer Department of Biology Faculty of Science Universiti Putra Malaysia (Internal Examiner) Nik Marzuki Sidik, PhD Associate Professor Pusat Pengajian Biosains dan Bioteknologi Fakulti Sains dan Teknologi Universiti Kebangsaan Malaysia (External Examiner)

________________________________ HASANAH MOHD. GHAZALI, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Faridah Abdullah, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Chairman) Mohd Arif Abd Manaf, PhD Senior Research Officer Advanced Breeding and Biotechnology Center Malaysian Palm Oil Board (Member) Tan Soon Guan, PhD Professor Faculty of Science Universiti Putra Malaysia (Member) ______________________ AINI IDERIS, PhD Professor and Dean

School of Graduate Studies Universiti Putra Malaysia Date: 15 November 2007

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DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

______________________________________

ZETTY NORHANA BINTI BALIA YUSOF

Date: 20 September 2007

TABLE OF CONTENTS

Page ABSTRACT ii ABSTRAK iv ACKNOWLEDGEMENTS vi APPROVAL viii DECLARATION x LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xvii CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 5

2.1 The Oil Palm (Elaeis guineensis Jacq.). 5 2.1.1 Origin 5

2.1.2 Oil Palm Industry in Malaysia 6 2.2 The Botany of Oil Palm 8

2.3 Fungal Diseases of Oil Palm 8 2.4 Basal Stem Rot (BSR) 9

2.4.1 Disease Occurence 10 2.4.2 Ganoderma species 11

2.4.3 Disease Symptoms 12 2.5 Disease Control and Management Strategies 13

2.5.1 Agronomic Practices 13 2.5.2 Surgery and Soil Mounding 14 2.5.3 Fungicides 15 2.5.4 Replanting Technique 16 2.5.5 Development of Resistant Varieties 17 2.5.6 Biological Control Agents 19

2.6 Fungi and the Cell Wall Structure 19 2.7 Defense Responses in Higher Plants 20 2.8 Molecular Mechanisms 23 2.9 Antifungal Proteins 27 2.10 Pathogenesis-Related (PR)-proteins 28

2.10.1 PR-1 proteins 30 2.10.2 PR-2 proteins (β-glucanases) 30 2.10.3 PR-3 proteins (chitinases) 31 2.10.4 PR-4 (chitin-binding) proteins 32 2.10.5 PR-5 (TL) proteins 34 2.10.6 Defensins 35

2.11 Application of Identified Defense-Related Proteins 36

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2.12 Application of Biotechnology in Oil Palm Disease 37 Management

2.12.1 Types of Gene Expression Approaches 38 2.12.2 Suppression Subtractive Hybridization (SSH) 39 2.12.3 Subtractive Cloning – The Basics of SSH 41 2.12.4 Application of SSH 42 2.12.5 Identification of Differentially Expressed Gene 43

Transcripts by SSH 3 MATERIALS AND METHODS 45

3.1 Plant Materials 45 3.2 Fungal Materials 45 3.3 Artificial Inoculation of G. boninense 45 3.4 Sampling 46 3.5 Total RNA Extraction and Quantification 47 3.6 Agarose Gel Electrophoresis 48 3.7 Purification of Poly A+ RNA from Total RNA 49 3.8 Suppression Subtractive Hybridization (SSH) 50

3.8.1 First-strand cDNA Synthesis 51 3.8.2 Second-strand cDNA Synthesis 51 3.8.3 RsaI Digestion 53 3.8.4 Adaptor Ligation 53 3.8.5 First Hybridization 55 3.8.6 Second Hybridization 55 3.8.7 PCR Amplification 56

3.9 Cloning into a TA vector 58 3.10 Plasmid Isolation 59 3.11 EcoRI Digestion 60 3.12 Size Fractionation 60 3.13 Purification of DNA Fragment from Agarose Gel 61 3.14 Quantitating the DNA with Ethidium Bromide Plate Assay 61 3.15 Sequencing of cDNA 62 3.16 Membrane Preparation for Reverse Northern Blot 62 3.17 Synthesis of Single-Stranded cDNA for Probe Labeling 63 3.18 Labeling of Single Stranded cDNA with 32P-dCTP 65 3.19 Reverse Northern Hybridization 65 3.20 Film Developing and Fixing 66 3.21 Northern Blot 67 3.22 Preparation of Probes 69 3.23 Hybridization and Washing of Membranes 69 3.24 Reverse Transcription-PCR 71 3.25 Analysis of DNA Sequence 72

4 RESULTS 74

4.1 Artificial Inoculation of G. boninense 74 4.2 Total RNA Extraction and Quantification 76 4.3 Purification of Poly A+ RNA from Total RNA 79 4.4 Subtractive Library Construction 79

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4.5 Sequence Analysis 83 4.5.1 Homology Comparisons and Sequence Analysis 85 4.6 Reverse Northern Analysis 92 4.7 Northern Analysis 93 4.8 RT-PCR 97

5 DISCUSSION 99 6 CONCLUSION 110 BIBLIOGRAPHY 112 APPENDICES 128 BIODATA OF THE AUTHOR 134

LIST OF TABLES

Table Page 1 Classification of Pathogenesis-Related Proteins (PR Proteins) 22

according to Van Loon et al., 1994 and Van Loon et al., 1999. 2 Oil palm tissue samples and its OD260/280 ratios obtained 78

from the extracted RNA. 3 Genes that were postulated to be pathogenesis-related in 86

the G. boninense-inoculated oil palm.

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LIST OF FIGURES

Figure Page 1 An illustration of the main features involved in a of 26

host-pathogen interaction. (Modified from http://www.staff.kvl.dk/~dacoj3/resource/model-colour.htm.)

2 Isolation of G. boninense from oil palm root at 2 months 75

after artificial inoculation on GSM. Roots were labeled 1 to 6 according to their sequence from the inoculation point up to the basal stem. The presence of brown halo and mycelia could be found around roots numbered 1 and 2.

3 Visualization of the 28S and 18S RNA on RNA gel where 77

lane 1 is the selected total RNA sample prepared from basal stem tissues and lane 2 is the selected total RNA sample prepared from spear leaf tissues. RNA marker (Promega) is loaded in lane M.

4 Various stages of hybridization as analyzed on 2% agarose 81

gel. Lane M1 represents pBR322 DNA/AluI marker, lanes 1-6 represent results from the hybridization steps of basal stem tissue samples, lanes 7-12 represent results from the hybridization steps of spear leaf tissue samples and lane M2 represents lambda DNA/HindIII marker.

5 Blue and white colonies on LB agar after incubation at 37 82

°C overnight. 6 Restriction enzyme digestion of plasmids on 1% (w/v) 82

agarose gel. Lane M represents GeneRulerTM DNA Ladder Mix, odd numbered lanes represent digested plasmids while even numbered lanes represent undigested plasmids.

7 A pie chart showing the percentage of the DEGs obtained 84 according to their respective types.

8 The nucleotide sequence alignment of putative chitinase 88 genes MAG44, MAG59, MAG72, and MAG87 from T1.

Identical nucleotides sequences are shown boxed.

9 The nucleotide sequence alignment of putative endochitinase 91 genes MAG208, MAG211, MAG225, MAG229, MAG237, MAG252, and MAG254 from T1. Identical nucleotides sequences are shown boxed.

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10 Reverse northern analysis of the selected cDNA inserts. Lane 1 93 contained the cDNA insert of clone MAG 43. Lane 2 contained the cDNA insert of clone MAG 59. Lane 3 contained MAG 10, lane 4 contained MAG 40, lane 5 contained MAG 225, lane 6 contained MAG 70 and lane 7 contained MAG 286.

11 Northern blot analysis of total RNA. Total RNA from T1 94

and T2 were transferred to Hybond N+ membrane and was then hybridized with radiolabeled cDNA inserts namely MAG 43, MAG 59, MAG 225 and MAG 286.

12 Northern blot showing the time course of selected cDNAs 96 expression in T1. Total RNAs were prepared from G. boninense-inoculated basal stem and spear leaf tissue samples for the indicated times (week(s) post-inoculation). Each lane contains 5 µg of total RNA. Lanes 1 and 2 contain 1 week post-inoculation basal stem tissue, lanes 3 and 4 contain 2 weeks post-inoculation basal stem tissue, lanes 5 and 6 contain 4 weeks post-inoculation basal stem tissue, lanes 7 and 8 contain 1 week post-inoculation spear leaf tissue, lanes 9 and 10 contain 2 weeks post-inoculation spear leaf tissue, lanes 11 and 12 contain 4 weeks post-inoculation spear leaf tissue and lanes 13 and 14 contain 48 weeks post-inoculation spear leaf tissue. Northern blots were hybridized with radiolabeled MAG 43 (A), MAG 59 (B) and MAG 225 (C) cDNA fragments.

13 RT-PCR analysis of differentially expressed genes obtained 98 from the SSH technique. Lane M: GeneRulerTM DNA Ladder Mix; lane 1: amplification of MAG 43 from T2 RNA; lane 2: amplification of MAG 43 (480 bp) from T1 RNA; lane 3: amplification of MAG 59 from T2 RNA; lane 4: amplification of MAG 59 (320 bp) from T1 RNA; lane 5: amplification of MAG 225 from T2 RNA; and lane 6: amplification of MAG 225 (600 bp) from T1 RNA.

LIST OF ABBREVIATIONS

% percent

~ about / roughly

< less than

> more than

°C degree Celsius

µg microgram

µJ microjoule

µl microliter

µM micromolar

32P radioactive isotope of phosphorus

α alpha

β beta

β-NAD beta-nicotinamide adenine dinucleotide

ATP adenosine triphosphate

avr-R avirulence-resistance

BLAST Basic Local Alignment Search Tool

BSA bovine serum albumine

BSR basal stem rot

Ca2+ calcium ion

cDNA complementary DNA

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cm centimeter

cm2 centimeter square

CPKO crude palm kernel oil

CPO crude palm oil

D × P Dura × Pisifera

dATP 2’-deoxyadenosine 5’-triphosphate

dCTP 2’-deoxycytidine 5’-triphosphate

DEGs differentially expressed genes

dGTP 2’-deoxyguanosine 5’-triphosphate

dH2O distilled water

DNA deoxyribonucleic acid

dNTP deoxyribonucleotide triphosphate

DOA Department of Agriculture

dTTP 2’-deoxythymidine 5’-triphosphate

EDTA ethylenediaminetetraacetic acid

EtBr ethidium bromide

g gram

GRPs glycine-rich proteins

GSM Ganoderma selective medium

h hour

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H2O water

H2O2 hydrogen peroxide

kb kilo base pairs

KCl kalium chloride

kDa kiloDalton

LB Luria-Bertani

LiCl lithium chloride

LTPs lipid-transfer proteins

M molar

MARDI Malaysian Agriculture Research and

Development Institute

MgCl2 magnesium chloride

min minute

ml mililiter

mM milimolar

MPOB Malaysian Palm Oil Board

MPOB-UKM Malaysian Palm Oil Board-Universiti Kebangsaan

Malaysia

MPOC Malaysian Palm Oil Council

MPOCF Malaysian Palm Oil Wildlife Conservation Fund

mRNA messenger RNA

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NaCl natrium chloride

ng nanogram

nm nanometer

OAS oral allergy syndrome

OD optical density

PCR polymerase chain reaction

PORIM Palm Oil Research Institue of Malaysia

PR proteins pathogenesis-related proteins

RIPs ribosome-inactivating proteins

RNA ribonucleic acid

rpm revolutions per minute

rRNA ribosomal RNA

SAR systemic acquired resistance

SDS sodium dodecyl sulfate

SSC sodium chloride-sodium citrate

SSH suppression subtractive hybridization

T1 treatment 1

T2 treatment 2

T3 treatment 3

TAE tris-acetate-EDTA

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TL proteins thaumatin-like proteins

Tris-HCl tris hydrochloride

U unit

UV ultraviolet

V/cm volt per centimeter

v/v volume per volume

w/v weight per volume

CHAPTER 1

INTRODUCTION

Oil palm (Elaeis guineensis) is one of the most important oil tree species in

tropical regions because of the raw materials it produces, palm oil and palm

kernel oil. However, fungal diseases of the oil palm can cause very serious

losses in production. Important diseases of oil palms are reported as vascular

wilt caused by Fusarium oxysporum, basal stem rot (BSR) (G. boninense), red

ring disease (Rhadinaphelenchus cocophilus), sudden wilt (Phytomonas staheli)

and spear rot (unknown pathogen) (Turner, 1981; Ariffin, 2000). As far as the

disease problem to oil palm in Malaysia is concerned, the most serious disease

is BSR and it requires an urgent solution.

BSR is not new to Malaysia as it has been known to attack oil palm since the

early years when the crop was introduced into this country. The disease was

first reported in 1931 (Thompson, 1931). Losses due to BSR is not only through

the direct reduction in oil palm stands, but also through a decrease in fruit bunch

number and weight from diseased standing palms as well as those with sub-

clinical infections (Turner, 1981). The disease can result in the death of more

than 80% of the plants by the time they are half-way through their normal

economic life and losses reaching 30% have quite frequently occurred (Turner,

1981). A survey indicated that BSR was present in more than 50% of the oil

palm fields in Peninsular Malaysia (Idris et al., 2001).

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The threat of G. boninense to the oil palm industry in this country warrants new

and more aggressive approaches in finding solution to the disease. It would

benefit the industry very much if a rapid and accurate diagnostic technique for

detecting G. boninense in oil palms at an early stage of infection is available.

Studies by De Franqeville et al. in 2001 showed that there were differences in

the susceptibility of germplasm materials from different genetic origins to BSR.

This provides hope in the screening for oil palm varieties with reduced levels of

susceptibility using existing genetic materials. The development of diagnostic

tools based on PCR primers have also provided new dimension for early

detection of the pathogen in oil palms (Bridge et al., 2001). Another new area of

investigation is in the altered expression of several classes of genes in plants

when infected by pathogens. These include genes associated with cell

maintenance and development, genes involved in the biosynthesis of lignin and

phenolics and genes implicated in oxidative burst, programmed cell death or

hypersensitive response (Schenk et al., 2000). Thus, the isolation and

characterization of disease resistance or stress responsive genes through

molecular biology approach are the initial steps in the commencement of a

molecular biology approach towards an understanding of the defense and stress

response mechanisms in oil palms.

The discovery of novel genes has traditionally been a laborious task. The

conventional breeding techniques to produce disease resistant and stress tolerant

lines are limited, time consuming and also laborious. Consequently, the

2