universiti putra malaysia genetic …psasir.upm.edu.my/7545/1/abs_----__fs_2009_27.pdfto my babies...

UNIVERSITI PUTRA MALAYSIA

GENETIC CHARACTERIZATION OF GANODERMA sp. USING INTERFERTILITY AND MOLECULAR METHODS

HUSRITA BINTI HUSSIN

FS 2009 27

GENETIC CHARACTERIZATION OF GANODERMA sp. USING

INTERFERTILITY AND MOLECULAR METHODS


Thesis Submitted in Fulfilment of the Requirement for the Degree of Master of Science, Universiti Putra Malaysia

August 2009

500 bp

II




Thesis Submitted in Fulfilment of the Requirements for the Degree of Master of Science, Universiti Putra Malaysia

August 2009

ii

Abstract of the Thesis Submitted to the Senate of Universiti Putra Malaysia in Fulfilment of the Requirement for the Degree of Master of Science.



By

HUSRITA HUSSIN

August 2009

Chairman: Professor Faridah Abdullah, PhD

Faculty : Science

Laccate polypores of the genus Ganoderma are wide spread but species

identification cannot be easily done based only on traditional methods. The

species of Ganoderma boninense is of economic importance to Malaysia as it

causes the disease Basal Stem Rot (BSR) of oil palms. In this study, 53

Ganoderma were isolates from different hosts. Which are 23 isolates from

infected oil palms (Elaeis guineensis), 12 from various non-Elaeis palmae hosts,

18 Ganoderma from non-palm woody hosts and 2 non-Ganoderma as an outgroup

specimens were used to conduct interfertility studies, Random Amplified

Microsatellite DNA (RAMS) and Internal Transcribe Spacer (ITS1). The

interfertility studies showed that 34 laccate Ganoderma that collected from

various palmae hosts determined as G. boninense whereas another 12 laccate

Ganoderma samples and all 7 non-laccate Ganoderma specimens were non-

iii

boninense. The compatible dikaryotic pairs were further validated through

sporophore induction studies of which all those tested as G. boninense produced

viable fruiting bodies. Molecular studies using Random Amplified Microsatellite

(RAMS) generated a dendrogram of two major clusters. Cluster I, all the laccate,

non-laccate Ganoderma and two non-Ganoderma samples which are determined

as non-boninense G. by interfertility study were grouped together except two

isolates which is determined as G. boninense (FA3026 and FA5014). This showed

that RAMS not totally support interfertility studies. Clusters II consists of all

laccate Ganoderma which are determined as G. boninense in interfertility study.

The dendrogram constructed from gene sequence data of ITS 1 region of the

rDNA produced three major clusters. Major Cluster I consisted of outgroups

samples PLP, while Major Cluster II was WRR. Major Cluster III separated into

two sub-cluster IIIA and IIIB, sub-cluster IIIA were consisted of all laccate

Ganoderma samples and this sub-cluster were separated into two, 34 samples are

determined as G. boninense and 12 samples are non-boninense G. Sub-cluster IIIB

consisted of 7 non-laccate Ganoderma and determined as non-boninense G.

BLAST analysis showed that all 34 determined as G. boninense, 4 were G.

neojaponicum, 2 isolates were G. formosanum, 3 were G. lucidum, 2 G. tsugae, 2

G. resinaceum, 2 G. cupreum, 3 G. adspersum and 4 isolates as a G. australe. This

investigation found that ITS not only analysed at genus level but also able to

identified at species level. The phylogenetic analysis by ITS regions showed

agreement with the interfertility data but not with RAMS analysis. The clustering

of Ganoderma isolates in ITS and RAMS are difference because different

approaches were use. With a different types of primers used in the two different

DNA-based methods, the banding sites in fungal genome would also be different

iv

which could lead to different clustering of the isolates in the cluster analysis. In

this study found that G. boninense is a single species which causes the basal stem

rot disease on oil palm in Malaysia. G. boninense also can effect to non-palmae

woody host, which found on Caesalpinia sappan.

v

Abstrak Tesis Yang Dikemukan Kepada Senat Universiti Putra Malaysia Sebagai Memenuhi Keperluan Untuk Ijazah Sarjana Sains

CIRI-CIRI GENETIK GANODERMA sp. MENGGUNAKAN KESERASIAN

PENGAWANAN DAN KAEDAH MOLEKULAR

Oleh

HUSRITA HUSSIN

Ogos 2009

Pengerusi: Profesor Faridah Abdullah, PhD

Fakulti : Fakulti Sains

.

Spesies Ganoderma berwarna kemerahan dengan permukaan dorsal berkilat

banyak ditemui tetapi sehingga kini pengenalpastian spesiesnya tidak mudah

dilakukan hanya dengan berasaskan kaedah tradisional. Spesies Ganoderma

boninense mempengaruhi kepentingan ekonomi di Malaysia kerana ia

menyebabkan penyakit reput pangkal batang kelapa sawit. Di dalam kajian ini,

sebanyak 53 isolat Ganoderma yang dikutip dari pelbagai perumah. Sebanyak 23

isolat dikutip dari pokok kelapa sawit yang dijangkiti (Elaeis guineensis), 12 isolat

Ganoderma dari pokok spesies palma, 18 isolat Ganoderma dari pokok berkayu

dan 2 isolat bukan Ganoderma dijadikan sebagai sampel luaran untuk dijalankan

kajian keserasian pengawanan, ‘random amplified microsatellite DNA (RAMS)

dan ‘internal transcribe spacer (ITS1)’. Ujian keserasian pengawanan

vi

menunjukkan sebanyak 34 isolat Ganoderma kemerahan yang telah dikutip dari

pelbagai perumah palma adalah G. boninense manakala 12 isolat Ganoderma

kemerahan dan 7 isolat bukan Ganoderma kemerahan merupakan bukan G.

boninense. Pasangan dikarion yang mengawan akan dilakukan pengesahan lanjut

dengan melakukan aruhan sporofor untuk menghasilkan jasad buah dan didapati

semua pasangan yang mengawan menghasilkan jasad buah. Kajian molekular

menggunakan ‘random amplified microsatellite’ (RAMS) menghasilkan

dendrogram yang terdiri daripada dua major kluster. Kluster I mengandungi

semua Ganoderma kemerahan, bukan Ganoderma kemerahan dan dua isolat

bukan Ganoderma yang mana semuanya dikenalpasti sebagai bukan G. boninense

oleh ujian keserasian pengawanan kecuali dua isolate yang dikenalpasti sebagai G.

boninense (FA3026 dan FA5014) terkelompok bersama. Ini menunjukkan RAMS

tidak sepenuhnya menyokong keputusan ujian keserasian pengawanan. Kluster II

mengandungi semua Ganoderma kemerahan yang mana telah dikenalpasti sebagai

G. boninense oleh ujian keserasian pengawanan. Dendrogram yang terbina dari

data jujukan gen lokus ITS 1 rDNA menghasilkan tiga major kluster. Major

kluster I mengandungi isolat luar (bukan Ganoderma) iaitu PLP, manakala major

kluster II pula WRR. Major kluster III membahagi kepada dua sub-kluster iaitu

IIIA dan IIIB, sub-kluster IIIA terdiri daripada semua isolat Ganoderma

kemerahan dan sub-kluster ini membahagi lagi kepada dua, sebanyak 34 isolat

dikenalpasti sebagai G. boninense dan 12 isolat bukan G. boninense. Sub-kluster

IIIB terdiri daripada 7 isolat Ganoderma bukan kemerahan dan dikenalpasti

sebagai bukan G. boninense. Analisis BLAST menunjukkan 34 isolat adalah G.

boninense, 4 adalah G. neojaponicum, 2 isolat adalah G. formosanum, 3 adalah G.

lucidum, 2 adalah G. tsugae, 2 G. resinaceum, 2 G. cupreum, 3 G. adspersum dan

vii

4 isolat G. australae. Kajian ini mendapati kajian ITS tidak hanya dapat

menganalisis organisms pada tahap genus tetapi juga sehingga ke tahap spesies.

Analisis filogenetik lokus ITS menyetujui keputusan ujian keserasian pengawanan

tetapi tidak menyokong keputusan RAMS. Pengklusteran isolate Ganoderma oleh

analisis ITS dan RAMS adalah berbeza kerana berbeza pendekatan yang

digunakan. Dengan perbezaan jenis jujukan primer yang digunakan oleh kedua-

dua kaedah DNA ini, jalur-jalur yang dihasilkan juga akan berbeza yang mana

menyebabkan pengkelasan isolate di dalam analisis pengklusteran juga berbeza.

Kajian ini mendapati G. boninense merupakan spesies tunggal yang menyebabkan

penyakit reput pangkal batang kelapa sawit di Malaysia. G. boninense juga

menjangkiti perumah bukan palma iaitu pokok berkekayu yang mana dijumpai

pada pokok Caesalpinia sappan.

viii

ACKNOWLEDGEMENTS

I wish to express my sincere thanks and gratitude to the chairman of the

supervisory committee, Prof. Dr. Faridah abdullah, for her invaluable advice,

guidance, constant encouragement and endless support throughout the duration of

the study and for her critical comments and constructive suggestions during the

preparation of my thesis.

I also wish to express my appreciation and sincere gratitude to the supervisory

committee members, Prof. Dr. Tan Soon Guan and Dr. Faridah Qamaruzzaman

for their advice, guidance and helpful suggestions throughout the course of my

study and in the preparation of my thesis. I am also indebted to Dr. Ho Chai Ling

from the Department of Cell and Molecular Biology, Faculty of Biotechnology

and Biomolecular Sciences, Universiti Putra Malaysia who has given

encouragement and endless support as well as financial support, especially

towards the last few months of my studies. Grateful acknowledgements are also

extended to the staff of the Department of Biology, Faculty of Science, Universiti

Putra Malaysia, for their help and cooperation.

Very special thanks are due to my beloved family, father Hussin Bin Ali, mother

Esun Binti Ismail and also my beloved husband Sallehuddin Bin Hj. Arshad and

to my babies Adam Izzrein and Nurkhalishah Syazwina (Ibu love both of you so

much), for their support, encouragements, understanding and sacrifices which

inspired and motivated me during the duration of my study.

ix

Finally to my post-graduate friends at Mycology Lab, Department of Biology,

Universiti Putra Malaysia, Noor Haida, Hefni, Nazif, Shafiq, Susen and also from

Genetic Lab, Boon Peng, Hafiz and Mr. Yuzine, thank you for your friendship,

support and laughter which contributed to the successful accomplishment of this

work.

x

I certify that an Examination Committee has met on 29th August 2008 to conduct the final examination of Husrita binti Hussin on her degree of Master of Science thesis entitled ‘Genetic Characterisation of Ganoderma sp. Using Interfertility And Other Molecular Methods’ in accordance with Universiti Pertanian Malaysia (Higher degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulation 1981. The Committee recommends that the student be awarded the degree of Master of Science. Members of the Examination Committee were as follows: MUSTAFA KAMAL BIN ABDUL SATAR, PhD Lecture Faculty of Agriculture Universiti Putra Malaysia (Chairman) MOHD SAID BIN SAAD, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) SITI KHADIJAH BINTI DAUD, PhD Associates Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) SITI AZIZAH BINTI MOHD NUR, PhD Associates Professor School of Biology Universiti Sains Malaysia (External Examiner) ________________________________ HASANAH MOHD. GHAZALI, Ph.D Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date :

xi

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilement of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows: Faridah Binti Abdullah, PhD Professor Faculty of Science Universiti Putra Malaysia (Chairman) Tan Soon Guan, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Faridah Binti Qamaruz Zaman, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Member)

________________________________

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

Date : 16 October 2009

xii

DECLARATION

I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that is has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.

_______________________


DATE: 18 August 2009

xiii

TABLE OF CONTENTS

Page

ABSTRACT II

ABSTRAK V

ACKNOWLEDGEMENT VIII

APPROVAL X

DECLARATION XII

LIST OF TABLES XVI

LIST OF FIGURES XVII

LIST OF ABBREVIATIONS XX

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW

2.1 Ganoderma boninense and basal stem rot of oil palm 6

2.2 Problem with Ganoderma taxonomy 9

2.3 Biological species concept 11

2.3.1 Nuclear migration in Basidiomycetes 12

2.3.2 Mating system in Basidiomycetes 13

2.4 Molecular studies

2.4.1 Polymerase chain reaction (PCR) 15

2.4.2 Random amplified polymorphic DNA 16

2.4.3 Random amplified microsatellite DNA (RAMS) 18

2.4.4 Restriction fragment length polymorphisms (RFLPs) 20

2.4.5 Internal transcribe spacer (ITS) region of the rDNA 22

3 DETERMINATION OF BIOLOGICAL SPECIES OF Ganoderma boninense (PATOULLARD, 1887), BY HOMOKARYOTIC INTERFERTILITY STUDIES

3.0 Introduction 26

3.1 Material and methods

3.1.1 Source of Ganoderma isolate 29

3.1.2 Isolation of fungal mycelia 34

3.1.3 Sporophore induction 35

3.1.4 Spore collection 36

xiv

3.1.5 Spore germination 37

3.1.6 Single spore monokaryon culture 37

3.1.7 Monokaryon mating test 38

3.1.8 Selection for tester strain 38

3.1.9 Testing of conspecificity within Ganoderma samples 39

3.2 Results

3.2.1 Ganoderma colony cultures 40

3.2.2 Basidiomata characteristics from in vitro cultures 42

3.2.3 Basidiospore characteristics 43

3.2.4 Selection and confirmation of tester strains 45

3.2.5 Testing of conspecificity of sample to G. boninense 47

3.2.6 Testing for genetic filtress of compatible G. boninense dikaryon 54

3.3 Discussion 57

4 GENETIC CHARACTERISATION OF Ganoderma boninense USING RANDOM AMPLIFIED MICROSATELLITE (RAMS)

4.0 Introduction 62


4.1.1 Fungal cultures 64

4.1.2 Preparation of mycelia 64

4.1.3 DNA extraction by Phenol-Chloroform method 65

4.1.4 DNA visualisation 67

4.1.5 DNA quantification 67

4.1.6 Random amplified microsatellite DNA amplification 68

4.1.7 Scoring and genetic distance analysis 70

4.2 Results

4.2.1 DNA visualisation 71

4.2.2 Optimisation for RAMS analysis 72

4.2.3 Random amplified microsatellite DNA analysis 72

4.2.4 Cluster analysis based on RAMS data 78

4.3 Discussion 85

xv

5 CHARACTERISATION OF THE INTERNAL TRANSCIBED SPACER 1 (ITS1) REGION OF Ganoderma

5.0 Introduction 91


5.1.1 DNA extraction of samples 95

5.1.2 Primers 95

5.1.3 PCR amplification of the ITS 1 regions of rDNA 95

5.1.4 Electrophoresis 96

5.1.5 Direct sequencing of PCR product 97

5.1.6 Sequence analysis 98

5.2 Results

5.2.1 Size of PCR-Amplified ITS1 regions 101

5.2.2 Blast analysis 105

5.2.3 Cluster analysis 108

5.3 Discussion 110

6 GENERAL DISCUSSION AND CONCLUSION

General discussion 115

Conclusion 121

BIBLIOGRAPHY 123

APPENDICES 135

BIODATA OF STUDENT 157

xvi

LIST OF TABLES

Table Page

3.1 Sources and origins of Ganoderma isolates collected from infected oil palms (Elaeis guineensis)

30

3.2 List of sources and origins of Ganoderma isolates that were collected from non-Elaies Palmae

31

3.3 Source and origins of laccate Ganoderma isolates that were collected from various non-palm woody hosts

32

3.4 Sources and origins of non-laccate Ganoderma that were collected from various non-palm woody hosts

33

3.5 Monosporus cultures and their compatible partner derived from the first round of mating

45

3.6 Comfirmation of tester strains by mating amongst FA5011 testers

46

3.7 Ganoderma samples biologically determined as G. boninense

49

3.8 Laccate Ganoderma samples biologically determined as non-boninense Ganoderma based on interfertility tests

50

3.9 Non-laccate Ganoderma samples that were determined as non-boninense Ganoderma based on interfertility tests

50

4.1 The 8 primers and their nucleotide sequences used in RAMS analysis of Ganoderma DNA.

69

4.2 The optimization conditions for RAMS analysis using 8 primers for RAMS analysis

72

4.3 The 8 oligonucleotide primers and the band characteristics of 53 Ganoderma produced

73

4.4 Genetic distances among Ganoderma isolates based on Random amplified microsatellite marker (RAMS)

83

4.5 Major and minor clusters of Ganoderma isolates and other hosts as illustrated in the dendrogram for RAMS analysis

84

5.1 Published nucleotide sequences of the ITS1 region from the difference Ganoderma species obtained from Genbank.

107

5.2 Major and minor clusters of Ganoderma isolates and other hosts as illustrated in the phylogeny for ITS 1 rDNA region analysis.

109

xvii

LIST OF FIGURES

Figure Page

2.1 The typical bracket-shaped sporophores of Ganoderma, with a shiny upper surface and distinct whitish border

8

2.2 Symptom of basal stem rot at an advanced stage. In addition of multiple spear leaves the oldest fronds have died and collapse

8

2.3 Clamp connection formation

13

2.4 Schematic diagram of the locations of rDNA

23

3.1 Map of Peninsular Malaysia showing the locations of the sampling sites

34

3.2 Direct isolation of Ganoderma fruiting bodies on Potato Dextrose Agar (PDA)

35

3.3 An actitively sporulating mature sporophores

36

3.4 The placing of two inoculum disc for compatibility mating tests

38

3.5 Schematic diagram of the placement of unknown Ganoderma monokaryon against each of the 4 testers FA5011

40

3.6 Ganoderma (FA5011) on PDA

41

3.7 Ganoderma (FA5014) on PDA

41

3.8 Induced sporophore of isolate FA5014 from infected oil palm

42

3.9 Colonization of rubber wood block with Ganoderma at week 1 and 8 weeks after inoculation

42

3.10 Artificially induced basidiomata of Ganoderma FA5011 on rubber wood substrate and formation of mature basidiomata of the same initials, 8 weeks later

43

3.11 Basidiospores of Ganoderma boninense (FA5011) showing ellipsoidal structure

44

3.12 Spore of G. boninense (FA5011) showing a double wall membrane and a hilar plaque at the apex

44

3.13 Spore of G. boninense (FA5011) showing a double wall membrane and a truncated apex

44

xviii

3.14 Dikaryotic hypha of Ganoderma, showing clamp connection at septum and monokaryotic hypha of Ganoderma, showing no clamp connection at septum

47

3.15 Induced sporophore of isolate PP28 determined as G. boninense collected from infected oil palm

51

3.16 Compatible reactions showing no barrage lines between dual cultures of each of the 4 monokaryotic tester strains of G. boninense with PP28 on the upperside and reverse side colonies

51

3.17 Induced sporophore of isolate GJ determined as non-boninense G.

52

3.18 Incompatible reactions showing a barrage lines between dual cultures of each of the 4 monokaryotic tester strains of G. boninense with GJ on the upperside and reverse side colonies

52

3.19 Induced sporophore of isolate FA3024 determined as non-boninense G. collected from infected oil palm

53

3.20 Incompatible reactions showing a barrage lines between dual cultures of each of the 4 monokaryotic tester strains of G. boninense with FA3024 on the upperside and reverse side colonies

53

3.21 Induced sporophores of isolates FA5502 from infected coconut determined as non-boninense G.

54

3.22 Induced sporophores from FA5012 and induced of FA5012 with T4 monokaryon hybrid

54


55


55


55


56


56

4.1 Culture of Ganoderma on Yeast Extract Sucrose Broth at 4 days after inoculation

65

xix

4.2 Single banding patterns of 16 Ganoderma isolates showing positively extracted DNA

71

4.3 RAMS banding patterns produced by primer LR1. Sample 1 to 16

74


74


75

4.6 RAMS banding patterns produced by primer BP8. Sample 1 to 16

75


76


76


77

4.10 RAMS banding patterns produced by primer P1A. Sample 1 to 16

77

4.11 RAMS dendrogram from UPGMA analysis using genetic distance based on Simple Matching Coefficients derived from Nei and Li (1979) – original

80

4.12 RAMS dendrogram from UPGMA analysis using genetic distance based on Simple Matching Coefficients derived from Nei and Li (1979)

81

5.1 Banding pattern of the amplified ITS-1 region. Lane 1 to 15.

102

5.2 Banding pattern of the amplified ITS-1 region. Lane 16-26.

103

5.3 Banding pattern of the amplified ITS-1 region. Lane 27-31.

103

5.4 Banding pattern of the amplified ITS-1 region. Lane 35-39

104

5.5 Banding pattern of the amplified ITS-1 region. Lane 40-55 104

5.6 Gene phylogeny fron rDNA sequences of ITS-1 region of Ganoderma isolates.

108

xx

LIST OF ABBREVIATIONS

BSR Basal Stem Rot

bp Base pairs

µg Microgram

ITS Internal Transcribed Spacer

M Molar

µl Microliter

ml Millimeter

NTSYS-pc Numerical Taxonomy and Multivariate Analysis System

PCR Polymerase Chain Reaction

PAUP Phylogenetic Analysis Using Parsimony

PDA Potato Dextrose Agar

RAMS Random Amplified Microsatellite DNA

RAPD Random Amplified Polymerase DNA

RFLP Restriction Fragment Length Polymerase

rpm Revolutions per minutes

UPGMA Unweight Paired Group Matching Analysis

V Volt

W Watt

IFFPRI Institute of Forestry and Forest Research Product, Japan

MPOB Malaysian Palm Oil Board

R FRIM Forestry Research Institute Malaysia

UPM Universiti Putra Malaysia

BRE Building Research Establishment

TFRI Taiwan Forest Research Institute

sp. Species

NaOCl Natrium hipochloride

TBE Tris Borate EDTA

Tris HCl Tris(hydroxymethyl)aminutesomethanehydrochloride

EDTA EDTA, Disodium Salt (Dihydrate)

SDS Sodium Dodecyl sulfate

NaCl2 Natrium Chlorida

CHAPTER 1

INTRODUCTION

The disease basal stem rot of oil palm (Elaeis guineensis Jacq.) caused by

Ganoderma in Malaysia was first recorded in Malaysia by Thompson in 1931.

Turner (1981) also believed that several species of Ganoderma or various strains

of the same species may associate with oil palms in Malaysia. Turner (1981)

reported the number of Ganoderma species associated with basal stem rot as

fifteen. However, Ho and Nawawi (1985) reported that basal stem rot was caused

by only one single species, and that is G. boninense, based on morphological

characteristics of the sporosphores. The name G. boninense was given by

Patouillard in 1887 from describing the collection of Ganoderma from the Bonin,

Nouka-hiva and Marquesa Islands in the Pacific Oceans. He described them

without establishing formal taxonomic distinctions (Commonwealth Agricultural

Bureaux, 1975).

The taxonomy of the whole Ganoderma genus is somewhat problematic at

present. This is due to a number of reasons, including the number of

heterogeneous forms and a large number of criteria upon which different

classification approaches have been based on. Species identification, which is

usually based on morphological characteristics, can be misleading and have

resulted in the description of over 250 species, with frequent synonymy as a

result. The situation is further complicated by the description of a number of

species complexes by various authors. As a consequence, the identification and

2

distribution of tropical species of Ganoderma remains unclear. A comprehensive

revision of the taxonomy has been recommended by a number of authors for

several decades (e.g. Steyaert, 1975b). Corner (1983) reviewed Steyaert’s

classification systems for Ganoderma, and made conclusion that gradation

occurred in all morphological features used to describe species. A number of

authors have thus concluded that the use of basidiomatal morphology, spore

characteristics and cutis anatomy alone were insufficient for delimitation of

species (Atkinson, 1908; Haddow, 1931; Furtado, 1965, Steyaert, 1980; Furtado

1981; Bazzalo & Wright, 1982; Corner, 1983 and Adaskaveg and Gilbertson,

1986), but their studies failed to define unambiguously the observed structure.

Murrill (1902, 1908, and 1915) considered host specificity, geographical

distribution and macromorphology of basidiomes as primary taxonomic

characters. As stated by Moncalvo & Ryvarden (1997), among many described

species, several are represented by one or few collections, and the type specimens

have sometimes been lost or lack of modern descriptions.

The method for an early diagnosis of basal stem rot (BSR) by sampling

diseased material of oil palms with a wood drill have been developed: (a) a

colorimetric method, using ethylenediaminutesetetraacetic acid (EDTA) to detect

G. lucidum in coconut where the causal agent of the ‘Thanjavur’ wilt disease was

used by Natarajan et al,. (1986) and (b) the use of semiselective media for

cultivating Ganoderma on agar plates (Darus et al,. 1991). However, all

conventional methods are time consuming and of a rather low accuracy.

universiti putra malaysia genetic …psasir.upm.edu.my/7545/1/abs_----__fs_2009_27.pdfto my babies...

Documents