UNIVERSITI PUTRA MALAYSIA

MORPHOLOGICAL, BIOCHEMICAL AND DNA STUDIES FOR THE DEVELOPMENT OF MARKERS FOR THE SELECTION OF Theobroma cacao L.

CLONES RESISTANT TO VASCULAR STREAK DIEBACK DISEASE

ROSMIN KASRAN

FSAS 1999 1

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MORPHOLOGICAL, BIOCHEMICAL AND DNA STUDIES FOR THE

DEVELOPMENT OF MARKERS FOR THE SELECTION OF Theobroma cacao L. CLONES RESISTANT TO VASCULAR STREAK DIEBACK DISEASE

ROSMIN KASRAN

DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

October 1999

MORPHOLOGICAL, BIOCHEMICAL AND DNA STUDIES FOR THE DEVELOPMENT OF MARKERS FOR THE SELECTION OF Theobroma cacao L.

CLONES RESISTANT TO VASCULAR STREAK DIEBACK DISEASE

By

ROSMIN KASRAN

Thesis Submitted in Fulfilment of the Requirements for the Degree of Doctor of Philosophy in the Faculty of Science and Environmental Studies Universiti

Putra Malaysia

October 1999

Dedicated to my family

ACKNOWLEDGEMENTS

I would like to express my special thanks and appreciation to my

supervisory committee, Prof. Dr. Marziah Mahmood, Assoc. Prof. Dr. Abdullah

Sipat and Assoc. Prof. Dr. Radzali Muse for their support and professional

guidance during the course of this academic and research program.

My appreciation is recorded to all academic advisory and staff in the

Department of Biochemistry and Microbiology, Faculty of Science and

Environmental Studies, Universiti Putra Malaysia for their kind cooperation.

I am greatly indebted to the Malaysian Agricultural Research and

Development Institute (MARDI) for granting the financial support and study leave.

My experiment would not have been successful without the efficient and

dedicated help of Mr. Rajinder Singh (Plant Science and Biotechnology, PORIM).

To Mr. Aziz, Mr. Azlan, Mr. Sumber, Mr. Tee, Miss Asnita, Miss Hafizah, Miss Iteu,

Mrs. Janna, Miss Radziah, Miss Ramani, Miss Suzita and Miss Zuraidah, I would

l ike to thank them for their kind cooperation.

Last but not least I would l ike to express my deepest gratitude to my family

for their constant support and inspiration throughout the study.

i i i

TABLE OF CONTENTS Page

ACKNOWLEDGEMENT . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . i i i LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . , ............. , ...... , ............. , ............ , ...... vii LIST OF FIGURES ........................................................ , ...... , ............. viii LIST OF PLATES ........................... , ............. , ...... , ............. , ...... , ... ... ... ... ix LIST OF ABBREVIATIONS .... , ................................. , '" ........................ x ABSTRACT .......... , ...... , ........................................ " ..... , ......... '" .,. ... .. xi ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

CHAPTER

INTRODUCTION ..... , ............. , ...... , ................................. 1

II LITERATURE REViEW..... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..... 5

Economic Importance of Cocoa ....... ,. '" .. , ... '" ... '" ....... ,. ... .. 5 Cocoa Industry in Malaysia............ ...... ... ... ...... ......... ... ... .... 7 Morphological Variability of Cocoa Trees... ............ .............. 11 Fungal Diseases of Cocoa ......... '" .. , ..... , ... ... ... ... ... ... ... ... ... 14 Vascular Streak Dieback Disease ....................................... 15

Morphology of Oncobasidium theobromae .. . . , ...... , ...... , 15 Culturing and Pathogenicity Tests ............................. 16 Disease Symptoms ....... ,. '" .. , ................... , ...... ,. ... .. 17 Epidemiology and Impact of Disease ............ '" .... ... ... 19

Biochemical Responses of Plants to Pathogens ............. '" .... 20 Changes in Phenol LeveL ........................................ 22 Changes in Polyphenol Oxidase Activity... . . . . . . . . . . . . .. . . .. 24 Changes in Peroxidase Activity ................................. 26 Changes in Chitinase and B-1,3-Glucanase Activity ...... 29 Changes in Phytoalexin LeveL .................................. 33

Approach in Controlling Oncobasidium theobromae... . . . ... ... ... 34 Importance of DNA Markers in Plant Selection... ...... ...... .... ... 36 Genome Size of Theobroma cacao ..................................... 42

III MATERIALS AND METHODS.. ...... ................. ... ......... ....... 43

Field Plots ...................................................................... 43 Morphological Characteristics of Cocoa Clones and Their Relationships to VSD Disease Susceptibility ..... , ... ... ... ... ... .... 43

Plant Materials ......................................... , ............ 43 Morphological Data Co"ected ........................ '" ........ 44

Variation of Soluble Phenol, Enzyme Level and Isozyme Pattern in the Leaves of Several Cocoa Clones Used as Specific Indicators for Susceptibility to VSD Disease .............. 45

Plant Materials ............... '" .................................... 45 Leaf Samples ...................... , .................... , ... ... .... 45 Determination of Total Soluble Phenol ................ '" .... 45

iv

Determination of Peroxidase and Polyphenol Oxidase Activity ..................................... , ........... , ... 47 Determination of Chitinase and B-1,3-Glucanase Concentration ....................................................... 49 Polyacylamide Gel Electrophoresis ............................ 52 Statistical Analysis ............................................. " .. 56

Light and Electron Microscopy Study of the Leaves of Cocoa from Resistant and Susceptible Plants After Infection with Oncobasidium theobromae ................................ . ........ 57

Isolation of Fungal Pathogen and Production of Culture .............................................................. 57 Inoculation of Bud Grafted Young Plants ..................... 57 Samples Preparation for Light Microscopy .................. 58 Samples Preparation for Electron Microscopy .............. 61

Genetic Polymorphism of Cocoa Clones Revealed by AFLP Analysis .................................................................... , ....... 63

Plant Materials .......................................................... 63 DNA Extraction .................................................. , ... 63 Quantification and Purity of DNA ............................... 64 Determination of Total Leaf DNA .............................. 64 AFLP Assay ........................................................... 65 Data Analysis .......................................................... 67

The Use of a Unique AFLP DNA Fragment as a Molecular Marker. .................................................... .... ......... . . . . . . . . 68

Plant Materials, DNA Extraction and AFLP Analysis ...... 68 Band Excision, Amplification and Purification ............... 68 Cloning of PCR Product.. ......................................... 69 Preparation of Miniprep Plasmid ............................... 71 Sequencing of Plasmid DNA .................................... 72 Labeling of DNA Probe ............................................ 73 Southern Blotting, Hybridization and Detection of DNA Bands ........................................................... 73

IV RESULTS AND DISCUSSiON ............................................. 76 Morphological Characteristics of Cocoa Clones and Their Relationships to VSD Disease Susceptibility ...................... , .. 76 Variation of Soluble Phenol, Enzyme Level and Isozyme Pattern in the Leaves of Several Cocoa Clones as Specific Indicators for Susceptibility to VSD Disease .......................... 83

Concentration of Total Soluble Phenols ... , ....... " ......... 83 Activity of Peroxidase ....................................... , ...... 85 Activity of Polyphenol Oxidase ............ , .................. '" 88 Concentration of Chitinase and 13-1,3-Glucanase ......... 90 Protein Pattern, Peroxidase, Polyphenol Oxidase, Chitinase and \3-1,3- Glucanase Isozyme Patterns ........ 94

v

Microscopy Study of the Cocoa Leaves from Resistant and Susceptible Clones After Infection with Oncobasidium Theobromae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

Genetic Polymorphism of Cocoa Clones Revealed by AFLP Analysis ............... , ....................... , ....... , ............ 106

The Use of a Unique DNA Fragment as a Molecular Marker ........... , ............................... '" .................... , .... 116

V SUMMARy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

VI CONCLUSION . . . . ... . . . . . . ... . . . . . . . . . . . . . . . . , ............................ 124

REFERENCES .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

APPENDICES . . . . . . . . , ... '" ............ '" ................. , ....... , .... 152 Appendix A - Preparation of Reagent for Biochemical,

Microscopy and Molecular Biology Studies . . . . . . 153 Appendix B - Procedures for Labeling of DNA Probe,

Southern Blotting, Hybridization and Detection of DNA Bands . . . '" ... '" .......... , ............... , .... 161

Appendix C - Similarity Index Based on the Proportion of Shared Fragments Using Specific Primer. . . . . . . . 164

VITA . . . . . , . . . '" . , . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 75

vi

Table

1

2

3

4

5

6

LIST OF TABLES

Page

Leaf Characteristics of Resistant and Susceptible Cocoa Clones to a VSD Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Pod Characteristic of Resistant and Susceptible Cocoa Clones to a VSD Disease ..................................... .............. 79

Bean Characteristic of Resistant and Susceptible Cocoa Clones to a VSD Disease .................................................. 81

Concentrations of Total Soluble Phenol, Specific Activities of Peroxidase and Polyphenol Oxidase in Youngest Expanded Leaf of Specific Cocoa Clones .............................. 84

Concentration of Chitinase and J3-1 ,3-Glucanse in Youngest Expanded Leaf of Specific Cocoa Clones .............................. 91

Similarity Matrix Based on the Proportion of Shared bands ...... 112

vii

Figures

1

2

3

LIST OF FIGURES Page

The four major steps of the AFLP procedure using one primer pair ................................. '" . . ... . .. .. .. ... .. . . .. . .... . . .. 41

UPGMA dendrogram of cocoa derived by use of genetic distances based on the similarity indices of Nei and Li (1979) . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 113

Sequence of a purified unique band from the primer combination of EcoRI + AAC/Msel + CAG ......................... 120

viii

Plates

1

2

3

4A

48

5

6

7

8

9

1 0

1 1

1 2

LIST OF PLATES

Page

Typical electrophoretogram of protein extracted from cocoa leaf of different clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

Typical zymogram of peroxidase extracted from cocoa leaf of different clones . . . . . . . . . . . . . . . . . . . . . . . . '" . . . . , . . . . . . . . . . . . . . . . . . 96

Typical zymogram of polyphenol oxidase extracted from cocoa leaf of different clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Growth of Oncobasidium theobromae in the xylem of resistant cocoa clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Growth of Oncobasidium theobromae in the xylem of susceptible cocoa clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . , . . . . . 1 00

Stomata response to varying degree of infection by Oncobasidium theobromae in resistant cocoa cl one KKM 25 and susceptible cocoa clone PA 7 . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 1

Xylem structure of leaf tissues from resistant cocoa clone KKM 25 and susceptible clone PA 7 with varying degree of infection by Oncobasidium theobromae . . . . . . . . . . . . . . . . . . . . . . . . . 1 03

Electron micrograph of leaf parenchyma cel ls from resistant cocoa clone KKM 25 and susceptible cocoa clone PA 7 with varying degree of infection of Oncobasidium theobromae . . . 1 04

The extracted genomic DNA of cocoa isolated from second youngest expanded leaflet. . . . . . . . . . . . '" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 07

AFLP profi le i n ten cocoa clones obtained by primer combination . . . . . . . . . . . , . , . ' " . . . . . . ' " . . . . . . . . . . . . . . . ' " . . . . . , . . . . . . . . . . . . . 1 1 0

Amplification products generated by primers combination of A}EcoR1 + AAC/Mse1 + eTA B}EcoR1 + AAC/Mse1 + CAT C)EcoR1 + AAC/Mse 1 + CAG D) EcoR1 + AAC/Mse1 + CAC E)EcoR1 +AAC/Mse1 +CAA . . . . . . . . . . . . ... . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7

PCR product before and after cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 8

Detection of genomic DNA on a Southern blot. . . . . . . . . . . . . . . . . . . 1 1 8

ix

LIST OF ABBREVIATIONS

AFLP = Amplified Fragment Length Polymorphism BAL = Borneo Abaca Limited BSA = Bovine Serum Albumin CBD = Coomassie Brilliant Blue Dye CCM = Corticium Culture Medium DMRT = Duncan's Multiple Range Test CPB = Cocoa Pod Borer IPM = Integrated Pest Management MARDI = Malaysian Agricultural Research and Development

Institute PCR = Polymerase Chain Reaction PPD-PC = p-phenylenediamine-pyrocatechol PR = Pathogenesis-related RAPD = Random Amplified Polymorphic DNA RFLP = Restriction Fragment Length Polymorphism SAS = Statistical Analysis System SEM = Scanning Electron Microscope SRFA = Selective Restriction Fragment Amplification SSRs = Simple-sequence-repeats STS = Sequence-tagged-site TEM = Transmission Electron Microscope UPGMA = Unweighted Pair Group Method with Arithmetic Averaging UV = Ultra-violet VSD = Vascular Streak Dieback YAC = Yeast Artificial Chromosome

Ilm = micrometer Ilg = microgram )11 = microliter kbp = kilo base pair mbp = mega base pair kDa = kilo Dalton m m = milimeter nm = nanometer mg = miligram ml = mililiter pg = picogram

x

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Doctor of Philosophy

MORPHOLOGICAL, BIOCHEMICAL AND DNA STUDIES FOR THE DEVELOPMENT OF MARKERS FOR THE SELECTION OF Theobroma cacao L.

CLONES RESISTANT TO VASCULAR STREAK DIEBACK DISEASE

Chairperson

Faculty

By

ROSMIN BIN KASRAN

October 1999

Professor Marziah Mahmood , Ph.D

Science and Environmental Studies

Vascular streak dieback (VSO) is a serious disease of Theobroma cacao

L. (cocoa) plant and it is attributed to the fungal pathogen Oncobasidium

theobromae. Control of the disease is largely dependent on the use of systemic

fungicides. Resistance to the pathogen had been found in the cocoa planting

materials but as yet there was no marker to select for the resistant clones. In

relation to this, this study was conducted to develop suitable markers for selection

of cocoa clones resistant to a VSO disease. These include morphological,

biochemical, enzyme activity, isozyme pattern, light and electron microscopy, and

DNA markers.

The cocoa clones chosen represented three levels of resistance to VSD

disease. The reported resistant clones were MHP80, SCA6, AMA 15-15, ICS95,

PA13, KKM25 and SCA9; moderately resistant clones were PBC140, KKM22

and MHP136, while the susceptible clones were MJS21, ICS84, NA32, NA33,

UA13, MHP14, MHP3?, PAY, IMC6? and EET 399.

The results showed that the morphological characteristics of leaf (shape,

color and size), pod (color, shape and surface) and bean (shape, size and

and cotyledon color) could not be used to distinguish between the cocoa

xi

clones resistant and susceptible to VSD disease.

The results on the biochemical and enzyme parameters indicated that the

activity of polyphenol oxidase and concentration of chitinase in cocoa leaf tissues

were related to resistance to VSD disease. The level of both enzymes were

significantly higher (P�O.05) in the resistant cocoa clones compared to the

susceptible clones. Others biochemical parameters such as the concentration of

total soluble phenol and P-1,3-glucanase, activity of peroxidase, proteins and

isozyme patterns could not distinguish the resistance of cocoa clones to VSD

disease.

Studies on light microscopy showed that the growth of Oncobasidium

theobromae in the leaves of resistant clones resulted in less hyphae compared

to those from the susceptible clones. However, the results from the electron

microscope studies showed that the infected tissues of both resistant and

susceptible cocoa clones showed similar damage symptoms such as cell

shrinkage and plasmolysis.

The results on Amplified Fragment Length Polymorphism (AFLP)

technique to distinguish between cocoa clones that are resistant and susceptible

to a VSD disease using selected primer combinations showed polymorphisms

among cocoa clones. However, the similarity index and cluster analysis obtained

from the data could not classify the cocoa clones as being resistant or susceptible

to VSD disease. Meanwhile, a cloned unique fragment of resistant cocoa clone

obtained from an AFLP analysis was able to differentiate between resistant

(ICS95, KKM25, MHP80, A MA15-15) and susceptible (NA32, PA7, MHP14,

MHP37) cocoa clones. This DNA marker was named VSDr1.

xii

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai me menu hi keperluan untuk ijazah Doktor Falsafah

KAJIAN MORFOLOGI, BIOKIMIA DAN DNA UNTUK MEWUJUDKAN PENAN DA-PENANDA BAGI MEMILIH KLON Theobroma cacao L. YANG

RINTANG TERHADAP PENYAKIT MATIROSOT JEJALU R VASKULAR

Oleh

ROSMIN KASRAN

Oktober 1999

Pengerusi Prof. Marziah Mahmood, Ph.D

Fakulti Sains dan Pengajian Alam Sekitar

Penyakit matirosot jejalur vaskular (VSD) ialah penyakit yang serius pada

tanaman Theobroma cacao L. (koko) dan ia disebabkan oleh sejenis kulat

patogen, Oncobasidium theobromae. Kawalan utamanya ialah dengan

penggunaan racun kulat sistemik. Bahan tanaman koko yang rintang terhadap

patogen telah dikenalpasti tetapi sehingga kini belum ada pen and a untuk memilih

klon yang rintang. Sehubungan dengan itu, kajian ini telah dijalankan untuk

mewujudkan penanda yang sesuai bagi memilih klon koko yang rintang terhadap

penyakit VSD. Ini termasuklah kajian morfologi, biokimia, aktiviti enzim, corak

enzim, mikroskop biasa dan elektron dan penanda DNA.

Klon-klon koko yang dipilih adalah mewakili tiga aras kerintangan

terhadap penyakit VSD. Klon yang dilaporkan rintang ialah M HP80, SCA6,

AMA15-1 5, ICS95, PA1 3, KKM25 dan SCA9; klon sederhana rintang ialah

PBC1 40, KKM22 dan M HP1 36, sementara klon rentan ialah MJS21 , ICS84,

NA32, NA33, UA13, MHP14, MHP37, PA7, IMC67 dan EET399.

Keputusan kajian mendapati bahawa ciri morfologi daun (bentuk, warna

dan saiz), buah (warna, bentuk atau kelicinan permukaan) dan biji (bentuk,

xiii

saiz atau warna koti/edon) tidak dapat membezakan klon koko yang rintang dan

rentan kepada penyakit VSD.

Keputusan mengenai parameter biokimia dan enzim pula menunjukkan

aktiviti politenol oksidase dan kepekatan kitinase di dalam daun koko mempunyai

kaitan kerintangan terhadap penyakit VSD. Kedua-dua aras enzim tersebut

adalah tinggi (P�O.05) pada klon koko yang rintang berbanding klon yang rentan.

Parameter biokimia lain seperti kepekatan jumlah fenol terlarut dan f3-1 ,3-

glukanase, aktiviti peroksidase, corak protein dan isozim didapati tidak boleh

membezakan kerintangan klon koko terhadap penyakit VSD.

Kajian mikroskop biasa menunjukkan pertumbuhan hifa Oncobasidium

theobromae pada daun koko yang rintang adalah sedikit berbanding dengan klon

yang rentan. Walau bagaimanapun, keputusan dari kajian mikroskop elektron

mendapati tisu yang dijangkiti pada kedua-dua jenis klon tersebut menunjukkan

simtom kerosakan yang sama seperti pengecutan dan plasmolisis.

Keputusan kajian menggunakan teknik 'Amplified Fragment Length

Polymorphism' (AFLP) untuk membezakan klon koko yang rintang dan rentan

menunjukkan polimorfisma di antara klon-klon koko dengan menggunakan

kombinasi primer pilihan. Walau bagaimanapun, indeks kesamaan dan analisis

kluster daripada data yang diperolehi tidak dapat mengkelaskan klon koko

kepada rintang dan rentan kepada penyakit VSD. Sementara itu, fragmen unik

DNA daripada analisis AFLP yang telah diklonkan didapati dapat membezakan di

antara klon yang rintang ( ICS95, KKM25, MHP80, AMA1 5-1 5) dan rentan

(NA32, PA7, MHP1 4, MHP37). Penanda ini dinamakan VSDr1 .

xiv

CHAPTER I

INTRODUCTION

Vascular streak dieback (VSO) disease, attributed to the fungal pathogen,

Oncobasidium theobromae, is a serious disease in cocoa plants (Keane and

Turner, 1972; Turner and Shepherd, 1978). The fungal basidiospores firstly

attaches itself to the leaves and then, progresses through the leaf petiole towards

the stem attacking the xylem vessels (Keane et al., 1972; Tey et al., 1985). The

current practice of identifying diseased plants is through observing the

symptoms. The first symptoms of infection are shown by chlorosis of one leaf

usually on the second or third flush behind the tip, enlarged lenticels are usually

evident on the stem immediately below the petiole of this leaf (Keane et al.,

1972). In the diseased region, the cambium turns rusty-brown rapidly when

exposed to air by stripping the bark (Prior, 1981; Tey et al., 1985). The disease

progression on a mature tree usually takes about five months from the initial leaf

infection to death of a branch up to one meter long (Keane et aI., 1972). A young

cocoa seedling, however, would succumb in a few weeks.

The control of VSO disease in the past is mainly dependent on the

application of systemic fungicides. However, these chemicals usually lose their

efficacy due to the evolution of the pathogen. Meanwhile, some cocoa clones

have been found to be resistant to the disease (Bong and Phua, 1984; Tey and

Musa, 1984), and thus present an alternative approach in dealing with the

1

2

disease. The conventional method to evaluate resistance has been the

selection for resistant plants after exposure to the natural inoculum of the

pathogen (Tey et al., 1 987b). Resistant clones were classified based on the

percentage of the plants not infected after 1 4 months of the trial period. This

method, however, is time consuming. Thus, it hypothesized that other

techniques could be used to identify the cocoa clones resistant to a VSD

disease.

An alternative method is to expose cocoa leaves to basiodiospore

suspension of Oncobasidium theobromae for a certain period and then

observing the development of the fungal pathogen in vitro. However, this

method has not been tested yet.

Several reports on the involvement of lytic enzymes (chitinase, 11-1 ,3-

glucanase) and enzymes that metabolize phenols (polyphenol oxidase,

peroxidase, phenylalanine ammonia-lyase, l1-glycosidase) during plant

defense against pathogens and parasites have been published (Van Loon,

1986; Barz et al., 1990; Hoagland, 1990). Increase in peroxidase, polyphenol

oxidase, chitinase and �-1 ,3-glucanase activities have been associated with

immunization in several plant-pathogen systems (Hammerschmidt et a/., 1 982;

Bajaj et a/., 1 986; Chai and Doke, 1 987; Dalisay and Kuc, 1 988; Metraux and

Boller, 1 988; Metraux et al ., 1 988; Tezun et al., 1 988; Pan et al., 1 99 1 ) .

Although the enzyme activities were usual ly increased i n the infected tissues,

this does not prove that they were involved in the disease resistance

3

(Lazarovits and Ward, 1 982). However, a strong correlation between the

activity of an enzyme before infection and certain disease resistance may

indicate such a role. A strict relationship between the enzyme levels of plants

and susceptibility to VSD disease in cocoa is yet to be established. In addition,

less attention has been paid to the variation in activity of these enzymes in

plants prior to infection and its role in resistance.

The availability of molecular techniques has opened new ways to

characterize germ plasm, to determine genetic diversity and to establish gene

l inkages of agronomic traits to molecular markers. Amplified Fragment Length

Polymorphism (AFLP) technique has been used to assess genetic

relationships, to quantify levels of genetic diversity and to identify cultivars of

agriculturally important plants (Zabeau and Vos, 1 993; Vos et al., 1 995; Hil l et

al., 1996; Maughn et al., 1 996; Powell et al., 1 996; Sharma et al., 1996; Donini

et a/. 1 997; Qi and Lindhout, 1 997; Van Toai et al., 1 997) . U nique DNA

fragments that are obtained in AFLP may correspond to unique locations in

the genome and, if associated with a particular phenotype, may be used as a

selection marker.

4

Objectives of Study

The objectives of this study are to develop markers suitable for the

selection of resistant cocoa clones to VSD disease. This involves a multi

parameter approach as follows:

1 . To study the morphology of leaf (shape. color and size). pod (color. shape

and surface) or bean (shape. size and cotyledon color) of various cocoa

clones and their relationships to VSD disease susceptibility.

2. To determine the variations in total soluble phenol. chitinase. �-1 .3-

glucanase. peroxidase and polyphenol oxidase activity in the leaves of

several cocoa clones and their relationships to VSD disease susceptibility.

3. To study the growth of Oncobasidium theobromae on the leaf of resistant

and susceptible cocoa clones after exposure to the fungal pathogen.

4. To determine whether the cocoa clones used that are known to be

resistant or susceptible to VSD disease could be differentiated using the

AFLP technique.

S. To clone a unique DNA fragment obtained by AFLP and to investigate its

use as a DNA probe for the selection of VSD resistance in cocoa.

CHAPTER I I

LITERATURE REVIEW

Economic Importance of Cocoa

Theobroma cacao L. (Sterculiaceae), an important tropical rain forest

species, is grown for its oil-rich seed, to produce cocoa and cocoa butter

(Figueira ef al., 1 993). Cocoa has a high food value, containing as much as 20

percent protein, 40 percent carbohydrate, and 40 percent fat (Whistler et al.,

1956). It is also mildly stimulating because of the presence of theobromine, an

alkaloid that is closely related to caffeine. The pulverized residue, which is

called cocoa, is used in beverages and as a flavoring. Chocolate is also

derived from cocoa beans.

Cocoa seeds are Gl major cash crop of the tropical world , but prices

fluctuate widely and economic hardships occur when prices are low. Despite

this, only about 1 0% by fresh weight of the fruit is commercialized, although

several promising commercial products could be obtained from the fruit

(Greenwood-Barton, 1965). Un utilized portions of cocoa pods contain many

potential new products that could provide extra income for cocoa growers . The

most promising products appear to be cocoa pulp and the gums from pod

husks (Figueira et al., 1 993). The high proportion of fat in the bean kernels

(cocoa butter) is used in medications, cosmetics, and soaps.

5

6

Cocoa pod husks contain 3 to 4% of potassium on a dry basis (Wood

and Lass 1 985). Pod husk ash has been used to make soap in Ghana and

Nigeria (Oduwole and Arueya 1 990; Arueya 1 991 ) .

A cocoa husk extract called cocoa pigment, which is a mixture of

condensed or polymerized f1avonoids (such as anthocyanidins, catechins,

leukoanthycyanidin), sometimes linked with glucose, has been utilized in

Japanese food industries (Kimura, 1 979). Recently this extract has been

shown to inhibit cytopathic effects of HIV in cell culture (Unten et a/., 1 99 1 ) .

The anti-HIV activity was attributable to interference with the virus adsorption,

rather than inhibition of the virus replication after adsorption.

I n cocoa, Iysigenous cavities filled with mucilaginous substances occur

in roots, stems, flowers, and leaves (Brook and Guard 1 952) as well as fruit

husks (Figueira et a/., 1 991 ) . Krishna Moorthy and Subba Rhao ( 1 976, 1978,

1 980) also isolated gums from the seed pulp. Polysaccharides of cocoa were

first characterized by Whistler et a/. (1 956), who found differences in hot

water-soluble polysaccharides between seed and pod husks. Blakemore et a/.

(1 966) examined the hot-water-soluble fraction of husk polysaccharide and

concluded that the major part of this fraction was a pectic material. Cocoa pod

husks were examined as a source of pectin by mild acid extraction by

Adomako (1 972) and Berbert ( 1 972), but yields were low and the pectin was

inferior to apple or citrus pectin in gel-forming ability. Krishna Moorthy and

Subba Rhao ( 1 978, 1 980) found that gums from seed pulp were effective in

7

low concentrations as a binder for pharmaceutical pills, and reported that

suspending properties were superior to tragacanth, sodium alginate, sodium

carboxy-methyl cellulose, and methyl cellulose. Figueira et al. ( 1 99 1 ). have

recently characterized cocoa gums from pod husks and stems to evaluate

their potential as a replacement for gum karaya or as a new commercial

product.

Cocoa Industry in Malaysia

The first commercial cocoa planting in Malaysia started in 1 950s in

Jerangau, Terengganu (Anon, 1 991 ) . This was followed by the Borneo Abaca

Limited (BAL Plantations Sdn Bhd) in Sabah. The initial planting material was a

mixture of Amelonado and Trinitario types. The programs on varietal

improvement have produced superior planting material , which are more

vigorous and tolerant to diseases. This led to the expansion in planting areas.

Availability of land and the high price of cocoa beans in the late 1 970s and

early 1 980s further boosted the expansion of cocoa cultivation throughout the

country. The introduction of clonal planting material and rehabilitation

programs also contributed markedly to this expansion. Some problems as

described below which can pose as threats to this industry have been

identified and steps towards solving them have been actively undertaken by

both the public and private sectors.

Unstable and low prices of cocoa dry bean, low productivity, pest and

disease problem, shortage and high cost of labor and competition for land use

from other crop have been identified as the major factors affecting the growth

8

of the cocoa planting industry. Whereas, the quality of cocoa beans and cocoa

products, limited cocoa product range and uses, and lack of processing

technology and facility are the major issues faced by the cocoa grinding and

manufacturing industry. These factors will influence the growth of the cocoa

planting and downstream industry in Malaysia.

As a result of the prolong low cocoa bean prices which are below

production costs to many growers, more cocoa areas have been either

abandoned or chopped down. In 1992, the total cultivated areas was

estimated at 388 700 ha, declined from about 405 000 ha in 1987 (Anon,

1992). The cultivated area was declined further in 1997 to about 156 682 ha

(Anon, 1997). For 1998, the reduction is marginal estimated at about 5% from

1997 to around 150 000 ha (Anon, 1998). With the recovery in prices, it is

expected that the cultivated cocoa areas will be stabilized at this level. The

practical solution to low prices of cocoa bean is to reduce the cost of

production through increase in productivity and efficiency, which can be within

reach of the cocoa growers. Development of new technology specifically to

address the various constrains would further enhance the competitive edge of

the cocoa industry that can generate profit even at low prices.

Low productivity in the small holder sector has been identified as a

problem. One of the causes is the premature senescence of young cocoa fruit

(cherelle wilt) that lead to sub-optimal productivity, particularly when losses are

already high due to fungal diseases and insect infestation. Lack of

9

input, inappropriate application of technology and improper management are

the major factors attributed to low productivity. The productivity of the small

holder sector could be increased through modification in the planting system,

use of high yielding cocoa clones and economical shade trees, proper ferti lizer

application and more efficient pest and disease management. In most cases,

the plants and/or the area need to be rehabil itated. Mature budding constitutes

the main technique of rehabilitation for these uneconomic farms besides

pruning and other cultural practices (Mohd Jelani , 1 985; 1 99 1 ). The suitabil ity

and improvement of various economic shade trees such as durian , rubber, oil

palm and bread fruit in order to increase the productivity are being extensively

evaluated (Denamany ef a/., 1 992; Mohd Jelani ef a/., 1 992; Sapiyah ef a/.,

1 992; Nawi, 1 992a,b).

High density planting of cocoa has been introduced in recent decades

in MalaYSia, and the prospect of higher early yield was antiCipated (David and

Pang, 1 989; Ab. Kahar ef a/., 1 991 ; Lam and Lim, 1 99 1 ) . It is important to

identify certain morphological traits in cocoa clones that are suitable to h igh

density, pest and disease management. The suitability of clones for high

density planting has been examined by Kasran ef a/. ( 1 993) and they

suggested that slow growing planting materials with high productivity to avoid

self-shading should be used. Many attempts have been made actively to

reduce tree vigor by utilizing dwarfing rootstocks to obtain slow growing

planting materials suitable for high density planting. However, the effect of

dwarfing rootstock on scion growth have not been detected thus far in cocoa