UNIVERSITI PUTRA MALAYSIA MORPHOLOGICAL ...

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


ROSMIN KASRAN

DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA October 1999


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 M use 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 M r. Rajinder Singh ( Plant Science and Biotechnology, POR I M). To Mr. Aziz, Mr. Azlan, Mr. Sumber, M r. Tee, Miss Asnita, Miss Hafizah, Miss Iteu, M rs

.

Janna, Miss Radziah, Miss Ramani, Miss Suzita and Miss Zuraidah,

I would

like to thank them for their kind cooperation.

Last but not least I would like to express my deepest gratitude to my family for their constant support and inspiration throughout the study.

iii

TABLE OF CONTENTS Page

ACKNOWLEDGEMENT . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. . . . . . . . . . . . . . . . . . . iii LIST OF TABLES . , .............,......, ............. , ............, ...... vii LIST OF FIGU RES........................................................,......, ............. viii . . . .

.

. . . . . . . . . . . . . . . . .

LIST OF PLATES..........................., .............,......, ............. , . ....., ... ... ... ... ix LIST OF ABBREVIATIONS....,................................. , '" ........................ x ABSTRACT..........,......, ........................................" ....., ......... '" .,. ... .. xi ABSTRAK xiii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C HAPTER INTRODUCTION....., .............,......, ................................. 1 II

LITERATURE REViEW..... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..... 5 Economic Importance of Cocoa.......,. '" .., ... '" ... '" ....... ,. ... .. Cocoa Industry in Malaysia............ ...... ... ... ...... ......... ... ... .... Morphological Variability of Cocoa Trees... ............ .............. Fungal Diseases of Cocoa ......... '" .. , ..... , ... ... ... ... ... ... ... ... ...

5 7 11 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 ...................................................................... Morphological Characteristics of Cocoa Clones and Their Relationships to VSD Disease Susceptibility ....., ... ... ... ... ... .... Plant Materials ......... ................................, ............ Morphological Data Co"ected........................ '" ........ Variation of Soluble Phenol, Enzyme Level and Isozyme

43 43 43 44

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 I nfection 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

VI

CONCLUSION

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REFERENCES . .

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125

A PPENDICES , ... '" ............ '" ................., .......,.... 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 . . .

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VITA

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

vi

LIST OF TABLES Table 1

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

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

77

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

3

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

4

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

5

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

6

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

vii

LIST OF FIGURES Figures 1

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

2

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

3

113

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Sequence of a purified unique band from the primer combination of EcoRI

+

AAC/Msel

viii

+

CAG.........................120

LIST OF PLATES Plates

Page

1

Typical electrophoretogram of protei n extracted from cocoa leaf of different clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

2

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

3

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

4A

Growth of Oncobasidium theobromae in the xylem of resistant cocoa clones

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99

48

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

5

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

6

Xylem structure of leaf tissues from resi stant cocoa clone KKM 25 and suscepti ble clone PA 7 with varying degree of infection by Oncobasidium theobromae . . . . . . .

7

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1 03

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

8

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

9

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

10

Amplification products generated by pri mers combination of

A}EcoR1 +AAC/Mse1 +eTA B}Eco R 1 +AAC/Mse1 +CAT C)Eco R 1 +AAC/Mse 1 +CAG D) Eco R 1 +AAC/Mse1 + CAC E)EcoR1 +AAC/Mse1 +CAA . . . . . . . . . . . . ......... . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7 11

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

12

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

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LIST OF ABBREVIATIONS AFLP BAL

BSA CBD CCM D M RT CPB IPM MARDI PCR PPD-PC PR RAPD RFLP SAS SEM S RFA SSRs STS TEM UPGMA UV VSD YAC Ilm Ilg )11 kbp mbp kDa mm nm mg

= = = = = = = = =

= = = = = = = = = = = = = = =

= = = = = = = = =

ml

=

pg

=

Amplified Fragment Length Polymorphism Borneo Abaca Limited

Bovine Serum Albumin Coomassie Brilliant Blue Dye Corticiu m Culture Medium Duncan's Multiple Range Test Cocoa Pod Borer Integrated Pest Management Malaysian Agricultural Research and Development Institute Polymerase Chain Reaction p-phenylenediamine-pyrocatechol Pathogenesis-related Random Amplified Polymorphic DNA Restriction Fragment Length Polymorphism Statistical Analysis System Scanning Electron Microscope Selective Restriction Fragment Amplification Simple-sequence-repeats Sequence-tagged-site Transmission Electron Microscope Unweighted Pair Group Method with Arithmetic Averaging Ultra-violet Vascular Streak Dieback Yeast Artificial Chromosome micrometer microgram m icroliter kilo base pair mega base pair kilo Dalton milimeter nanometer miligram mililiter picogram

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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 By

ROSMIN BIN KASRAN October 1999 Chairperson

Professor Marziah Mahmood , Ph.D

Faculty

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, AMA15-15, PA13,

KKM25 and SCA9;

ICS95,

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 and cotyledon color) could not be used

xi

to

distinguish

(shape,

size

and

between the cocoa

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 U NTUK M EWUJU DKAN PENAN DA-PENAN DA BAGI MEMILIH KLO N Theobroma cacao L. YANG RINTANG TERHADAP PENYAKIT MATIROSOT JEJALU R VASKULAR

Oleh ROSMIN KASRAN Oktober 1999 Pengerusi

Prof. Marziah M ahmood, Ph.D

Fakulti

Sains dan Pengajian Alam Sekitar

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

Theobroma cacao Oncobasidium

L. (koko) dan ia disebabkan oleh sejenis kulat

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 u ntuk memilih klon yang rintang. Sehubungan dengan itu, kajian ini telah dijalankan u ntuk 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

terhadap penyakit VSD. Klon yang dilaporkan rintang

tiga aras ialah

kerintangan

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 MJS2 1 , ICS84, NA32, NA33, UA13, M HP14, M HP37, 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 VS D. Keputusan mengenai parameter biokimia dan enzim pula menunjukkan aktiviti politenol oksidase dan kepekatan kitinase di dalam daun koko mempunyai kaitan kerintangan terhadap penyakit VSD. Ked ua-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 , 3glukanase, aktiviti peroksidase, corak protein dan isozim didapati tidak boleh membezakan kerintangan klon koko terhadap penyakit VSD. Kajian mikroskop biasa menunjukkan pertumbuhan hifa theobromae

Oncobasidium

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 VS D. Sementara itu, fragmen unik DNA daripada analisis AFLP yang telah diklonkan didapati dapat membezakan di antara klon yang rintang

(ICS95, KKM25, M H P80, AMA1 5-1 5) dan rentan

(NA32, PA7, M H P 1 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 VS D 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 , 3glucanase) and enzymes that metabolize phenols (polyphenol oxidase, peroxidase, phenylalanine a mmonia-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 usually 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 s usceptibility to VS D 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 germplasm, to determine genetic diversity and to establish gene linkages 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; Hill 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 .3glucanase. 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 II LITERATURE REVIEW Economic Importance of Cocoa

Theobroma cacao

L.

(Sterculiaceae), an important tropical rai n forest

species, is grown for its oil-rich seed, to produce cocoa and cocoa b utter (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, b ut prices

fluctuate widely and economic hardships occur when prices are low. D espite 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 (Oduwol e 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 techn ology and facility are the major issues faced by the cocoa g rinding and manufacturing industry. These factors will influence the growth of the cocoa plantin g 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 fertilizer application and more efficient pest and disease management. In most cases, the plants and/or the area need to be rehabilitated. Mature budding constitutes the main technique of rehabilitation for these uneconomic farms besides pruning and other cultural practices (Mohd J elani, 1 985; 1 99 1 ). The suitability 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 99 1 ; Lam and Lim, 1 99 1 ) . It is important to identify certain morphological traits in cocoa clones that are suitable to high density, pest and disease ma nag e m ent . 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