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UNIVERSITI PUTRA MALAYSIA

FUNGAL METABOLITES FOR ASSESSMENT OF GANODERMA BONINENSE PAT. INFECTION IN OIL PALM

ROGER LIEU TOH CHOON

ITA 2011 6

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FUNGAL METABOLITES FOR ASSESSMENT OF GANODERMA BONINENSE PAT. INFECTION IN OIL PALM

By

ROGER LIEU TOH CHOON

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

Science

September 2011

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the Degree of Master of Science

FUNGAL METABOLITES FOR ASSESSMENT OF GANODERMA BONINENSE PAT. INFECTION IN OIL PALM

By

ROGER LIEU TOH CHOON

September 2011

Chairman : Professor Sariah Meon, PhD

Institute : Tropical Agriculture

Basal Stem Rot (BSR) caused by Ganoderma boninense is a serious

disease in oil palm. Detection of BSR is based mainly on visible symptoms,

and confirmed by plating infected tissues on Ganoderma selective media.

Unfortunately, these symptoms are only visible when at least half of the basal

tissue has been infected. Current research on the plant-pathogen interaction

showed that invasion by pathogens can lead to increase of fungal biomass in

the healthy plant, making it detectable during the infection. Certain primary

fungal metabolites were targeted as they were produced abundantly during

the reproduction of mycelium. Therefore, based on the mode of infection and

spread of G. boninense on oil palm, the probable strategy is to unravel the

metabolic pathway of the plant upon infection and possibly use this approach

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to differentiate between healthy and infected palms at the early stage of

infection. The technique used for analysis and identification of the targeted

metabolites were Thin Layer Chromatography (TLC), Ultra Performance

Liquid Chromatography (UPLC), Gas Chromatography coupled with Mass

Spectrometry (GCMS) and Nuclear Magnetic resonance (NMR). This

research focused on the potential of detecting targeted metabolites as

indicators for Ganoderma infection in field palms. Metabolites from G.

boninense were extracted and used as indicator for TLC and UPLC analysis.

Eighty healthy oil palm seedlings were used as control where no artificial

infection applied while eighty seedlings for artificial infection. Metabolites for

healthy and infected palms were extracted from root tissues of the

experimental seedlings. Meanwhile, two random samplings for the healthy

and Ganoderma-infected stem tissues were carried out from MPOB

Experimental Station, Kluang, Johor and Sime Darby oil palm plantation from

Banting, Selangor. By using the same extraction method, these samples

were analyzed by TLC with optimum combination of solvent systems to

detect and the quantification of the metabolites was done with UPLC with

progressive development of BSR symptoms. G. boninense extract was used

as indicator for TLC and UPLC analysis to distinguish between healthy and

infected oil palm samples. Results showed that the best combination solvent

system which gave the clearest spot separation were acetone and hexane in

the ratio of 30:70. Round and clear separated spots representing different

metabolites were detected. Compared with pure G. boninense extract as

indicator, four clear spots were consistently detected in infected tissues

extracts which however were not detected in healthy tissues extracts. These

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spots could be used to distinguish between healthy and infected tissues. Two

clearest spots were chosen for further analysis named as metabolite A and

metabolite B. In UPLC, it showed that metabolites A and B were increased

exponentially and showed a positive correlation of the disease severity with

time. Metabolites A and B were purified and sent for GCMS for their

molecular weight followed by NMR analysis for chemical structure

identification. Based on the results from GCMS and NMR, it was concluded

that metabolite B was identified to be ergosterol which played an important

role in fungal cell wall development. Meanwhile, metabolite A was tentatively

suggested to be 2,4-ditert-butylphenol. According to the present work, it

could be concluded that Ganoderma infection in palms increase the

production of metabolites which could be used as indicators for early

detection of infection in oil palms. Further studies are needed to produce a

better implementation of this finding as disease diagnostic tool in the

commercialized oil palm field.

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

METABOLIT KULAT UNTUK PENTAFSIRAN JANGKITAN GANODERMA BONINENSE PAT. PADA KELAPA SAWIT

By

ROGER LIEU TOH CHOON

September 2011

Pengerusi : Profesor Sariah Meon, PhD

Institut : Pertanian Tropika

Penyakit reput pangkal (BSR) merupakan sejenis penyakit serius dalam

kelapa sawit yang disebabkan oleh Ganoderma boninense. Pengesanan

BSR biasanya berdasarkan kepada simptom luaran dan disahkan dengan

pengasingan Ganoderma daripada tisu berpenyakit atas media selektif.

Malangnya, simptom-simptom ini hanya menjadi nyata apabila sekurang-

kurangnya setengah daripada tisu pangkal telah dijangkiti oleh penyakit ini.

Kajian semasa atas interaksi tumbuhan-patogen menunjukkan bahawa

jangkitan patogen boleh menyebabkan penambahan biojisim kulat dalam

pokok yang sihat, menjadikannya senang dikesan apabila jangkitan berlaku.

Sesetengah metabolit kulat primer menjadi sasaran penunjuk apabila ianya

banyak dihasilkan semasa reproduksi miselium kulat. Oleh itu, berdasarkan

kepada cara jangkitan dan penyebaran oleh G. boninense dalam kelapa

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sawit, strategi yang sesuai adalah dengan melihat laluan metabolik pokok

semasa jangkitan dan menggunakan pendekatan ini untuk membezakan

pokok sihat dan pokok terjangkit pada tahap awal jangkitan. Teknik yang

digunakan dalam analisis dan pengenalpastian metabolit sasaran adalah

kromatografi lapisan nipis (TLC), kromatografi cecair prestasi ultra (UPLC),

kromatografi gas digabungkan dengan spektrometri jisim (GCMS) dan

resonansi magnetik nuklear (NMR). Kajian ini akan menumpu kepada

metabolit sasaran berpotensi sebagai penunjuk dalam mengesan jangkitan

Ganoderma dalam pokok kelapa sawit. Metabolit G. boninense diekstrak dan

dijadikan penunjuk untuk analisis TLC dan UPLC. Lapan puluh anak benih

kelapa sawit dijadikan sebagai kawalan di mana tiada jangkitan buatan

dilakukan sementara lapan puluh anak benih dijangkiti secara buatan.

Metabolit untuk pokok sihat dan pokok terjangkit diekstrak daripada tisu akar

daripada sampel anak benih yang dikaji. Sementara itu, dua kali

penyampelan tisu batang pokok kelapa sawit sihat dan terjangkit dijalankan

di stesen ujikaji MPOB, Kluang, Johor dan ladang kelapa sawit Sime Darby,

Banting, Selangor. Dengan menggunakan cara ekstrak yang sama, sampel-

sampel ini dianalisis dengan TLC dengan kombinasi sistem pelarut optimum

untuk mengesan dan mengenalpasti kuantiti metabolit atas kertas TLC.

Penentuan kepekatan metabolit dan pembentukan progresif simptom

penyakit BSR dilakukan dengan UPLC. Extrak kulat G. boninense dijadikan

penunjuk dalam analisis TLC dan UPLC untuk membezakan sampel kelapa

sawit yang sihat dan berjangkit. Keputusan menunjukkan kombinasi sistem

pelarut terbaik yang memberikan bintik pemisahan yang jelas adalah

asetone: heksana dengan nisbah 30:70. Bintik- bintik bulat yang jelas

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terpisah mewakili metabolit yang berlainan dikesan. Berbanding dengan

ekstrak tulen G. boninense sebagai penunjuk, empat bintik jelas dikesan

dalam ekstrak tisu terjangkit yang tidak dikesan dalam ekstrak tisu yang

sihat. Bintik-bintik ini mungkin boleh membezakan tisu-tisu sihat dan

terjangkit. Dua bintik yang paling jelas dipilih untuk analisis seterusnya

dinamakan sebagai metabolit A dan metabolit B. UPLC menunjukkan

bahawa metabolit A dan B meningkat dan memberikan korelasi positif antara

keparahan penyakit dan masa. Metabolit A dan B yang tulen dihantar untuk

GCMS analisis untuk mendapatkan jisim molekul bagi metabolit. Ini diikuti

dengan analisis NMR untuk menentukan struktur kimia metabolit A dan B.

Berdasarkan kepada keputusan GCMS dan NMR, metabolit B dikenalpasti

sebagai ergosterol yang memainkan peranan penting dalam pembentukan

dinding sel-sel kulat. Sementara, metabolit A dinamakan sebagai 2,4-ditert-

butylphenol. Menurut kajian semasa, dapatlah disimpulkan bahawa jangkitan

Ganoderma dalam pokok kelapa sawit boleh menghasilkan metabolit yang

boleh digunakan sebagai penunjuk untuk mengesan jangkitan dalam kelapa

sawit pada tahap awal. Kajian lanjut perlu dijalankan untuk mendapatkan

implimentasi yang lebih baik menggunakan maklumat kajian ini sebagai alat

diagnosis penyakit dalam ladang kelapa sawit komersial.

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ACKNOWLEDGEMENTS

I would like to extend my heartfelt thanks and gratitude to my supervisor,

Prof. Dr. Sariah Meon for her valuable guidance, advice and suggestions

towards the completion of this project. Not forgetting my supervisory

committee member, Dr. Siti Mariam Mohd Nor for her constructive comments

and truthful helps during the on-going research work and the preparation of

the thesis.

To all staffs of the laboratory of Institute of Tropical Agriculture (ITA), a

million thanks for their assistance and suggestions and helped me for

overcoming the problem I encountered.

And finally, thanks to my family for their moral supports and love throughout

the period of my study in UPM. To all my laboratory mates, I conveyed my

gratefulness for their loves, supports, encouragements and the wonderful

memory I shared with them. Needless to say, their friendships will be in my

mind eternally.

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I certify that a Thesis Examination Committee has met on 27 September 2011 to

conduct the final examination of Roger Lieu Toh Choon on his thesis entitled

“Fungal Metabolites for Assessment of Ganoderma boninense Pat. Infection in Oil

Palm” in accordance with the Universities and University College Act 1971 and the

Constitution of the Universiti Putra Malaysia [P. U. (A) 106] 15 March 1998. The

committee recommends that the student be awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

Mohamed Hanafi bin Musa, PhD

Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

Zainal Abidin bin Mior Ahmad, PhD

Associate Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Internal Examiner)

Muskhazli bin Mustafa, PhD

Associate Professor

Faculty of Science

Universiti Putra Malaysia

(Internal Examiner)

Sepiah Muid, PhD

Senior Lecturer

Faculty of Technology and Science Resource

Universiti Sarawak Malaysia

(External Examiner)

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 22 November 2011

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The thesis was submitted to the Senate of Universiti Putra Malaysia and has

been accepted as fulfillment of the requirements for the degree of Master of

Science. The members of the Supervisory committee were as follow:

Sariah Meon, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman)

Siti Mariam Mohd. Nor, PhD Lecturer Faculty of Science Universiti Putra Malaysia (Member)

BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and

citations, which have been duly acknowledged. I also declare that it has not

been previously, and is not concurrently, submitted for any other degree at

UPM or Universiti Putra Malaysia or other Institutes.

ROGER LIEU TOH CHOON

Date: 27th September 2011

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TABLE OF CONTENTS

Page

ABSTRACT ii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL ix DECLARATION xi LIST OF TABLES xiv LIST OF FIGURES xv LIST OF ABBREVIATIONS xvii CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 2.1 Oil Palm

2.1.1 Introduction 6 2.1.2 Economic Importance 7

2.2 Basal Stem Rot (BSR) in Oil Palm 2.2.1 Causal Pathogen 9 2.2.2 Mode of Spread of BSR Disease 11 2.2.3 Economic Impact 13 2.2.4 BSR Disease Symptoms 15 2.2.5 Current Management of BSR Disease 17

2.3 Current Detection Methods 2.3.1 Symptomatology 24 2.3.2 Selective Media Culture 25 2.3.3 Polymerase Chain Reaction (PCR) 25 2.3.4 Elisa Assay 27

2.4 Fungal Metabolites 2.4.1 Primary Metabolites 29 2.4.2 Role and Importance of Primary Fungal

Metabolites 30 2.4.3 Secondary Metabolites 32 2.4.4 Secondary Metabolites in Disease

Diagnosis 33

3 MATERIALS AND METHODS 3.1 Media for Mycelial Growth of Ganoderma boninense 37 3.2 Extraction and Detection of Metabolites

3.2.1 Ganoderma boninense Mycelium 38 3.2.2 TLC Detection 39

3.3 Oil Palm Stem Sections 39 3.4 Oil Palm Seedlings

3.4.1 Preparation of Ganoderma Inoculum 40 3.4.2 Inoculation of Oil Palm Seedlings with

Ganoderma boninense colonized RWB 42

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3.4.3 Quantification of Metabolites with UPLC 45 3.4.4 Disease Assessment 46

3.5 Mature Standing Oil Palm 49 3.6 Extraction of Metabolites from Other Fungal

Pathogens 50 3.7 Identification of Chemical Structure Using GCMS

and NMR 50

4 RESULTS AND DISCUSSION 4.1 Media for Mycelial Growth of Ganoderma boninense 52 4.2 Detection of Metabolites from Ganoderma boninense

Mycelium 53 4.3 Detection of Metabolites from Oil Palm Sections 56 4.4 Detection and Quantification of Metabolites in

Artificially Infected Seedlings 4.4.1 Disease Assessment 60 4.4.2 TLC Detection of Metabolites 66 4.4.3 Quantification of Metabolites with UPLC 70

4.5 Detection and Quantification of Metabolites from Infected Field Palm with Different External Symptom of BSR 76

4.6 Detection of Metabolites in other Fungal Pathogens 83

4.7 Identification of the Metabolites 4.7.1 Metabolite A 86 4.7.2 Metabolite B 88

5 SUMMARY, GENERAL CONCLUSION AND RECOMMENDATION FOR FUTURE RESEARCH 91

REFERENCES 94 APPENDICES A1 BIODATA OF STUDENT B1