UNIVERSITI PUTRA MALAYSIA NUSAIBAH BINTI SYD ALI ITA 2012 9 METABOLITE PROFILING AND DEFENSE GENE EXPRESSION OF SUSCEPTIBLE AND TOLERANT OIL PALM SEEDLING PROGENIES AT EARLY STAGE OF GANODERMA BONINENSE INFECTION
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UNIVERSITI PUTRA MALAYSIA
NUSAIBAH BINTI SYD ALI
ITA 2012 9
METABOLITE PROFILING AND DEFENSE GENE EXPRESSION OF SUSCEPTIBLE AND TOLERANT OIL PALM SEEDLING PROGENIES
AT EARLY STAGE OF GANODERMA BONINENSE INFECTION
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METABOLITE PROFILING AND DEFENSE GENE EXPRESSION OF
SUSCEPTIBLE AND TOLERANT OIL PALM SEEDLING PROGENIES AT
EARLY STAGE OF GANODERMA BONINENSE INFECTION
By
NUSAIBAH BINTI SYD ALI
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfilment of the Requirements for the Degree of Doctor of Philosophy
December 2012
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Dedicated to my parents, my husband and Boney
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Abstract of thesis submitted to the Senate of Universiti Putra Malaysia in fulfilment of
the requirements for Doctor of Philosophy
METABOLITE PROFILING AND DEFENSE GENE EXPRESSION OF
SUSCEPTIBLE AND TOLERANT OIL PALM SEEDLING PROGENIES AT
EARLY STAGE OF GANODERMA BONINENSE INFECTION
By
NUSAIBAH BINTI SYD ALI
December 2012
Chairman: Associate Professor Datin Siti Nor Akmar Abdullah, PhD
Institute: Institute of Tropical Agriculture
Knowledge on the localized and systemic defense mechanism during Ganoderma
boninense-oil palm interaction is of primary importance for detecting basal stem rot
disease. Thus the objectives of this study include i) to establish and confirm G.
boninense infection on oil palm root seedlings using artificial inoculation method, ii)
to identify secondary metabolites accumulated in oil palm roots at early stages of G.
boninense infection and assess their antifungal activities and iii) to profile expression
of key genes for the biosynthesis of the identified compounds involved in defense
mechanism. In this study, oil palm seedlings were artificially infected with G.
boninense infested rubber wood blocks. Establishment of G. boninense infection at
early stages in tolerant and susceptible oil palm seedling progenies were examined
through scanning electron microscopy (SEM) and transmission electron microscopy
(TEM). Metabolites present in oil palm roots during oil palm-G. boninense
pathogenic interactions were investigated via gas chromatography-mass spectrometric
(GC-MS) analysis. Antifungal assays were carried out to associate the involvements
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of total metabolites and single metabolite from inoculated oil palm roots extracts in
defense mechanism against G. boninense infection. Gene expression analysis of
anthranilate synthase α- subunit1 (EGASα-1) and stearoyl-ACP-desaturase3 (SAD3)
genes from tryptophan and fatty acid pathways respectively were conducted with
quantitative reverse transcriptase-polymerase chain reaction. A rapid outer layer
colonization of hyphae was observed in the susceptible progeny compared to tolerant
progeny through SEM. Cell wall degradation was observed through TEM as early as
24 hpi before penetration of G. boninense hyphae. Total metabolite in vitro study
evaluating inhibitory activity of oil palm root methanolic against G. boninense
showed that tolerant progeny extracts gave a higher inhibition rate with 100 % at 72
and 96 hpi, where else in susceptible extracts, 100% inhibition were only achieved at
96 hpi. An alkaloid metabolite, quinoline was found to have a much more rapid and
elevated accumulation in the roots of tolerant progeny (56.4% at 72 hpi) compared to
susceptible progeny (43.9% at 144 hpi) at early stages of G. boninense infection.
Quinoline gave an EC50 of 0.211μg/ml and showed an increasing antifungal activity
with increasing quinoline concentration against G. boninense. The level of EGASα-1
gene expression reached a maximum at 120 hpi in tolerant and susceptible progeny,
whereby it was 3.0-fold and 1.5-fold higher respectively. Expression of SAD3 gene in
infected roots was 5.3-fold and 1.7-fold in susceptible and tolerant progeny
respectively at 120 hpi. This indicates SAD3 gene is constitutively expressed in oil
palm roots and their expression levels were influenced by biotic stress. These findings
showed that oil palm-G. boninense interaction induces biochemical defense and
activates pathogenesis related genes as a form of early defense mechanism.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Doktor Falsafah
PEMPROFILAN METABOLIT DAN PENGEKSPRESAN GEN BAGI
PROGENI KELAPA SAWIT YANG TIDAK RENTAN DAN RENTAN PADA
PERINGKAT AWAL JANGKITAN GANODERMA BONINENSE
Oleh
NUSAIBAH BINTI SYD ALI
Disember 2012
Pengerusi: Professor Madya Datin Siti Nor Akmar Abdullah, PhD
Institut: Institut Pertanian Tropika
Pengetahuan dalam mekanisme pertahanan penyakit yang setempat dan sistemik
semasa interaksi G. boninense-anak benih kelapa sawit adalah sangat penting untuk
mengesan penyakit BSR. Oleh itu, objektif-objektif bagi kajian ini termasuklah; i)
untuk menghasilkan jangkitan Ganoderma boninense dan pengesahkannya pada akar
anak benih kelapa sawit dengan menggunakan teknik inokulasi artifisial, ii) untuk
mengidentifikasi metabolit-metabolit sekundari yang terkumpul di dalam akar anak
benih kelapa sawit pada peringkat awal jangkitan G. boninense serta mengukur
aktiviti antikulat metabolit-metabolit tersebut dan iii) untuk memprofilkan
pengekspresan gen-gen utama yang terlibat dalam sintesis metabolit-metabolit
pertahanan yang telah diidentifikasi. Dalam kajian ini, blok kayu getah yang telah
dikolonisasi oleh G. boninense telah digunakan untuk menjangkiti anak benih kelapa
sawit. Peringkat awal jangkitan G boninense pada anak benih kelapa sawit telah
diperiksa melalui mikroskopi pengimbasan electron (SEM) dan mikroskopi transmisi
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elektron (TEM). Pengesanan dan pengecaman metabolit yang hadir dalam akar anak
benih kelapa sawit semasa interaksi anak benih kelapa sawit dengan G. boninense
telah dilakukan menggunakan alat gas kromatografi-spektrometrik jisim (GC-MS).
Analisis anti-kulat telah dijalankan untuk mengaitkan penglibatan metabolit total dan
metabolit tunggal dalam ekstrak akar anak benih kelapa sawit yang dijangkiti dalam
mekanisme pertahanan terhadap jangkitan G. boninense. Analisis pengekspresan gen
anthranilate α-subunit 1 (EGASα-1) dan stearoyl-ACP-desaturase3 (SAD3) daripada
laluan triptofan (Trp) dan asid lemak masing-masing telah dijalankan dengan kaedah
kuantitatif transcriptase berbalik-tindakbalas rantaian polimerase (qRT-PCR).
Pengkolonian hifa yang lebih banyak pada lapisan luar akar anak benih kelapa sawit
yang tidak rentan berbanding yang rentan telah dibuktikan melalui analisis SEM.
Selain itu, TEM telah mengungkai bahawa sebelum penetrasi hifa G. boninense ke
dalam dinding sel akar anak benih kelapa sawit, degradasi dinding sel telah berlaku
seawal 24 hpi. Oleh itu, kajian anti-kulat ekstrak metanol metabolit total yang berbeza
melalui faktor progeni dan pelbagai rawatan menunjukkan bahawa kadar perencatan
kulat G. boninense yang dipamerkan oleh ekstrak daripada progeni yang rentan lebih
tinggi berbanding ekstrak yang tidak rentan. Keputusan kajian in vitro ekstrak
metabolit total menunjukkan kadar perencatan pertumbuhan G. boninense adalah
100% oleh ekstrak progeni rentan (72 dan 96 hpi) dan ekstrak progeni tindak rentan
(96 hpi). Salah satu daripada metabolit alkaloid, quinoline telah didapati mempunyai
pengumpulan lebih pesat dan maksimum dalam akar anak benih yang rentan (56.4%
pada 72 hpi) berbanding progeni yang tidak rentan (43.9% pada 144 hpi). Quinoline
memenuhi semua kriteria sebagai alkaloid phytoalexin dan memberikan EC50 0.211
μg/ml serta mempamerkan aktiviti perencatan yang semakin meningkat dengan
peningkatan kepekatan quinoline terhadap G. boninense. Tahap pengekspresan gen
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EGASα-1 mencapai kadar maksimum pada 120 hpi dalam progeni rentan dan tidak
rentan, di mana ia adalah 3.0-kali ganda dan 1.5-kali ganda masing-masing.
Pengekspresan gen SAD3 dalam akar yang dijangkiti adalah 5.3-kali ganda dan 1.7-
kali ganda dalam progeni yang rentan dan tidak rentan masing-masing pada 120 hpi.
Penemuan-penemuan dalam kajian ini menunjukkan interaksi anak benih kelapa sawit
dengan G. boninense mendorong tindak balas pertahanan biokimia di dalam akar anak
benih kelapa sawit yang telah dijangkiti sebagai salah satu bentuk mekanisme
pertahanan.
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I certify that an Examination Committee has met on date of viva voce to conduct the
final examination of Nusaibah Binti Syd Ali on Doctor of Philosophy degree thesis
entitled "Metabolites profiling and defense gene expression of susceptible and
tolerant oil palm seedling progenies at early stage of Ganoderma boninense
infection" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act
1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The
Committee recommends that the student be awarded the Doctor of Philosophy.
Members of the Examination Committee were as follows:
Mohamed Hanafi bin Musa, PhD
Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Chairman)
Norhani bt Abdullah, PhD
Professor
Faculty of Biotechnology and Bimolecular Sciences
Universiti Putra Malaysia
(Internal Examiner)
Wong Mui Yun, PhD
Senior Lecturer
Faculty of Agriculture
Universiti Putra Malaysia
(Internal Examiner)
Monica L. Elliott, PhD
Professor
University of Florida Ifas
United States
(External Examiner)
Seow Heng Fong , PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfilment of the requirement for the degree of Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Datin Siti Nor Akmar Abdullah, PhD
Associate Professor
Institute of Tropical Agriculture
Universiti Putra Malaysia
(Chairman)
Idris Abu Seman, PhD
Principle Research Officer
Biology Division
Malaysian Palm Oil Board
(Member)
Sariah Meon, PhD
Professor
Faculty of Agriculture
Universiti Putra Malaysia
(Member)
Mohammad Pauzi Zakaria, PhD Professor
Faculty of Environmental Sciences
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 Universiti Putra Malaysia
or at any other institution.
NUSAIBAH BINTI SYD ALI
Date: 4 December 2012
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TABLE OF CONTENTS
ABSTRACT
ABSTRAK
ACKNOWLEDGEMENTS
APPROVAL
DECLARATION
LIST OF TABLES
LIST OF FIGURES
LIST OF ABBREVIATIONS
CHAPTER
1. INTRODUCTION
2. LITERATURE REVIEW 2.1 Oil palm in Malaysia
2.2 Basal stem rot disease on oil palm
2.3 Plant-pathogen interaction
2.4 Ganoderma as a comprehensive enzyme factory
2.5 Hypersensitive response
2.6 Systemic acquired resistance
2.7 Gene-for-gene resistance
2.8 Induced metabolic defence pathways
2.9 Secondary metabolites in plants
2.10 The tryptophan pathway
2.10.1 Anthranilate synthase
2.11 Role of fatty acids in plant defense
2.11.1 Stearoyl-ACP-desaturase
2.12 African origin Ganoderma tolerant oil palm materials
3. ARTIFICIAL INOCULATION OF OIL PALM SEEDLINGS FOR
STUDYING EARLY STAGES OF OIL PALM-GANODERMA
BONINENSE INTERACTION
3.1 Introduction
3.2 Materials and Methods/Methodology
3.2.1 Maintenance of fungal isolate
3.2.2 Plant materials
3.2.3 Rubber wood block preparation
3.2.4 Artificial inoculation
3.2.5 Time-course sampling
3.2.6 Scanning Electron Microscopy
3.2.7 Transmission Electron Microscopy
3.2.8 Experimental design and statistical analysis
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3.3 Results
3.3.1 Artificial inoculation with time-course sampling
3.3.2 Scanning Electron Microscope
3.3.3 Transmission Electron microscopy
3.4 Discussion
4. METABOLITE PROFILING: AT EARLY STAGES
INFECTION OF GANODERMA BONINENSE INOCULATION
ON OIL PALM SEEDLINGS 4.1 Introduction
4.2 Materials and Methods
4.2.1 Maintenance of fungal isolate
4.2.2 Plant material
4.2.3 Rubber wood-block preparation
4.2.4 Inoculation
4.2.5 Time-course sampling and statistical analysis
4.2.6 Plant root metabolite extraction
4.2.7 GC-MS analysis
4.2.8 Identification of compounds
4.2.9 Extract assay- antifungal activity time-course vs progeny
4.2.10 Determination of plant metabolite extract sensitivity
4.2.11 Ganoderma. boninense culture preparation
4.2.12 Ganoderma boninense metabolite extraction
4.2.13 Ganoderma boninense GC-MC analysis
4.3 Results
4.3.1 GC-MS analysis
4.3.2 Fatty acid detection and identification
4.3.3 Phytosterol detection and identification
4.3.4 In vitro antifungal assay
4.3.5 Detection and identification of compounds present in G.
boninense mycelium
4.4 Discussion
5. QUINOLINE AS AN ALKALOID PHYTOALEXIN IN
GANODERMA BONINENSE INFECTED OIL PALM SEEDLINGS
5.1 Introduction
5.2 Materials and Methods
5.2.1 Fungal strain
5.2.2 Plant materials
5.2.3 Rubber wood block preparation
5.2.4 Inoculation
5.2.5 Metabolite extraction
5.2.6 GC-MS analysis
5.2.7 Identification of compounds in sample extracts
5.2.8 Determination of 50% effective concentration (EC50) of
Quinoline against G. boninense
5.2.9 Statistical analysis
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5.3 Results
5.3.1 GC-MS analysis
5.3.2 Determination of 50% effective concentration (EC50) of
Quinoline compound against G. boninense
5.4 Discussion
6. EXPRESSION OF ANTHRANILATE SYNTHASE α-1 AND
STEAROYLACP-DESATURASE GENES AT EARLY STAGES OF
G. BONINENSE INFECTION OF OIL PALM SEEDLINGS 6.1 Introduction
6.2 Materials and Methods/Methodology
6.2.1 Fungal strain
6.2.2 Plant materials
6.2.3 Experimental design and statistical analysis samples
6.2.4 Extraction of total RNA from oil palm roots using Qiagen
RNeasy Plant Mini Kit
6.2.5 Determination of RNA quality and quantity
6.2.6 Primer design
6.2.7 Expression analysis by Reverse transcriptase-PCR
6.2.8 Quantitative analysis of gene expression
6.2.9 Sequencing
6.3 Results
6.3.1 Expression of EGASα-1 gene associated with Trp pathway
in tolerant and susceptible oil palm roots artificially
inoculated with G. boninense
6.3.2 Lane-based array analysis of EGASα-1 genes differentially
expressed upon treatment through qRT-PCR products
6.3.3 Expression of SAD3 gene associated in fatty acid pathway
in treated tolerant and susceptible oil palm roots
inoculated with G. boninense
6.3.4 Lane-based array analysis of SAD3 genes differentially
expressed upon treatment through qRT-PCR products
6.4 Discussion
6.4.1 Activation of the Trp pathway in response to G. boninense
infection
6.4.2 Stearoyl- ACP Desaturase (SAD) activity in oil palm
defense mechanism
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7. SUMMARY, CONCLUSION AND RECOMMENDATIONS FOR
FUTURE RESEARCH
REFERENCES
APPENDICES
BIODATA OF STUDENT
LIST OF PUBLICATIONS
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