UNIVERSITI PUTRA MALAYSIA EFFECTS OF ARBUSCULAR ...

UNIVERSITI PUTRA MALAYSIA

EFFECTS OF ARBUSCULAR MYCORRHIZAL FUNGI AND Trichoderma harzianum ON GROWTH AND BASAL STEM ROT DISEASE

SUPPRESSION IN OIL PALM

MOHD NAZIF SAIFUDDIN BIN ABD MALIK

FS 2012 46

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MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA

2012

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By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for Degree of Master of Science

March 2012

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DEDICATIONS

االلهھم صلى على محمد ووعلى االل محمد

This thesis is dedicated to my beloved parents, sisters, family and friends.

&

Al-Fatihah to Allahyarhamah Professor Dr. Faridah Abdullah.....

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for degree of Master of Science



By


March 2012

Chairman : Associate Professor Radziah Othman, PhD Faculty : Science The Malaysian oil palm industry is a major sector that plays an important role in

supporting Malaysia’s economy. This industry faced huge challenges to increase oil

palm yields due to the limited land area for oil palm cultivation and the basal stem

rot (BSR) disease that attacks oil palm. Chemical fertilizers and chemical fungicides

have been used to overcome these problems, but the results have not been

satisfactory. Thus, biological approaches using beneficial microorganism such as

arbuscular mycorrhiza and Trichoderma harzianum, could be another alternative to

complement the role of conventional fertilizers and fungicides. Arbuscular

mycorrhiza (AM) and T. harzianum have been well known as plant growth enhancer

and protector against diseases in various plant species. Therefore, both of these fungi

were used in the present study to determine their effects on oil palm. The objectives

of this study were: i) to compare the effects of AM and T. harzianum (isolate FA

1132) as single and mixed inoculums on oil palm growth in nursery trial, ii) to

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compare the effects of AM and T. harzianum (isolate FA 1132) as single and mixed

inoculums on growth of oil palms in the field, and iii) to compare the effects of AM

and T. harzianum (isolate FA 1132) as single and mixed inoculums to suppress basal

stem rot (BSR) disease in oil palm seedlings. Three different studies were conducted

to fulfill these objectives. Experiment 1 was conducted in a nursery trial with seven

treatments, which were single AM species (SAM), multiple AM species (MAM),

Trichoderma harzianum (isolate FA 1132) infused-compost (TC), mixture of SAM

with TC (SAM+TC), mixture of MAM with TC (MAM+TC), chemical fertilizers (F)

and the control (C). Oil palm vegetative parameters were observed six times over 24

weeks. Leaf and soil samples were analyzed for chemical nutrients. Experiment 2

was conducted under field conditions at Mungka plantation, Segamat, Johore. There

were five treatments, which were MAM, TC, MAM+TC, F and C. Vegetative

growth parameters were observed every six weeks until week 30. Leaf and soil

samples were also analyzed for chemical nutrients. Experiment 3, the disease

resistance study, was conducted in a glasshouse with oil palms artificially infected

with Ganoderma boninense. Seven treatments as in Experiment 1 were given after

the artificial infection. Trichoderma harzianum (isolate FA 1132) infused-compost

(TC) oil palms showed the best overall growth performance in Experiment 1. Oil

palms receiving TC had the highest mean shoot dry weight (15.74 g), leaf area

(1181.65 cm2) and potassium (K) content (2.68 %). Oil palms receiving TC,

SAM+TC and MAM+TC also had higher stem girth and frond numbers compared to

other treatments. Higher chlorophyll a (2.51 mg g-1), chlorophyll b (0.84 mg g-1) and

total chlorophyll (3.35 mg g-1) were also recorded in TC treated oil palms.

Application of TC also increased the mean pinnae number (15.30) better than in F

and control oil palms. Oil palms treated with TC had higher nitrogen, N (3.79 %) and

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phosphorus, P (0.14 %) contents. Overall, the performance of the treatments in

decreasing order of growth performance in the nursery trial was TC > SAM+TC >

MAM+TC > MAM > SAM > F > C. The results of the field trial (Experiment 2)

were similar to Experiment 1, and TC treatments gave the best overall growth

performance. Oil palms receiving the TC treatment had the best stem girth (80.2 cm),

frond length (193.50 cm), pinnae number (112.3) and fruit bunch number (1.90).

Higher frond numbers (20.3), chlorophyll a (3.70 mg g-1), chlorophyll b (1.72 mg g-1)

and total chlorophyll (5.42 mg g-1) contents were recorded in TC treated oil palms.

The contents of N (3.12 %), P (0.26 %), K (2.39 %) and calcium, Ca (0.65 %)

recorded were also higher in TC treated oil palms. Overall, the performance of the

treatments in decreasing order of growth performance in the field trial was TC >

MAM+TC > MAM > F > C. Results of Experiment 3 showed that BSR disease was

reduced by 100.00 % in TC treated oil palms, while the reduction was 91.17 % in

SAM+TC and MAM+TC oil palms. However, oil palms treated with MAM (50.00

%) and SAM (43.75 %) had higher percentage of disease severity compared to the oil

palms treated with chemical fertilizers (33.30 %). Application of TC and the mixture

of AM and TC showed satisfactory results in suppressing BSR disease in oil palms

compared to AM inoculum alone. In conclusion, oil palms treated with Trichoderma

harzianum (isolate FA 1132) showed the best growth performance both in the

nursery and field trials. Trichoderma harzianum (isolate FA 1132) was also an

effective antagonist against Ganoderma boninense that causes BSR disease in oil

palm seedlings.

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

KESAN-KESAN KULAT MIKORIZA ARBUSKUL DAN Trichoderma harzianum TERHADAP PERTUMBUHAN DAN PENINDASAN PENYAKIT

REPUT PANGKAL BATANG DALAM KELAPA SAWIT

Oleh


Mac 2012

Pengerusi : Profesor Madya Radziah Othman, PhD Fakulti : Sains Industri kelapa sawit Malaysia merupakan sektor utama yang memainkan peranan

penting dalam menyokong ekonomi Malaysia. Industri ini berhadapan cabaran besar

untuk meningkatkan hasil kelapa sawit kerana kawasan tanah yang terhad bagi

penanaman kelapa sawit dan penyakit reput pangkal batang (RPB) yang menyerang

kelapa sawit. Baja kimia dan racun kulat kimia telah digunakan untuk mengatasi

masalah-masalah ini, tetapi keputusannya tidak memuaskan. Oleh itu, pendekatan

biologi menggunakan mikroorganisma bermanfaat seperti mikoriza arbuskul dan

Trichoderma harzianum, boleh menjadi alternatif lain bagi melengkapkan peranan

baja dan racun kulat konvesional. Mikoriza arbuskul (AM) dan Trichoderma

harzianum telah dikenali sebagai peningkat pertumbuhan pokok dan pelindung

terhadap penyakit dalam pelbagai spesies tumbuhan. Oleh itu, kedua-dua kulat ini

telah digunakan dalam kajian ini untuk menentukan kesan-kesan mereka terhadap

kelapa sawit. Objektif-objektif kajian ini adalah i) untuk membandingkan kesan-

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kesan AM dan T. harzianum (isolat FA 1132) sebagai inokulum tunggal dan

campuran terhadap pertumbuhan kelapa sawit dalam percubaan nurseri, ii) untuk

membandingkan kesan-kesan AM dan T. harzianum (isolat FA 1132) sebagai

inokulum tunggal dan campuran terhadap pertumbuhan kelapa sawit di lapangan, dan

iii) untuk membandingkan kesan AM dan T. harzianum (isolat FA 1132) sebagai

inokulum tunggal dan campuran untuk menindas penyakit reput pangkal batang

(RPB) dalam anak pokok kelapa sawit. Tiga kajian berbeza telah dilaksanakan untuk

memenuhi objektif-objektif ini. Eksperimen 1 telah dilaksanakan dalam percubaan

nurseri dengan tujuh rawatan, iaitu satu spesies AM (SAM), pelbagai spesies AM

(MAM), Trichoderma harzianum (isolat FA 1132) terapan-kompos (TC), campuran

SAM dengan TC (SAM+TC), campuran MAM dengan TC (MAM+TC), baja kimia

(F) dan kawalan (C). Parameter vegetatif kelapa sawit telah diperhatikan enam kali

dalam 24 minggu. Sampel daun dan tanah telah dianalisis untuk nutrien kimia.

Eksperimen 2 telah dilaksanakan di bawah kondisi lapangan di ladang Mungka,

Segamat, Johor. Terdapat lima rawatan, iaitu MAM, TC, MAM+TC, F dan C.

Parameter pertumbuhan vegetatif telah diperhatikan setiap enam minggu sehingga

minggu ke-30. Sampel daun dan tanah telah juga dianalisis untuk nutrien kimia.

Eksperimen 3, kajian rintangan penyakit, telah dilaksanakan di dalam sebuah rumah

kaca dengan menjangkitkan kelapa sawit dengan Ganoderma boninense secara

buatan. Tujuh rawatan seperti dalam eksperimen 1 telah diberikan selepas jangkitan

secara buatan. Kelapa sawit Trichoderma harzianum (isolat FA 1132) terapan-

kompos (TC) menunjukkan prestasi pertumbuhan keseluruhan yang terbaik dalam

Eksperimen 1. Kelapa sawit yang menerima TC mempunyai min berat kering pucuk

(15.74 g), luas daun (1181.65 cm2) dan kandungan kalium, K (2.68 %) yang

tertinggi. Kelapa sawit yang menerima TC, SAM+TC dan MAM+TC juga

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mempunyai lilitan batang dan bilangan pelepah yang lebih tinggi berbanding dengan

rawatan lain. Klorofil a (2.51 mg g-1), klorofil b (0.84 mg g-1), jumlah klorofil (3.35

mg g-1) yang lebih tinggi juga telah direkodkan dalam kelapa sawit dirawat dengan

TC. Aplikasi TC juga meningkatkan min bilangan pinnae (15.30) lebih baik daripada

kelapa sawit F dan kawalan. Kelapa sawit yang dirawat dengan TC mempunyai

kandungan nitrogen, N (3.79 %), dan fosforus, P (0.14 %) yang lebih tinggi. Secara

keseluruhan, prestasi rawatan-rawatan dalam turutan menurun bagi prestasi

pertumbuhan dalam percubaan nurseri adalah TC > SAM+TC > MAM+TC > MAM

> SAM > F > C. Keputusan percubaan lapangan (Eksperimen 2) adalah sama dengan

Eksperimen 1, dan rawatan TC memberikan prestasi pertumbuhan keseluruhan yang

terbaik. Kelapa sawit yang menerima rawatan TC mempunyai lilitan batang (80.2

cm), panjang pelepah (193.50 cm), bilangan pinnae (112.3) dan bilangan tandan buah

(1.90) yang terbaik. Bilangan pelepah (20.3), kandungan klorofil a (3.70 mg g-1),

klorofil b (1.72 mg g-1) dan jumlah klorofil (5.42 mg g-1) yang lebih tinggi dicatatkan

dalam kelapa sawit dirawat dengan TC. Kandungan N (3.12 %), P (0.26 %), K (2.39

%) dan kalsium, Ca (0.65 %) yang direkodkan juga lebih tinggi dalam kelapa sawit

dirawat dengan TC. Secara keseluruhan, prestasi rawatan-rawatan dalam turutan

menurun bagi prestasi pertumbuhan dalam percubaan lapangan adalah TC >

MAM+TC > MAM > F > C. Keputusan dalam Eksperimen 3 menunjukkan penyakit

RPB telah dikurangkan sebanyak 100.00 % dalam kelapa sawit yang dirawat dengan

TC, manakala pengurangan sebanyak 91.17 % dalam kelapa sawit SAM+TC dan

MAM+TC. Walau bagaimanapun, kelapa sawit yang dirawat dengan MAM (50.00

%) dan SAM (43.75 %) mempunyai peratusan keparahan penyakit yang lebih tinggi

berbanding kelapa sawit yang dirawat dengan baja kimia (33.30 %). Aplikasi TC dan

campuran AM and TC telah menunjukkan keputusan memuaskan dalam menindas

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penyakit RPB dalam kelapa sawit berbanding inokulasi AM sendirian.

Kesimpulannya, kelapa sawit yang dirawat dengan Trichoderma harzianum (isolat

FA 1132) telah menunjukkan prestasi pertumbuhan terbaik dalam kedua-dua

percubaan nurseri dan lapangan. Trichoderma harzianum (isolat FA 1132) juga

merupakan antagonis yang berkesan terhadap G. boninense yang menyebabkan

penyakit RPB dalam anak pokok kelapa sawit.

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ACKNOWLEDGEMENTS

بسم هللا االرحمن االرحيیم

Alhamdulillah, praise be to Allah for all His kindness, blessings and mercy in

allowing me to complete my master thesis successfully. Throughout these years, I

have received much support from my supervisors, lecturers, family and friends. It

would be possible for me to complete my research and thesis without their helps.

Therefore, in this opportunity, it is my pleasure to express my sincere gratitude to all

of them.

Most of all, with immense pleasure, I would like to express my heartfelt appreciation

to Allahyarhamah Professor Dr. Faridah Abdullah, who was the former Chairman of

my supervisory committee for all her precious supervision, guidance and advice

throughout the difficult times in completing my research. I am indebted for her

invaluable knowledge, constant encouragement and her patience with me for all over

the years. May Allah bless you and place your soul among the Solihin. Amin.

In this opportunity, I would like to express my sincere and deepest gratitude to

Associate Professor Dr. Radziah Othman, chairman of my supervisory committee for

her invaluable supervision, never ending assistance and constant encouragement

throughout the research and writing stage. My deepest appreciation goes to Professor

Dr. Umi Kalsom Yusuf, member of supervisory committee for her helpful guidance

and suggestion, continuous motivation, and endless support throughout my studies,

thesis preparation and my career path.

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Special appreciation goes to my mother (R. Zainab R. Aziz), father (Abd Malik

Ismail), sisters, brother and family for their endless love, continuous support,

encouragement and sacrifice. Thank you for not giving up on me and always being

there for me, especially during the hardest times.

Special thanks to Dr. Jugah Kadir for his guidance and explanation in designing the

research experimental designs. I am greatly indebted to EPA Management Sendirian

Berhad who allowed me to run the field experiment in Mungka Plantations, Segamat,

Johore. Thank you for the support and cooperation that have been given during those

periods. Special thanks also to Sime Darby Berhad for sponsoring the oil palm

seedlings and Myagri® for sponsoring MYCOgold®. Thank you to Mr. Norazlan

Mohammad for driving the members of mycology laboratory and I to the planting

site. Thanks also to all the staff of Department of Biology, Faculty of Science, UPM

for their help and cooperation.

I am also thankful to all the members of Mycology Laboratory (Husrita Hussin,

Mohd Hefni Rusli, Noor Haida Sebran, Shafique Siddique, Allicia Jack, Norabhar

Khalid, Tee Sue Tsean, Nurazila, Nurliyana and Arbaayah) for all the support and

knowledge sharing. Not forgetting to all the postgraduate members, thank you for the

friendship, support and good times we have had together.

Special thanks to Bokhary Zainal, Khairul Azri, Ahmad Syukri, Zul Husni and

Salumiah. Not forgetting to Akhmal Ayob, Irwan Rani, Firdaus Abdullah,

Allahyarham Farid Zakaria, Adlan, Irwan Ariffin, Suffian, Idzwan, Mubaraq, Nader

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Ismail, Khairil Azhar, Azmey, Izar Azmi, Ihab Ismail, Ismawie, Ismat, Jeefferie Abd

Razak, Salfarina Ramli, Saidatul Husni, Farizan Tohfah, Fatma Azwani, Hisham,

Shahrizim, Sapari, Amirul Zaidi, Zul Aizzi, Khairul Sekawi and Eswandi. Thank you

for your friendship, support, encouragement and for being there for me even the

world is pulling me down. Your friendship makes my life cheerful and keeping me

sane throughout the difficult times.

It is possible for me to complete my research and thesis without the help of many

people and it would be impossible to individually thank of all those who deserve it.

Therefore, I would like to apologize if I forgot to mention few names right here.

However, I would like to acknowledge my debt once again to all those people who

made this thesis possible and enriched my life in the past few years.

Thank you...!

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I certify that a Thesis Examination Committee has met on 6th March 2012 to conduct the final examination of Mohd Nazif Saifuddin bin Abd Malik on his thesis entitled "Effects of Arbuscular Mycorrhizal Fungi and Trichoderma harzianum on Growth and Basal Stem Rot Disease Suppression in Oil Palm" in accordance with the Universities and University Colleges 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 Examination Committee were as follows: Norhani Abdullah, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Sariah Meon, PhD Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Hishamuddin Omar, PhD Lecturer Faculty of Science Universiti Putra Malaysia (Internal Examiner) Latiffah Zakaria, PhD Associate Professor School of Biological Sciences Universiti Sains Malaysia (External Examiner) __________________________ SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 23 July 2012

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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 Master of Science. The members of the Supervisory Committee were as follows: Radziah Othman, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Umi Kalsom Yusuf, PhD Professor 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 Universiti Putra Malaysia or at any other institution. ____________________________________________ MOHD NAZIF SAIFUDDIN BIN ABD MALIK Date: 6th March 2012

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TABLE OF CONTENTS

Page ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS x APPROVAL xiii DECLARARTION xv LIST OF TABLES xix LIST OF FIGURES xxii LIST OF PLATES xxiii LIST OF ABBREVIATIONS xxiv CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 2.1 Oil Palm 5 2.1.1 Status of oil palm in Malaysia 5 2.1.2 Threats to oil palm industry 7 2.1.3 Basal stem rot disease in oil palm 8 2.1.4 Treatment to control basal stem rot 9 2.1.5 Biological control agents of basal stem rot disease 11 2.2 Mycorrhiza 13 2.2.1 Arbuscular Mycorrhiza (AM) 13 2.2.2 Benefits and mechanism of AM in increasing

nutrient uptake in plants 17

2.2.3 Benefits and mechanism of AM as biological control

20

2.2.4 Benefits and mechanism of AM: tolerance to adverse environments

23

2.3 Trichoderma spp. 24 2.3.1 Benefits and mechanism of Trichoderma spp. as

biological control agent 24

2.3.2 Benefits and mechanism of Trichoderma spp. as plant growth enhancer

29

2.4 Effect of AM and Trichoderma spp. combination as a mixed inoculums

32

3 MATERIALS AND METHODS 3.1 Experiment 1: Effects of arbuscular mycorrhiza (AM) and

Trichoderma harzianum as single and mixed inoculums on oil palm growth in nursery trial

34

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3.1.1 Source of biological fertilizers and oil palm seedlings

34

3.1.2 Experimental layout of nursery trial 35 3.2 Experiment 2: Effects of arbuscular mycorrhiza (AM) and

Trichoderma harzianum as single and mixed inoculums on growth of oil palms in the field

37

3.2.1 Source of biological fertilizers and oil palm seedlings

37

3.2.2 Experimental layout of field trial 37 3.3 Experiment 3: Effects of arbuscular mycorrhiza (AM) and

Trichoderma harzianum as single and mixed inoculums in suppressing basal stem rot in oil palm seedlings

38

3.3.1 Source of biological fertilizers and fungi 38 3.3.2 Preparations of Ganoderma boninense inoculums 39 3.3.3 Artificial infection and experimental layout 39 3.3.4 Assessment of BSR disease developments in oil

palms 40

3.4 Laboratory procedures 42 3.4.1 Leaf area and dry weight determination 42 3.4.2 Chlorophyll content determination 42 3.4.3 Determination of leaf nutrient content 43 3.4.4 Determination of soil nutrient content 43 3.4.5 Determination of arbuscular mycorrhiza (AM)

spore numbers 44

3.4.6 Colonization of AM in oil palm roots 45 3.4.7 Soil pH determination 46 3.4.8 Trichoderma harzianum population in the soil 46 3.5 Assessment of overall best growth performance and

nutrient content 47

3.6 Statistical analysis 48 4 RESULTS 4.1 Experiment 1: Effects of arbuscular mycorrhiza (AM) and


49

4.1.1 Vegetative growth parameters 49 4.1.2 Dry weight and leaf area 53 4.1.3 Chlorophyll contents 54 4.1.4 Leaf nutrient content 55 4.1.5 Assessment of overall growth performance and

nutrient content by growth index based on ranking by mean

57

4.1.6 Arbuscular mycorrhiza and Trichoderma harzianum populations

60

4.1.7 Soil analysis 61 4.2 Experiment 2: Effects of arbuscular mycorrhiza (AM) and

Trichoderma harzianum as single and mixed inoculums on growth of oil palms in the field

62

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4.2.1 Vegetative growth parameters 62 4.2.2 Fruit bunch number 67 4.2.3 Chlorophyll contents 67 4.2.4 Leaf nutrient content 68 4.2.5 Assessment of overall growth performance and

nutrient content by growth index based on ranking by mean

69

4.2.6 Arbuscular mycorrhiza (AM) and Trichoderma harzianum populations

72

4.2.7 Soil analysis 73 4.3 Experiment 3: Effects of arbuscular mycorrhiza (AM) and

Trichoderma harzianum as single and mixed inoculums in suppressing basal stem rot in oil palm seedlings

74

4.3.1 Disease progression in oil palm seedlings 74 4.3.2 Dry weight 76 4.3.3 Leaf nutrient content 76 4.3.4 Arbuscular mycorrhiza and Trichoderma

harzianum populations 77

4.3.5 Soil analysis 78 5 DISCUSSION 5.1 Experiment 1: Effects of arbuscular mycorrhiza (AM) and


80

5.2 Experiment 2: Effects of arbuscular mycorrhiza (AM) and Trichoderma harzianum as single and mixed inoculums on growth of oil palms in the field

87

5.3 Experiment 3: Effects of arbuscular mycorrhiza (AM) and Trichoderma harzianum as single and mixed inoculums in suppressing basal stem rot in oil palm seedlings

91

6 SUMMARY, CONCLUSION AND RECOMMENDATIONS

FOR FUTURE RESEARCH 97

REFERENCES 99 APPENDICES 121 BIODATA OF STUDENT 132 LIST OF PUBLICATIONS 133

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