UNIVERSITI PUTRA MALAYSIA

FORMULATION OF MYCOINSECTICIDES AS WATER-DISPERSIBLE

GRANULES FOR VEGETABLE INSECT PEST CONTROL

IVY CHAI CHING HSIA

FP 2011 44

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FORMULATION OF MYCOINSECTICIDES AS WATER-DISPERSIBLE

GRANULES FOR VEGETABLE INSECT PEST CONTROL

By

IVY CHAI CHING HSIA

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements of the Degree of Doctor of Philosophy

February 2011

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Dedicated to my beloved parents.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Doctor of Philosophy

FORMULATION OF MYCOINSECTICIDES AS WATER-DISPERSIBLE

GRANULES FOR VEGETABLE INSECT PESTS CONTROL

By

IVY CHAI CHING HSIA

February 2011

Chairman: Professor Dzolkhifli Omar, PhD

Faculty: Agriculture

Microbial pesticides have been widely accepted as a biological alternative to chemical

insecticides in controlling insect pests. Where disease-causing (entomopathogenic) fungi

are being manipulated and used inundatively for insect pest control, such microbial

pesticides are known as mycoinsecticides. The infective propagules known as conidia

produced by the entomopathogenic fungi was mass-produced using semi-solid

fermentation to be used as an active ingredient in a mycoinsecticidal product. The aims of

this study was to formulate the conidia as water-dispersible granules (WG), to delve into

WG formulation methods, and to evaluate the factors that influence the biological and

physical performances of the WG-conidia formulations in both laboratory and glasshouse

conditions.

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The study initiated with the screening for the best entomopathogenic fungal isolate to be

used as active ingredient. Five fungal isolates i.e. Metarhizium anisopliae (MPs, MaBg

and MaCc1a), Beauveria bassiana (BbGc) and Paecilomyces fumosoroseus (PfPx) were

screened by exposing each isolate’s conidia to wet-heat and oven-heat stress through a

series of temperatures. Isolate MPs showed the best tolerance to the heat stresses and was

selected as the active ingredient in all WG-conidia formulations. Inert ingredients and/or

additives were then selected based on the ability of the conidia of MPs to survive more

than 80% germination upon contact with each ingredient at different temperatures and

exposure duration. Following the selection of inert ingredients and/or additives, ternary

phase diagrams were constructed to obtain five ‘Surfactant systems’, in which a total of

22 different WG-conidia formulations were prepared. All 22 formulations were tested

and rated for their physical and biological performances using four parameters i.e.

conidial fresh viability, viability after 7 days in storage, suspensibility, and dispersibility.

Five formulations were rated as ‘Good’, 12 formulations as ‘Satisfactory’ and five was

‘Unsatisfactory’. Six WG-conidia formulations containing additives were selected and

tested for their storage stability at different temperatures and exposure period. Results

showed that shelf life of WG-conidia formulations were greatly reduced when storage

temperature and exposure period increased. Shelf life improved when additive(s) was

added to the formulations. Formulations containing sodium alginate and sodium acetate

showed better conidial germination (80%), 15 days after formulation (DAF) at 15°C, than

formulations without additives. While the formulation containing sodium alginate

showed 27% conidial germination at 30DAF, 15°C, most WG-conidia formulations did

not store well beyond this period and temperature.

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The effectiveness of the WG-conidia formulation was evaluated through a time-mortality

response bioassay against the insect pests of economic importance in Malaysia, Plutella

xylostella and Epilachna indica. The WG-conidia formulation, 3B5gK, was

comparatively satisfactory to unformulated conidia in causing death to both insect pests

with median lethal time (LT50) of 6.5 days and 5 days for P. xylostella and E. indica,

respectively. Relative potency of unformulated conidia was 1.3-1.6 times than that of

3B5gK. In a pre-field trial conducted in the glasshouse to investigate the effects of

different volume application rates of WG-conidia formulations on the larvae of P.

xylostella, the formulation containing sodium alginate, 7A5gBAl caused the quickest

mortality of larvae (LT50=72 hours) at high volume application rate compared to three

other WG-conidia formulations and unformulated conidia. Most WG-conidia

formulations showed potential to be further developed as mycoinsecticidal products due

to their good biological potency and physical performances in both laboratory conditions

and pre-field application. However, further studies should be done to improve shelf lives

of the product and their applicability in reduced cost, which are imperative factors for any

microbial products to be acceptable by end users.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

FORMULASI KULAT SEBAGAI RACUN ‘WATER-DISPERSIBLE GRANULES’

UNTUK KAWALAN SERANGGA PEROSAK TANAMAN

Oleh

IVY CHAI CHING HSIA

Februari 2011

Pengerusi: Profesor Dzolkhifli Omar, PhD

Fakulti: Pertanian

Penggunaan racun perosak dalam bentuk mikroorganisma telah lama diterima-guna

pakai sebagai alternatif kepada racun kimia dalam mengawal serangga perosak. Kulat

yang boleh menyebabkan penyakit kepada serangga perosak, atau lebih dikenali sebagai

kulat entomopatogen boleh dimanipulasikan untuk dijadikan racun serangga atau

mikoinsectisid. Bahan jangkitan/sebaran atau konidia yang dikeluarkan oleh kulat

entomopatogen boleh dimanipulasikan dan hasilkan secara besar-besaran melalui

fermentasi separa-pepejal untuk dijadikan bahan aktif racun serangga. Objektif kajian ini

adalah untuk merumus konidia ini sebagai bahan aktif racun serangga dalam bentuk

granul tersebar-air (WG), iaitu bentuk formulasi butiran kering yang terbaru dalam

pasaran. Kajian ini juga akan menyelami cara dan proses konidia diformulasikan sebagai

WG-konidia, serta penilaian kepada faktor-faktor yang mempengaruhi prestasi biologi

dan fisikal formulasi-formulasi di makmal dan rumah kaca.

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Percubaan pertama adalah pemilihan kulat yang terbaik antara lima isolat kulat yang

tersedia, iaitu MPs, MaBg, MaCc1a daripada kulat Metarhizium anisopliae, BbGc

(Beauveria bassiana) dan PfPx (Paecilomyces fumosoroseus) untuk dijadikan bahan aktif

racun. Isolat-isolat didedahkan kepada suhu tinggi dalam bentuk wap basah dan wap

kering (oven). Isolat kulat MPs menunjukkan toleransi tertinggi kepada suhu dan

seterusnya konidianya dipilih sebagai bahan aktif untuk kesemua fomulasi WG-konidia.

Kemudiannya, bahan-bahan lengai dan aditif-aditif untuk diformulasikan bersama

konidia dipilih berdasarkan keupayaan konidia MPs untuk hidup >80% apabila terdedah

kepada bahan lengai dan aditif tersebut untuk sesuatu masa dan suhu tertentu. Selepas

bahan-bahan lengai dan aditif–aditif telah dipilih, diagram fasa-tiga darjat diolah supaya

lima ‘Sistem Surfaktan‘ didirikan dan 22 formulasi WG-konidia disediakan. Setiap

formulasi WG-konidia itu diuji dan dipangkatkan berdasarkan prestasi biologi dan fisikal

mereka, iaitu percambahan segar konidia, percambahan konidia selepas 7 hari distorkan,

keupayaan untuk mengampai, dan keupayaan untuk melerai apabila dimasukkan ke

dalam air. Lima formulasi telah dikategorikan sebagai ‘Baik‘, 12 formulasi dikategorikan

sebagai ‘Memuaskan‘, manakala 5 lagi ‘Tidak Memuaskan‘. Enam formulasi terpilih

kemudiannya diuji untuk kestabilan-dalam-simpanan dengan mendedahkan formulasi-

formulasi kepada suhu dan tempoh yang berbeza. Ujian menunjukkan bahawa kestabilan

kesemua WG-konidia menurun dengan mendadak apabila suhu dan tempoh dedahan

meningkat. Jika dibuat bandingan, kestabilan WG-konidia yang mengandungi aditif

adalah lebih tinggi daripada formulasi yang tiday dirumus dengan aditif. Formulasi yang

mengandungi kombinasi aditif - sodium alginat dan sodium asetat merekodkan

percamabahn konidia sebanyak 80% selepas 15 hari terdedah kepada suhu 15°C.

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Sementara itu, formulasi yang mengandungi sodium alginat merekodkan 27% germinasi

selepas 30 hari terdedah kepada suhu 15°C, walaupun pada suhu tersebut, kebanyakan

formulasi WG-konidia telahpun menjadi tidak stabil melebihi 30 hari dalam simpanan.

Keberkesanan formulasi-formulasi WG-konidia ditentukan melalui ujikaji masa-respon

terhadap serangga perosak yang penting di Malaysia iaitu Plutella xylostella dan

Epilachna indica. Keupayaaan membunuh median (LT50) bagi formulasi WG-konidia,

3B5gK, didapati memuaskan dengan LT50 = 6.5 hari dan LT50 = 5 hari untuk P.

xylostella dan E. indica, masing-masing. Potensi relatif konidia segar adalah 1.3-1.6 kali

daripada 3B5gK. Dalam ujikaji pra-lapangan di rumah kaca untuk mengetahui kesan

isipadu aplikasi seburan berbeza (VAR) kepada larva P. xylostella, boleh dirumuskan

bahawa formulasi WG-konidia 7A5gBAl menyebabkan kematian yang paling pantas

iaitu 72 jam menggunakan VAR tinggi, berbanding 3 formulasi WG-konidia lain dan

konidia segar. Formulasi-formulasi WG-konidia telah menunjukkan potensi untuk

dikembangkan sebagai produk mikoinsecticid kerana menunjukkan prestasi biologi dan

fisikal yang memuaskan di makmal dan lapangan. Namun, kajian yang lebih harus

dilakukan untuk memperbaiki kestabilan dalam simpanan dan pengurangan kos

pembuatan dan penggunaan, iaitu dua faktor terpenting dalam penerimaan-guna pakai

produk mikrobial oleh para pengguna.

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ACKNOWLEDGEMENTS

I would like to express my deepest gratitude and appreciation to my supervisor Prof.

Dzolkhifli bin Omar, that throughout my Ph.D, candidacy had provided me not only

guidance but important feedbacks in order to complete this dissertation and research. He

was very supportive of my opinions and ideas and there was definitely no one as patient

as him in assessing my work. My mum and dad should be put in the highest honour for

firstly, allowing me to pursue a subject so unusual yet endearing to my heart. I dedicate

this doctorate to them for they have not only endured but respected my pursue of this

degree. This journey would not have come to a closure without the generous financial

assistance of the Kuok Foundation Berhad who was very supportive of my research

throughout my 3-year tenure. Not forgetting the members of the Faculty of Agriculture,

UPM, especially the officers in the Department of Plant Protection who have handled all

my vital administrative matters. Not forgetting the lab mates and lab assistant from

Toxicology laboratory (year 2005-2010) - Arshia Hematpoor, Norhayati Ngah, Mahbub

Morshed, Mogeret Sidi, Dahlia Shahbuddin, Ahmad Termizi, Aizat, Zaki, En. Jagasi, and

lab assistants from Microbiology Lab who have strongly supported me technically and

given me the priceless joy I felt while working in the lab. Deepest appreciation also to

members of the faculty that have advised and motivated me directly - Dr. Tan Yee How,

Prof. Yusof Ibrahim, Dr. Rita Muhammad, and Dr. Kamaruzaman Sijam.

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I am thankful for the time and delight that was shown throughout my journey in UPM.

Without these people and also others that I’ve not mentioned, I would not have been what

I am today.

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I certify that an Examination Committee met on 23 February 2011 to conduct the final

examination of Ivy Chai Ching Hsia on her Doctor of Philosophy thesis entitled

“Formulation of Mycoinsecticides as Water-Dispersible Granules for Vegetable

Insect Pests Control” in accordance with Universiti Pertanian Malaysia (Higher Degree)

Act 1980 and Universiti Pertanian Malaysia (High Degree) Regulations 1981. The

Committee recommends that the candidate be awarded the Doctor of Philosophy.

Members of the Examination Committee were as follows:

LAU WEI HONG, Ph.D.

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

RITA MUHAMAD, PhD

Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Internal Examiner)

AHMAD SAID SAJAP, PhD

Professor

Faculty of Forestry

Universiti Putra Malaysia

(Internal Examiner)

ROY PETER BATEMAN, PhD

International Pesticide Application Research Consortium (IPARC)

Imperial College London, Silwood Park Campus, Ascot

United Kingdom

(External Examiner)

________________________

Shamsuddin Sulaiman, Ph.D.

Professor/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 fulfillment of the requirement for the degree of Doctor of Philosophy. The

members of the Supervisory Committee were as follows:

DZOLKHIFLI B OMAR, PhD

Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Chairman)

YUSOF B IBRAHIM, PhD

Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Member)

TAN YEE HOW, PhD

Associate Professor

Faculty of Agriculture

Universiti Putra Malaysia

(Member)

_________ _____________________

HASANAH MOHD GHAZALI, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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

been duly acknowledged. I also declare that it has not been previously or is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or other

institution.

________________________

IVY CHAI CHING HSIA

Date:

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS ix

APPROVAL xi

DECLARATIONS xiiii

LIST OF TABLES xiv

LIST OF FIGURES xvii

LIST OF ABBREVIATIONS/NOTATIONS/GLOSSARY OF TERMS xix

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 6

2.1 Entomopathogens and biopesticides 6

2.2 Entomopathogenic fungi 8

2.2.1 Beauveria bassiana (Balsamo) Vuellemin 10

2.2.2 Paecilomyces (=Isaria) fumosoroseus Wize 11

2.2.3 Metarhizium anisopliae (Metchnikoff) Sorokin 12

2.3 Formulation of biopesticides 14

2.3.1 Principles and background 14

2.3.2 Types of biopesticide formulation 17

2.4 Entomopathogenic fungi conidia in water-dispersible

granules (WG) 19

2.4.1 Background 19

2.4.2 Development of WG-conidia 21

2.5 Application of mycoinsecticides 31

2.6 Vegetable pests 37

2.6.1 Plutella xylostella Linnaeus 37

2.6.2 Epilachna indica Mulsant 40

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3 CULTURE AND MAINTENANCE OF

ENTOMOPATHOGENIC FUNGI 43

3.1 Introduction 43

3.2 Fungal culture and maintenance 43

3.3 Quantifying conidial viability 45

3.4 Mass production of fungal conidia 47

4 SCREENING OF FUNGAL ISOLATES AND INERT

INGREDIENTS SELECTION 50

4.1 Introduction to the screening of fungal isolate 50

4.1.1 Methodology 51

4.1.2 Results 54

4.1.3 Discussion 63

4.1.4 Conclusion 66

4.2 Introduction to the selection of inert ingredients 67

4.2.1 Methodology 68

4.2.2 Results 72

4.2.3 Discussion 78

4.2.4 Conclusion 82

5 PREPARATION OF WG-CONIDIA 84

5.1 Introduction 84

5.2 Methodology 86

5.2.1 Ingredients used in WG-conidia formulation 86

5.2.2 Three-component blending 86

5.2.3 Response region within the ternary phase diagram 88

5.2.4 The Surfactant System 88

5.2.5 Processes in formulating WG-conidia 89

5.2.7 Effects of formulation process on conidial viability 92

5.3 Results 95

5.3.1 Ingredients selected for WG-conidia formulation 97

5.3.2 Determination of Point A and Point B 97

5.3.3 Surfactant system of the WG-conidia formulations 98

5.3.4 Conidia viability for different diluents 101

5.3.5 Conidia viability during each WG formulation process 102

5.4 Discussion 104

5.5 Conclusion 105

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6 PERFORMANCES AND SHELF LIFE OF WG-CONIDIA

FORMULATIONS 106

6.1 Introduction 106

6.2 Materials and methods 107

6.2.1 Physical performances of WG-conidia formulations 107

6.2.2 Biological performances of WG-conidia formulations 109

6.2.3 Quantifying the performances of WG-conidia

formulations 110

6.2.4 Study of the shelf lives of WG-conidia formulations 111

6.3 Results 115

6.3.1 Evaluation of all 22 WG-conidia formulations 115

6.3.2 Shelf lives of WG-conidia formulations 122

6.4 Discussion 127

6.5 Conclusion 133

7 EFFECTIVENESS OF WG-CONIDIA FORMULATIONS

AGAINST INSECT PESTS 135

7.1 Introduction 135

7.2 Methodology 136

7.2.1 Laboratory bioassay against Plutella xylostella L. and

Epilachna indica Muls. 136

7.2.2 Effects of different volume application rates (VARs)

of WG-conidia formulations on P. xylostella 141

7.3 Results 146

7.3.1 Laboratory bioassay against P. xylostella and

E. indica 146

7.3.2 Effects of different volume application rates (VARs)

on P. xylostella 148

7.4 Discussion 150

7.5 Conclusion 154

8 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATION FOR FUTURE RESEARCH 156

REFERENCES 160

BIODATA OF STUDENT 174