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UNIVERSITI PUTRA MALAYSIA

EFFECTS OF BACILLUS LICHENIFORMIS BLI008 ON RHIZOCTONIA ROOT ROT DISEASE AND PLANT GROWTH IN CHILLI

KANDANGAMUWA PATHIRANNAHALAGE SOMACHANDRA

FP 2011 15

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EFFECTS OF BACILLUS LICHENIFORMIS BLI008 ON RHIZOCTONIA ROOT

ROT DISEASE AND PLANT GROWTH IN CHILLI

By

KANDANGAMUWA PATHIRANNAHALAGE SOMACHANDRA

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

Master of Science

June 2011

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DEDICATION

Special dedication to:

“JANAKIE”

My ever-loving wife. I live far away from you but I realize how much you

sacrificed.

“DINITHI, MIHIRI, and DINIDU”

My loving daughters and son. Your endless love keeps me alive.

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

fulfillment of the requirements for the degree of Master of Science

EFFECTS OF BACILLUS LICHENIFORMIS BLI008 ON RHIZOCTONIA ROOT ROT DISEASE AND PLANT GROWTH IN CHILLI

By

KANDANGAMUWA PATHIRANNAHALAGE SOMACHANDRA

June 2011

Chair: Associate Professor Jugah b. Kadir, PhD

Faculty: Agriculture

Rhizoctonia root rot disease caused by Rhizoctonia solani Kühn is economically

important on chilli (Capsicum annuum L.). Due to the increasing public concerns

of fungicide usage, unavailability of resistant varieties and the limitations of

cultural methods, biological control is emerging as a promising alternative to

control R. solani in an environmentally-friendly way. Locally isolated Bacillus

strain, Bacillus licheniformis BLI008, was tested against R. solani in-vitro to find out

the antagonistic activity and the mechanism of antagonism. Strong mycelial

growth reduction (74.79±0.48%) associated with frequent induction of a variety

of morphological abnormalities and changes in mycelial color was observed in

dual culture assay. Similar growth reduction (44.99±1.65%) and delaying of

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sclerotial germination occurred with the volatile compounds produced by the

bacterium. Several methods of seed bacterization were tested under pot culture

conditions in the presence of R. solani. A two days old nutrient agar culture of the

bacterium applied as slurry on sterilized chilli seeds subsequently coated with

CaCO3 provided best protection against damping-off. This method of seed

bacterization significantly (P=0.05) increased seed germination (86%) and

reduced post emergence damping-off (22.78%) over untreated seeds, and

performed equally as chemical seed treatment. The control of root disease and

bacterium mediated plant growth promotion was observed in greenhouse pot

experiment. Measurements on root disease severity and growth parameters were

taken 30 and 75 days after transplantation. The highest disease suppression was

achieved when B. licheniformis BLI008 was inoculated (1x109 CFUs/mL) at the

time of seed sowing as a drench to the nursery medium. Bacillus licheniformis

BLI008 inoculation significantly increased shoot length (26.26%), root length

(22.98%) total biomass (173.53%), and leaf chlorophyll content (13.64 mg/g of

fresh weight) of chilli seedlings over the control at 30 days after transplantation.

Those plants flowered earlier producing higher number (27.25) of flowers. In the

presence of R. solani, bacterium mediated growth promotion was observed only

on plants raised in bacterized nursery medium. They showed 12.46% increase in

shoot length, 17.77% in root length and 99.00% in total biomass compared to

untreated control. However, the responses varied with time, and were not

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consistent up to maturity. Among bacterized treatments challenged with R.

solani, the highest (11.21 mg/g fresh weight) and lowest (8.98mg/g fresh weight)

leaf chlorophyll values were reported in plants raised in bacterized medium and

bacterized after four days of planting, respectively. The time taken for flower

initiation and the number of flowers produced by bacterized treatments

challenged with R. solani, except plants inoculated after four days of

transplantation were similar to the control. The results of this study showed that

the bacterial biocontrol agent tested has a great potential in controlling

Rhizoctonia damping-off and root rot of chilli. Its plant growth enhancement can

be considered as an added advantage in crop production.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

KESAN BACILLUS LICHENIFORMIS BLI008 KEATAS REPUT AKAR

RHIZOCTONIA DAN PERTUMBUHAN POKOK CILI

Oleh

KANDANGAMUWA PATHIRANNAHALAGE SOMACHANDRA

Jun 2011

Pengerusi: Profesor Madya Jugah b. Kadir, PhD

Fakulti: Pertanian

Penyakit reput akar Rhizoctonia yang berpunca daripada Rhizoctonia solani Kuhn

merupakan penyakit yang penting daripada aspek ekonomi kepada tanaman cili

(Capsicum annuum L.). Faktor-faktor seperti peningkatan kesedaran orang ramai

terhadap penggunaan racun kulat, ketiadaan varieti yang tahan penyakit dan kekangan

teknik kebiasaan penanaman menjadikan, kaedah pengawalan biologi muncul sebagai

alternatif yang berpotensi untuk mengawal R. solani secara mesra alam. Strain Bacillus

tempatan, Bacillus licheniformis BLI008, telah diuji terhadap R. solani secara in-vitro bagi

mengkaji aktiviti antagonistik dan mekanisme antagonistik. Pengurangan pertumbuhan

miselia secara mendadak (74.79±0.48%) dikaitkan dengan kekerapan ransangan

menyebabkan kecacatan morfologi dan perubahan warna miselia dapat diperhatihan

selepas ujian diwikultur. Komponen volatil yang dihasilhan oleh B. licheniformis

menyebabkan pengurangan pertumbuhan yang serupa pada kadar (44.99±1.65%) serta

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memperlahankan percambahan sklerotia. Beberapa teknik pembakteriaan benih telah

diuji dalam rumah kaca terhadap R. solani. Bakterium berusia 2 hari yang ditumbuhkan

pada agar nutrien telah dicairkan dan diaplikasikan kepada benih cili yang sudah

disteril kemudian diselaputi dengan CaCO3 untuk melindungi daripada penyakit

“lecuh”. Teknik pembakteriaan benih ini signifikan pada (P=0.05) dalam meningkathan

percambahan benih (86%) dan mengurangkan kemunculan “lecuh”(22.78%) berbanding

benih tidak dirawat, serta menghasilkan keputusan sama seperti penggunaan racun

kimia. Pengawalan terhadap penyakit akar dan ransangan pertumbuhan kepada

tanaman yang dirawat dengan penggunaan bakteria dapat diperhatikan pada ujian di

rumah hijau. Bacaan untuk kerosakan penyakit dan parameter pertumbuhan diambil 30

dan 75 hari selepas pemindahan tanaman. Pengawalan penyakit pada kadar tertinggi

terhasil apabila B. licheniformis BLI008 diinokulasi (1x109 CFUs/mL) pada media

tanaman ketika proses penyulaman benih sebagai rawatan kepada media tanaman.

Penginokulatan B. licheniformis BLI008 memberi kesan signifikan dengan meningkathan

pertumbuhan pucuk (26.26%) pertumbuhan akar (22.98%), berat keseluruhan (173.53%)

dan kandungan kolorofil pada daun (13.64 mg/g daripada berat segar) terhadap anak

pokok cili berbanding anak pokok cili yang tidak dirawat selepas 30 hari pemindahan

tanaman. Pokok cili yang dirawat berbunga lebih awal dan menghasilkan lebih jumlah

bunga pada keadaan kehadiran R. solani. Ransangan pertumbuhan hanya diperhatikan

pada pokok yang ditanam di media penanaman yang dirawat dengan bakteria.

Tanaman menunjukkan peningkatan 12.46% pertumbuhan pucuk, 17.77% pertumbuhan

akar dan 99% berat keseluruhan berbanding tanaman tidak dirawat.

Walaubagaimanapun, tindakbalas dihasilkan berbeza mengikut tempoh, dan tidak

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konsisten sehingga proses kematangan. Di antara rawatan bakteria terhadap R. solani,

nilai klorofil yang tertinggi (11.21 mg/g berat segar) dan terendah (8.98 mg/g berat

segar) dikatakan pada tanaman yang ditanam pada media yang dirawat dengan

bakteria dan dirawat dengan bakteria selepas empat hari ditanam. Pokok yang dirawat

dengan bakteria bagi mencegah jagkitan R. solani menghasilkan jumlah bunga serta

tempoh pembungaan yang sama dengan pokok yang tidak dirawat kecuali pokok yang

diinokulasi selepas 4 hari. Keputusan kajian menunjukkan bahawa penggunaan bakteria

sebagai agen kawalan biologi terbukti berpotensi dalam mengawal “lecuh” Rhizoctonia

dan kerosakan akar pada pokok cili. Pertambahan kadar pertumbuhan pada tanaman

boleh dijadikan sebagai nilai tambah kepada pengeluaran tanaman ini.

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ACKNOWLEDGEMENTS

The completion of this study would have been impossible without the support

from many people. First, I am deeply indebted to my supervisory committee

chairman and mentor, Associate Professor Dr. Jugah B. Kadir for his

encouragement, guidance, motivating discussions, understanding, patience and

his open door. I will forever appreciate the working environment that he

provided and his friendliness, which made my stay at UPM a joyful experience.

Equal great appreciation goes to my supervisory committee member Associate

Professor Dr. Halimi Mohd Saud for his perception, discussions and constructive

comments on my work, advice and help throughout my studies and in the

preparation of this final manuscript.

I am exceedingly grateful to Sri Lankan Government and Colombo Plan

Secretariat at Colombo for the financial support of this study.

In the course of my research, I have collaborated with the staff of the Plant

Pathology Laboratory, and I wish to extend my warmest thanks to all those who

lent me a helping hand. A special “thank you” goes to: Mr. Samsuddin, Mr.

Nazri, Mr. Johari and Mrs. Asmalina.

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Thanks are also due to my friends at UPM and in Sri Lanka, for their help,

support and encouragement. Thengoua Fabien Fonguimgo, my closest friend at

UPM, thank you very much for your great support in the preparation of the final

manuscript.

Finally, I take this opportunity to express my deepest gratitude to my father-in-

law, mother-in-law, brothers and sisters. The mountains and oceans would never

separate our hearts being close to each other. I thank you all for your love,

support and encouragement throughout my study in UPM and in my whole life.

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I certify that a Thesis Examination Committee has met on 7th June 2011 to conduct the final examination of Kandangamuwa Pathirannahalage Somachandra on his thesis entitled “Effect of Bacillus licheniformis BLI008 on

Rhizoctonia root rot disease and plant growth of chilli” 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 degree. Members of the Thesis Examination Committee were as follows: Ganesan a/l Vadamalai, PhD Faculty of Agriculture Universiti Putra Malaysia (Chairman) Kamaruzaman b Sijam, PhD, Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Zainal Abdin b Mior Ahmad, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Latiffah binti Zakaria, PhD Centre for Biological Science Studies Universiti Sains Malaysia (External Examiner) ------------------------------------

Bujang Kim Huat, 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 fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows:

Jugah B. Kadir, PhD Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman)

Halimi Mohd Saud, PhD

Associate Professor Faculty of Agricultural Technology Universiti Putra Malaysia (Member)

_________________________________ HASANAH MOHD GHAZALI, 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.

_____________________________________________________________ KANADANGAMUWA PATHIRANNAHALAGE SOMACHANDRA

Date: 7 June 2011

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TABLE OF CONTENTS

Page

ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix

APPROVAL xi DECLARATION xiii LIST OF TABLES xvi LIST OF FIGURES xvii LIST OF ABBREVIATIONS xx CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 4 2.1 Chilli (Capsicum annuum L.) 4

2.1.1 Taxonomy and Spread 4 2.1.2 Economic Importance 5 2.1.3 Problems Militating Improved Production 5 2.1.4 Rhizoctonia Diseases of Chilli 6 2.2 The Fungus Rhizoctonia solani Kuhn 7 2.2.1 History 7 2.2.2 Biology and Ecology 7 2.2.3 Epidemiology 9 2.2.4 Host Range 10 2.2.5 Control of Rhizoctonia 11 2.3 Biological Control 14 2.3.1 Introduction 14 2.3.2 Biocontrol Mechanisms 16 2.3.3 Biocontrol of Rhizoctonia solani 20

2.3.4 Bacillus Species as Biocontrol Agents 21 2.3.5 Bacillus licheniformis as a Biocontrol Agent 22

2.3.6 Bacillus licheniformis as a PGRP 23 2.4 Testing of Biocontrol Agents 23 3 MATERIALS AND METHODS 26

3.1 Antagonistic Bacterium and Pathogenic Fungus 26 3.2 In-vitro Assays 26

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3.2.1 Antagonistic Activity via Dual Culture 26 3.2.2 Antagonistic Activity via Spread Plate 28 3.2.3 Antagonistic Activity via Sealed Plate 28 3.2.4 Inhibition Effect of Volatiles on Sclerotial

Germination 29 3.2.5 Experimental Design and Statistical Analysis 30 3.3 Greenhouse Experiments 31

3.3.1 BLI008 Seed Treatments on the Pre and Post Emergence Damping-off of Chilli 31

3.3.2 Effect of BLI008 Inoculation on root rot and growth of Chilli 35

3.3.3 Determination of the Number of BLI008 CFUs in Soil 44

4 RESULTS AND DISCUSSION 46 4.1 Confirmation of Bacterial Purity 46

4.2 In-vitro Assays 47 4.2.1 Effect of BLI008 on Radial Gowth of R. solani 47 4.2.2 Antifungal Effect of Bacterial Metabolites on Fungal Pigments 47 4.2.3 Effect of BLI008 on Structural Deformations of

R. solani 54 4.2.4 Inhibition Effects of Volatiles on Sclerotial Germination 56 4.3 Greenhouse Experiments (in-vivo studies) 58 4.3.1 Effect of BLI008 Seed Bacterization on the Pre

and Post Emergence Damping-off of Chilli 58 4.3.2 Effect of BLI008 Inoculation at Different Time

Intervals on Root Rot Severity and Seedling Growth of Chilli 66

4.3 Longevity of BLI008 in Artificially Inoculated Soil 88 5 SUMMARY, CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH 91

REFERENCES 98 APPENDICES 129

BIODATA OF STUDENT 142 LIST OF PUBLICATIONS 143