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UNIVERSITI PUTRA MALAYSIA

APPLICATION OF BACILLUS SPHAERICUS UPMB10 FOR GROWTH ENHANCEMENT OF BANANA (MUSA SPP. VAR. BERANGAN) AND

ITS EFFECT ON FUSARIUM WILT

ILLANI ZURAIHAH BINTI IBRAHIM

FP 2008 32

APPLICATION OF BACILLUS SPHAERICUS UPMB10 FOR GROWTH

ENHANCEMENT OF BANANA (MUSA SPP. VAR. BERANGAN) AND ITS

EFFECT ON FUSARIUM WILT


MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA

2008

ii




By


Thesis submitted to the School of Graduate Studies, Universiti Putra

Malaysia, in Fulfillment of the Requirement for the Degree of Master of

Science

SEPTEMBER 2008

iii

DEDICATION

Special dedication to:

Zull and Danial

My lovely family…. For all the supports, endless loves and advices throughout

the long journey to complete my study…. Keeps me alive!

iv

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




By


September 2008

Chairman : Professor Zulkifli Hj Shamsuddin, Ph.D. Faculty : Agriculture

A locally isolated strain Bacillus sphaericus UPMB10, categorized as plant

growth promoting rhizobacteria (PGPR), was reported to have beneficial

effects in stimulating growth and yield of many agricultural crops. This study

explores the indirect mechanism of B. sphaericus UPMB10 that would support

the hypothesis that this PGPR could act as a biocontrol agent. Through in-

vitro study, B. sphaericus UPMB10 was observed to inhibit the growth of

Fusarium oxysporum f.sp cubense race 4 (FOCR4) mycelium by 70% in

culture medium. Malformation of the fungal hyphae by vacuole formation,

swelling and thickening of the hyphal strands occurred with the presence of B.

sphaericus UPMB10. In addition, B. sphaericus UPMB10 showed positive

v

results in the tests for production of cell wall-degrading enzymes and

phosphate solubilizing activity by producing clear zones or halos. It can also

produce a plant growth regulating hormone, indole acetic acid (IAA). An initial

glasshouse study showed that inoculation with B. sphaerius UPMB10

increased plant growth up to 30% in plant height and increased nutrient

uptake namely for N, P, K, and Ca, and also reduced the disease severity

through visual symptoms and vascular discoloration of the corm. In the last

glasshouse experiment, the appearance of the disease incidence was

observed. All of the treatments with FOCR4 application were infected by

FOCR4 including those treated with B. sphaericus UPMB10. Significantly, the

infection occurred at the late stage of the experiment indicating that B.

sphaericus UPMB10 could delay the infection process. Solid inoculum

inoculation with B. sphaericus UPMB10 showed a delayed onset of

symptoms, lower percentage in disease incidence and epidemic rate. As

liquid and solid substrate were selected for evaluation against the disease in

the glasshouse, it showed that soil application of B. sphaericus UPMB10 with

solid inoculum provided the best suppression of Fusarium wilt of banana. In

this study, an inoculum dose of 100 g of solid formulation per plant was

optimum, because it suppressed the disease to the same extent as higher

doses of the antagonist. This showed B. sphaericus UPMB10 has a high

potential for its use as a plant growth enhancer and biocontrol agent in the

disease management of Fusarium wilt in bananas.

vi

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

APLIKASI BACILLUS SPHAERICUS UPMB10 UNTUK MENINGKATKAN

PERTUMBUHAN PISANG (MUSA SPP. VAR. BERANGAN) DAN

KESANNYA KE ATAS PENYAKIT LAYU FUSARIUM

Oleh

ILLANI ZURAIHAH IBRAHIM

September 2008

Pengerusi : Professor Zulkifli Hj Shamsuddin, Ph.D.

Fakulti : Pertanian

Strain bakteria tempatan Bacillus sphaericus UPMB10 yang dikategorikan

sebagai rhizobakteria penggalak pertumbuhan pokok (PGPR) dilaporkan

mempunyai keupayaan dalam menggalakkan pertumbuhan dan hasil dalam

pelbagai tanaman pertanian. Kajian ini merupakan explorasi yang

menunjukkan secara tidak langsung mekanisme yang dapat menyokong

hipotesis PGPR ini untuk bertindak sebagai agen kawalan biologi. Melalui

kaijan in vitro, B. sphaericus UPMB10 telah diperhatikan dapat merencatkan

pertumbuhan Fusarium oxysorum f.sp.cubense race 4 (FOCR4) sebanyak

70% di dalam medium kultur. Ketidakseragaman pertumbuhan hifa fungi yang

disebabkan oleh pembentukan vakuol, pembengkakkan dan penebalan

vii

jaluran hifa telah berlaku dengan kehadiran B. sphaericus UPMB10.

Tambahan pula, B. sphaericus UPMB10 juga telah menunjukkan keputusan

yang positif di dalam ujikaji penghasilan enzim pelupus dinding sel dan ujikaji

aktiviti pelarutan fosfat dengan menunjukkan kesan zon jernih yang jelas atau

halo. Ia juga berupaya menghasilkan hormon pertumbuhan iaitu indole asetik

asid (IAA). Pada permulaan eksperimen rumah kaca menunjukkan yang

inokulasi dengan B. sphaericus UPMB10 telah meningkatkan pertumbuhan

pokok sebanyak 30% ketinggian pokok dan kadar pengambilan nutrient

terutamanya N, P, K dan Ca. Ia juga telah mengurangkan kadar keterukan

penyakit melalui pemerhatian simptom luaran pokok dan perwarnaan umbisi

pokok. Pada akhir eksperimen, kadar insiden penyakit ditunjukkan melalui

simptom luaran dengan menunjukkan kesemua rawatan yang melibatkan

Fusarium telah dijangkiti walaupun dirawat dengan B. sphaericus UPMB10.

Secara signifikannya, serangan hanya bermula lewat di akhir eksperiment

dan ini menunjukkan keupayaan B. sphaericus UPMB10 untuk melambatkan

serangan penyakit tersebut. Inokulum pepejal B. sphaericus UPMB10 telah

menunjukkan perlambatan kemunculan simptom, peratus insiden penyakit

dan kadar epidemik yang rendah. Aplikasi kepada tanah telah menunjukkan

B. sphaericus UPMB10 dengan cara pepejal telah memberikan ketahanan

terbaik kepada penyakit layu fusarium. Kombinasi samada menggunakan

aplikasi substrat cecair atau pepejal telah dipilih untuk penilaian terhadap

penyakit di eksperimen rumah kaca. Di dalam kajian ini, didapati bahawa

viii

inokulum dengan formulasi 100 g pepejal per pokok adalah optimum untuk

memberikan ketahanan terbaik pada tahap antagonis pada dos yang sama.

Kesimpulan menunjukkan B. sphaericus UPMB10 berpotensi tinggi untuk

digunakan sebagai penggalak pertumbuhan pokok dan agen kawalan biologi

dalam pengurusan penyakit layu Fusarium pada tanaman pisang.

.

ix

ACKNOWLEDGMENTS

All praises and thanks to Almighty Allah SWT, the beneficent and merciful for

the blessing and the strength in completion of my study. Firstly, I would like to

express my deepest and sincerest appreciations with a heartfelt gratitude to

Professor Dr. Zulkifli Hj Shamsuddin, the Chairman of Supervisory Committee

for his invaluable advice, constructive criticism, support, encouragement,

patience and understanding that made this study possible. Not forgetting to

Professor Dr. Sariah Meon and Associate Professor Dr. Zakaria Wahab as

members of the Supervisory Committee, for their guidance, suggestions and

invaluable advice and comments throughout the duration of this study and

preparation of this thesis.

Also with deep sense of honour, I wish to extend my sincere gratitude to all

staffs of the Department of Land Management especially in the Soil

Microbiology Laboratory and to Mrs Zarinah and Mr Dzulkifli Duaji for their

time and assistance in the preparation of materials. Heartfelt thanks are also

extended to my friends Wahida, Noraini, Maliza, Zakry, Prema, Negash, Ina,

Palie, Yani, Ziana, Siti and others.

I also would like to thank the Ministry of Science, Technology and Innovation

(MOSTI) for the research grant no. 01-02-04-PR0010/04-02 and financial

x

support through PASCA Scheme that provided me the opportunity to pursue

my master degree.

xi

I certify that an Examination Committee met on 25th September 2008 to conduct the final examination of Illani Zuraihah Ibrahim on her thesis entitled “Application of Bacillus sphaericus UPMB10 for Growth Enhancement of Banana (Musa spp.var. Berangan) and Its Effects On Fusarium Wilt“ in accordance with Universities and University Colleges Act 1971 and the Constitution of the Univeristi Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded Master of Science. Members of the Thesis Examination Committee were as follows: Zaharah Abdul Rahman, Ph.D. Professor Faculty of Agriculture Universiti Putra Malaysia (Chairman) Jugah Kadir, Ph.D. Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Halimi Mohd Saud, Ph.D. Associate Professor Faculty of Agriculture Universiti Putra Malaysia (Internal Examiner) Nik Masdek Nik Hassan, Ph.D. Lecturer Horticultural Research Centre Malaysian Agricultural Research and Development Institute (External Examiner)

BUJANG KIM HUAT, PhD Professor and Deputy Dean, School of Graduate Studies, Universiti Putra Malaysia. Date: 19 February 2009

xii

This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows:

Zulkifli Hj. Shamsuddin, Ph.D. Professor, Faculty of Agriculture, Universiti Putra Malaysia. (Chairman) Sariah Meon, Ph.D. Professor, Faculty of Agriculture, Universiti Putra Malaysia. (Member) Zakaria Wahab, Ph.D. Associate Professor, Faculty of Agriculture, Universiti Putra Malaysia. (Member)

HASANAH MOHD GHAZALI, Ph.D.

Professor and Dean, School of Graduate Studies, Universiti Putra Malaysia. Date: 9 April 2009

.

xiii

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

___________________________

Candidate: ILLANI ZURAIHAH IBRAHIM Date:

xiv

TABLE OF CONTENTS

PAGE iii iv vi ix xi xiii xvii xviii

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST PLATES LIST OF FIGURES LIST ABBREVIATION

xx xxi

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 2.1 Overview of banana industry 2.2 Fusarium wilt

2.2.1 Systematics and Characterization of Fusarium oxysporum

2.2.2 Characterization of Fusarium oxysporum f.sp.cubense

2.2.3 Fusarium Wilt of Banana 2.2.4 Disease Distribution and Losses 2.2.5 Epidemiology 2.2.6 Current Management of Fusarium Wilt

2.3 Beneficial Microbes as An Alternatives for Biocontrol 2.3.1 Bacillus sphaericus

2.4 Mechanisms of Biocontrol by PGPR 2.4.1 Antibiosis 2.4.2 Antifungal Metabolites 2.4.3 Enzymes 2.4.4 Competition 2.4.5 Plant Ethylene Levels 2.4.6 Induced Systemic Resistance 2.4.7 Direct Stimulation of Plant Growth

and Development 2.5 Microbial Inoculum Performance

6 6 7 7 9

10 13 15 17 19 21 24 25 25 27 28 32 33 35

36

xv

3 4

MATERIALS AND METHODS 3.1 Morphological and Metabolite Production

Characteristics, and Biological Activity of B. sphaericus UPMB10 3.1.1 PGPR Strain 3.1.2 Fungal Strain 3.1.3 Culture, Media and Growth Condition 3.1.4 Dual Culture Technique 3.1.5 Hyphal Morphology 3.1.6 Production of Cell-wall Degrading Enzyme 3.1.7 Test for Phosphate Solubilizing Activity 3.1.8 Production of Indole Acetic Acid (IAA)

3.2 B. sphaericus UPMB10 Inoculation on Growth of Banana Seedlings and Pathogenecity Toward Fusarium oxysporum 3.2.1 Tissue-cultured Banana Plantlets 3.2.2 Preparation of Fusarium Inoculant 3.2.3 Plant Tissue Analysis 3.2.4 Plant Growth Measurement 3.2.5 Disease Assessment

3.3 Infectivity Studies and Effect of Different Rates of B. sphaericus UPMB10 Inoculation on the Disease Incidence Caused by Fusarium oxysporum 3.3.1 Inocula Preparation and Application Rate 3.3.2 Plant Growth Performance (based on height

increment) 3.3.3 Evaluation on Different Rates of Inoculum on

Disease Assessment 3.4 Statistical Analysis

RESULTS AND DISCUSSION 4.1 Morphological and Metabolite Production

Characteristics, and Biological Activity of B. Sphaericus UPMB10 4.1.1 Dual Cultural Studies 4.1.2 Hyphal Morphology 4.1.3 Secondary Metabolite Production of B.

sphaericus UPMB10 4.1.3.1 Test for Phosphate Solubilizing Activity 4.1.3.2 Production of Indole Acetic Acid (IAA) 4.1.3.3 Production of Cell Wall-degrading Enzymes

38

38 38 38 39 40 41 41 42 42

43 43 43 44 44 45

48 48

49

50 53

54

54 54 56

60 60 61 63

xvi

4.2 Evaluation of B. sphaericus UPMB10 Inoculation on Plant Growth Performance 4.2.1 Evaluation on Nutrient Status of Different Plant

Components 4.2.1.1 Nitrogen (N) 4.2.1.2 Phosphorus (P) 4.2.1.3 Potassium (K) 4.2.1.4 Calsium (Ca)

4.2.1.5 Magnesium (Mg) 4.2.2 Evaluation on Plant Height Development

4.2.3 Evaluation on Disease Progress 4.3 Effect of Different Rates of B. sphaericus UPMB10

Inoculation on Disease Incidence of Fusarium oxysporum 4.3.1 Evaluation and Comparison Between Liquid vs.

Solid Inocula and Application Rate on Growth Performance

4.3.2 Evaluation on Disease Assessment (Disease Incidence and Symptom Development)

66

73 73 76 77 78 79 79 83

89

92

96

5 GENERAL DISCUSSION AND CONCLUSION 96

REFERENCES/BIBLIOGRAPHY APPENDICES BIODATA OF THE STUDENT

105 131 139

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LIST OF TABLES

No. Table

Page

1 S Some free-living rhizobacteria (PGPR) that are also plant growth-promotive. (Note: Not all strains of the microorganisms can act as PGPR)

20

2 Summary of beneficial and antagonistic characteristic of B. sphaericus UPMB10

65

3 The nutrient status in root, pseudostem, corm and leaves of banana plantlets inoculated with B. sphaericus UPMB10 after 12 weeks. (Means having the same letter (s) in a column of a parameter do not differ significantly at 0.05% level by Tukey Test)

74

4 Total nutrient accumulation in banana plantlets inoculated with B. sphaericus UPMB10 after 12 weeks. (Means having same letter (s) in a column do not differ significantly at 0.05 levels by Tukey’s Test)

75

5 Disease severity (vascular discoloration of corm) index (DSI) of banana seedlings var. Berangan cv. Intan challenged with FOCR4 at 90 days. Each value is the mean of five replicates

86

6 Area Under Disease Progress Curve (AUDPC) and epidemic rate of Fusarium wilt under different treatments

94

xviii

LIST OF PLATES

No. Plate

Page

1 Inoculated banana plants under evaluation for tolerance to Fusarium oxysporum in a glasshouse.

53

2 B. sphaericus UPMB10 colony in a ring inhibiting the growth of FOCR4 on NA plate after 5 days of incubation

55

3 5mm agar disc of FOCR4 on NA plate as the control after 5 days’ incubation

55

4 A streak colony of B. sphaericus UPMB10 bacteria inhibiting the growth of FOCR4 on NA plate after 5 days of incubation.

56

5 (a) (a) Light microscopy shows abnormalities of FOCR4 hyphae with B. sphaericus UPMB10 in the culture medium; thickened and vacuolar hyphal strand.

57

5 (b) Normal fungal growth with smooth hyphal wall (no swelling and vacuoles) in absence of B. sphaericus UPMB10.

58

5 (c) Photo from Chaurasia et al., 2005 showed (A) Normal hyphae macroconidia of F.oxysporum , (B) swelling of hyphal tip and deformation of conidia in F.oxysporum

58

6 Clear zone around B. sphaericus UPMB10 cultured on chitin agar plate after 5 days of incubation

63

7 Comparison of inoculation effect of B. sphaericus UPMB10 (T2) with control (T5) after 4 weeks of treatments.

68

8 Effect of B. sphaericus UPMB10 inoculation on plant height of banana tissue cultured plantlet at six weeks. Left B. sphaericus UPMB10 (T2) and right control (T5).

81

9 Infected banana plant (left) showing foliar symptoms of Fusarium wilt caused by FOCR4 compared to healthy control plant (right).

82

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10 Infected banana plant showing the development of foliar associated symptoms of Fusarium wilt of banana on cultivar INTAN caused by Fusarium oxysporum f. sp. cubense. Healthy plant (A); chlorosis or yellowing of lower leaves (B); desiccation of lower leaf and chlorosis of upper leaves (C); advanced stage of infection with severe chlorosis and wilting (D).

85

11 Disease severity index, (based on vascular discoloration of corm caused by FOCR4): (a) uninfected control, (b, c) less severe (inoculated with UPMB10) and (d) most severe (FOC4); of banana seedlings var. Berangan cv. Intan challenged with FOC4 after 60 days.

87

12 Culture of banana pseudostem on PCNB agar. New Fusarium colonies formed from tissue of an infected plant (a) and no fungal proliferation from tissue of a healthy plant (b).

88

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LIST OF FIGURES

No. Figure

Page

1 Effect of B.sphaericus UPMB10 inoculation on fresh weight of banana tissue culture plantlets (pseudostem and corm, roots, leaves) at 12 weeks of treatments. (Means having same letter (s) in a column of a parameter do not differ significantly at 0.05 level by Tukey’s Test)

67

2 Effect of B.sphaericus UPMB10 inoculation on dry weight of banana tissue culture plantlets (pseudostem and corm, roots, leaves) at 12 weeks of treatments. (Means having same letter (s) in a column of a parameter do not differ significantly at 0.05 level by Tukey’s Test)

69

3 The effect of B.sphaericus UPMB10 inoculation on root volume of banana tissue cultured after 12 weeks of treatments. (Means having same letter (s) between treatment not differ significantly at 0.05 level by Tukey’s Test)

71

4 Effect of different treatments of inoculation in plant height of banana plantlets. (Each value represent mean of 5 replicates). Vertical bars on the top indicate LSD for treatment comparisons at a given week of treatment. Vertical bars on the right indicate LSD for comparisons of growth height over weeks within a treatment.

80

5 Disease development on banana seedlings challenged with FOCR4 based on foliar symptoms. (Each value is the mean of 5 replicates).

84

6 Figure 6: Effect of different rates of B. sphaericus UPMB10 inoculation to FOCR4 challenge on growth height of banana plants. (Each value represent mean of 12 replicates). Vertical bars on the right indicate LSD for comparisons of growth height over weeks within a treatment

90

7 Effect of different treatments on the development of Fusarium wilt in banana based on foliar symptoms. Each value represent mean of 12 replicates.

93

xxi

LIST OF ABBREVIATIONS

ANOVA Analysis of variance

AUDPC Area under Disease Progress Curve

cfu Colony forming units

DI Disease incidence

DS Disease severity

DSI Disease severity index

FOCR4 Fusarium oxysporum f.sp. cubense race 4

FSM Fusarium selective media

FW Fresh weight

GA Gibberelic acid

IAA Indole acetic acid

M monit

PDB Potato Dextrose Broth

PGPR Plant growth promoting rhizobacteria

PIRG Percentage of inhibition of radial growth

PSB Phosphate solubilizing bacteria

RCBD Randomised Complete Block Design

rm Epidemic rate

TSA Tryptic Soy Agar

TSB Tryptic Soy Broth

CHAPTER 1

INTRODUCTION

Non-symbiotic rhizobacteria, associated with plant roots and able to

enhance plant growth, known as plant growth promoting rhizobacteria

(PGPR) has generated much interest in a wide range of research. It has

impacts in different fields of study because it provides enormous information

on different aspects of plant growth promotion and development while at the

same time promises possible biological control solutions to pytopathogens of

some plant diseases. At present, phytopathogens have caused a great loss in

crop productivity and had to depend largely on chemicals, fumigation, steam-

treatment and solarization of soils in order to control it (Gamliel and Katan,

1992). Earlier, it was believed that the advantages of chemical controls of

diseases could be obtained without any deleterious environmental effects.

Currently, researchers have realized that these chemical agents are both

hazardous to animals and humans because they can persist and accumulate

in natural ecosystems. Consequently, there is a strong desire to replace

chemicals with biological approaches that are potentially more “friendly” to the

environment in the long term. These biological approaches include the use of

transgenic plants, which are resistant to one or more plant pathogens

(Greenberg and Glick, 1993), or either to use of biocontrol plant growth-

2

promoting bacteria which can suppress or prevent the plant damage

(O’Sullivan and O’Gara, 1992; Sivan and Chet 1992; Sutton and Pegg, 1993;

Cook, 1993). Biocontrol seems to be a reliable alternative compared to

chemical fungicides, which have raised serious concerns of food

contamination and environmental pollution. Some saprotrophic bacteria can

serve as excellent biocontrol agents against plant pathogens (Fernando et al.,

2005).

The introduction of beneficial microbes as biological control of soil

borne plant pathogen as well as enhancement of plant growth has a

considerable potential in agriculture. Biological control using antifungal

rhizobacteria to suppress plant diseases offers a powerful alternative to the

use of synthetic chemicals. Numerous studies have demonstrated the ability

of several bacteria to suppress diseases caused by soil-borne and seed-

borne plant pathogens (Weller, 1988; O’Sullivan and O’Gara, 1992; Whipps,

1997). The anti-fungal abilities of these beneficial microbes have been known

since a few decades ago and there have been extensive efforts to use them

for plant disease control and more recently on a commercialized scale.

The uniqueness of microorganisms has never been understood until

their beneficial functions were discovered. It is a paradox that bacteria can be

both beneficial and antagonistic towards a plant pathogen to maintain its life

3

cycle. In this manner, it is possible to introduce the beneficial microorganism

as a biological control for soil borne plant pathogen as well as enhancement

of plant growth. This creates a great implication on plant growth development

through direct or indirect mechanisms (Glick et al., 1999). A direct promotion

of plant growth by PGPR is generally based on the ability of the bacteria to

synthesize and facilitate nutrient uptake from the environment. Meanwhile

indirect mechanism is through its effect on the phytopathogenic organism

(usually fungus) by one or several mechanisms such as production of

antibiotic and antifungal metabolite or enzyme that could lyse the fungal cell

wall. It was believed that this advantage would lead to the potential of PGPR

as a biocontrol agent besides stimulating plant growth. A local rhizobacterial

strain, isolated from oil palm roots and identified as Bacillus sphaericus

UPMB10 (B. sphaericus UPMB10), has shown great potential in promoting

plant growth development and has been characterized for its optimum growth

requirement, inoculum carrier (ground oil palm frond) and inoculum production

technique (Premalalatha, 2006). Bacillus sphaericus is categorized as a free-

living soil bacterium that can function as a plant growth promoting

rhizobacteria (Kloepper et al., 1989). A few studies have demonstrated that

inoculation with B. sphaericus UPMB10 enhanced plant growth and fixed

nitrogen with oil palm (Amir et al., 2001 and banana (Mia et al., 2000). The

current research interest is to assess the ability of B. sphaericus UPMB10 as

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