UNIVERSITI PUTRA MALAYSIA

BIOASSAY AND PARTIAL IDENTIFICATION OF NON-VOLATILE BIOACTIVE COMPOUNDS PRODUCED BY BACILLUS SUBTILIS

NALISHA ITHNIN

FS 2007 23

BIOASSAY AND PARTIAL IDENTIFICATION OF NON-VOLATILE BIOACTIVE COMPOUNDS PRODUCED BY BACILLUS SUBTILIS

By

NALISHA ITHNIN

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

March 2007

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

BIOASSAY AND PARTIAL IDENTIFICATION OF NON-VOLATILE BIOACTIVE COMPOUNDS PRODUCED BY BACILLUS SUBTILIS

By

NALISHA BINTI ITHNIN

March 2007

Chairman: Muskhazli Mustafa, PhD Faculty: Science Biological control of plant pathogens is an alternative to the strongly dependence of

modern agriculture on chemical fungicides. Extensive applications of chemical

control may lead to environmental pollution and development of resistant

phytopathogenic fungi strains. It is therefore necessary to develop alternatives to

synthetic chemical control to reduce the risks and raise consumer confidence. Bacillus

subtilis (BS) was used in this study as the biological control agent (BCA) against

Rhizoctonia solani (RS), Pythium ultimum (PU) and Sclerotium rolfsii (SR). The first

part of this study focus on optimizing BS as BCA by examining application

conditions using stability tests and bioassays. The effects of three variables namely

temperature (-20oC-100oC), pH (3-11) and light (sunlight, UV and darkness) on the

production of bioactive compounds were studied.

From the dual culture bioassay, BS was found to suppress the growth of PU better

than RS and SR. Temperature show a considerable effect on BS antifungal activity

with highest inhibition occur on SR at 80oC (58.30%), followed by PU at -20oC

treatment (38.68%) and RS at 30oC (35.39%). The optimal pH for antifungal

production was pH 3 for RS (51.12%), pH 11 for SR (28.33%) and pH 7 for PU

(28.73%). However, neither darkness nor UV treatment altered the antifungal activity.

Darkness treatment managed to subdue PU (57.16%), RS (58.30%) and SR (46.24%).

Thin layer chromatography (TLC) and high performance liquid chromatography

(HPLC) were used in the second part of this study in order to screen and isolate the

bioactive compound produced by BS. Methanolic extracts of BS was found to be the

best extraction method from which 2 anticipated peaks with inhibitory activity against

PU and Candida albicans (CA) were exhibited. However, the activity is more

significant when tested against CA compared to PU due to concentration limitation.

The TLC profiles of extracts revealed an identical chromatographic mobility to BS

iturin A (Rf 0.51) and surfactin (Rf 0.68). Meanwhile through HPLC, homologous

compound of fengycin and an iturinic compound were detected.

The final part of this study was to determine the effectiveness of supplementing

different carbon sources to BS on its antifungal activity and hydrolytic enzymes

production. Bioassay was again applied to record the inhibitory activities. By using

1% (w/v) of three different carbon sources namely oil palm root (OPR), Ganoderma

lucidum (GL) and ball-milled chitin (CHIT)], respectively, inhibitory activity of BS

was induced compared to BS grown in Nutrient Agar (NA). Inhibitory activities (cm

± SD) for each pathogen were as followed: for PU, OPR (3.688 ± 0.01) > CHIT

(2.304 ± 0.02) > GL (2.114 ± 0.1); for RS, CHIT (4.171 ± 0.05) > OPR (3.038 ± 0.66)

> GL (2.892 ± 0.06); while for SR, OPR (2.927 ± 0.02) > CHIT (2.854 ± 0.06) > GL

(2.843 ± 0.07).

The exposure of selected phytopathogenic fungi to the hydrolytic enzymes such as

chitinases, proteases or glucanases was found to degrade the structural matrix of

fungal cell walls. BS was found to produce high chitinase in the medium containing

CHIT (0.084 U/ml), followed by GL (0.056 U/ml) and OPR (0.051 U/ml),

respectively. Meanwhile for β-1,3 and β-1,6-glucanase production, both OPR (1.099

U/ml, 0.716 U/ml) and GL (0.820 U/ml, 1.165 U/ml) showed higher production than

CHIT (0.579 U/ml, 0.513 U/ml). The activity of protease was high when BS were

cultured with GL (2.579 U/ml), followed by OPR (2.547 U/ml) and CHIT

(2.548U/ml).

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

BIOASSAI DAN PENGENALPASTIAN SEPARA KOMPONEN BIOAKTIF TIDAK MERUAP DARI BACILLUS SUBTILIS

Oleh

NALISHA BINTI ITHNIN

Mac 2007

Pengerusi: Muskhazli Mustafa, PhD Fakulti: Sains Kawalan biologi bagi perosak tumbuhan merupakan suatu alternatif bagi

mengurangkan penggantungan pertanian moden kini terhadap racun perosak berunsur

kimia. Penggunaan bahan kawalan kimia sintetik yang berlebihan menyebabkan

pencemaran persekitaran dan penghasilan kulat yang sukar dikawal. Oleh itu adalah

satu keperluan untuk menghasilkan alternatif lain selain bahan kawalan kimia sintetik

bagi mengurangkan risiko dan meningkatkan kadar keyakinan pengguna. Bagi kajian

ini, Bacillus subtilis (BS) digunakan sebagai agen kawalan biologi (BCA) terhadap

Rhizoctonia solani (RS), Pythium ultimum (PU) dan Sclerotium rolfsii (SR). Bahagian

pertama kajian melibatkan penggunaan BS sebagai agen kawalan biologi dengan

menguji keadaan aplikasi dengan dalam ujian kestabilan dan bioassay. Kesan 3

parameter iaitu suhu (-20oC-100oC), pH (3-11) dan cahaya (cahaya matahari, UV and

gelap) terhadap penghasilan bahan bioaktif adalah dikaji.

Dari ujian bioassay, didapati BS mampu merencat pertumbuhan PU lebih baik dari

RS dan SR. Kesan suhu memainkan peranan terhadap aktiviti antikulat BS dengan

perencatan terbesar dapat dilihat pada SR dengan suhu 80oC (58.30%), diikuti dengan

PU pada -20oC (38.68%) dan RS pada suhu 30oC (35.39%). pH optimum bagi

penghasilan aktiviti antikulat adalah pada pH 3 bagi RS (51.12%), pH 11 bagi SR

(28.33%) dan pH 7 bagi PU (28.73%). Namun, kesan rawatan cahaya samada gelap

atau UV tidak merubah aktiviti antikulat BS secara keseluruhannya. Rawatan gelap

merencat PU (57.16%), RS (58.30%) dan SR (46.24%).

‘Thin Layer Chromatography’ (TLC) dan ‘High Performance Liquid

Chromatography’ (HPLC) telah digunakan pada bahagian kedua kajian ini bagi

memperinci dan mengasingkan bahan antikulat yang dihasilkan oleh BS. Didapati

ekstrak methanol adalah yang terbaik dimana melalui analisis HPLC, terdapat 2

puncak yang mempunyai aktiviti antikulat terhadap PU dan Candida albicans (CA).

Namun begitu, aktiviti antikulat ini hanya dapat dilihat dengan jelas terhadap CA

berbanding PU akibat kepekatan sampel yang terhad. Profil TLC bagi ekstrak BS ini

adalah sama seperti iturin A (Rf 0.51) dan surfactin (Rf 0.68). Manakala dengan

menggunakan HPLC, bahan seperti fengycin dan iturin dapat dikesan.

Bahagian terakhir kajian ini adalah bagi mengenalpasti keberkesanan penambahan

pelbagai sumber karbon kepada BS terhadap aktiviti antikulat dan penghasilan enzim.

Dimana sekali lagi ujian bioassay digunapakai bagi merekod aktiviti perencatan.

Dengan menggunakan 1% (mg/L) 3 sumber karbon yang berbeza iaitu akar kelapa

sawit (OPR), Ganoderma lucidum (GL) dan kitin (CHIT), didapati aktiviti perencatan

BS adalah lebih baik berbanding BS yang dikultur didalam Agar Nutrien (NA) sahaja.

Aktiviti perencatan (cm ± r.p) bagi setiap kulat adalah seperti berikut: bagi PU, OPR

(3.688 ± 0.01) > CHIT (2.304 ± 0.02) > GL (2.114 ± 0.1); bagi RS, CHIT (4.171 ±

0.05) > OPR (3.038 ± 0.66) > GL (2.892 ± 0.06); manakala bagi SR, OPR (2.927 ±

0.02) > CHIT (2.854 ± 0.06) > GL (2.843 ± 0.07).

Didapati pendedahan kulat perosak tumbuhan kepada enzim seperti chitinase,

protease atau glucanase mampu mengurangkan struktur matriks dinding sel kulat. BS

didapati menghasilkan enzim chitinase yang tertinggi didalam media mengandungi

CHIT (0.084 U/ml), diikuti dengan GL (0.056 U/ml) dan OPR (0.051 U/ml).

Manakala bagi penghasilan enzim β-1,3 dan β-1,6-glucanase, kedua-dua OPR (1.099

U/ml, 0.716 U/ml) dan GL (0.820 U/ml, 1.165 U/ml) menunjukkan penghasilan yang

lebih berbanding CHIT (0.579 U/ml, 0.513 U/ml). Aktiviti protease adalah tinggi

apabila BS dikultur dengan GL (2.579 U/ml), diikuti oleh OPR (2.547 U/ml) dan

CHIT (2.548U/ml).

ACKNOWLEDGEMENTS

I would especially like to thank my supervisor Dr Muskhazli Mustafa and Associate

Prof. Dr Radzali Muse for their guidance, support and encouragement. Without them I

would not have been able to complete my study.

I wish to thank all my friends in Biology Department for their friendship and

assistance throughout the length of my research. I would like to express my deepest

gratitude towards my family and dearest Siti Hajar for always giving me the love and

support I need.

Above all, I praise Allah for His guidance and continuous blessing upon my life.

THANK YOU

I certify that an Examination Committee has met on 14th March 2007 to conduct the final examination of NALISHA BINTI ITHNIN on her Master of Science thesis entitled “BIOASSAY AND PARTIAL IDENTIFICATION OF NON VOLATILE BIOACTIVE COMPOUND PRODUCED BY BACILLUS SUBTILIS” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulation 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Nor Aini Mohd Fadzillah, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Chairman) Faridah Abdullah, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Member) Shuhaimi Mustafa, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Uyup Abd Manaf, PhD Associate Professor Institute of Biological Science Universiti Sains Malaysia (Member) ________________________________

HASANAH MOHD GHAZALI, PhD Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirements for the degree of Master of Science. The members of the Supervisory Committee are as follows: Muskhazli Bin Mustafa, PhD Lecturer Faculty of Science Universiti Putra Malaysia (Chairman) Radzali Bin Muse, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) ______________________ AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia Date: 17th JULY 2007

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 no been previously or concurrently submitted for any other degree at UPM or other institutions. _____________________ NALISHA ITHNIN Date: 19th JUNE 2007

TABLE OF CONTENTS

Page

ABSTRACT ii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL ix DECLARATION xi LIST OF TABLES xv LIST OF FIGURES xvi LIST OF ABREVIATIONS xviii CHAPTER 1 INTRODUCTION 1 2 LITERATURE REVIEW 7 2.1 Bacillus subtilis 7 2.1.1 Taxonomy and Morphology 8 2.2 Bacillus subtilis as Biological Control Agent 11 2.3 Mechanism of Action 13 2.4 Antibiotic 14 2.5 Enzyme in Antagonistic Activity 17 2.5.1 Protease 17 2.5.2 Chitinase 18 2.5.3 ß-glucanase 19 2.6 Carbon Source for Enzyme Production 20 2.7 UV 21 2.8 Toxin 22 3 Bacillus subtilis; A Potential Biological Control Agent Against

Selected Plant Pathogen 24

3.1 Introduction 24 3.1.1 Objective 25 3.2 Materials and Method 26 3.2.1 Microorganisms 26 3.2.2 Bacterial Strain Inoculation 26 3.2.3 Bacterial Growth Measurement 26 3.2.4 Effects of B. subtilis on Seed Germination 27 3.2.5 Fungicide Tolerance Test 27 3.2.6 Bioassay Test 28 3.2.7 Stability Test 29 3.2.7.1 Temperature 29 3.2.7.2 pH 30 3.2.7.3 Light Condition 30 3.2.8 Statistical Analysis 30 3.3 Results and Discussion 31 3.3.1 Bacterial Growth Measurement 31 3.3.2 Effects of B. subtilis on Seed Germination 34

3.3.3 Fungicide Tolerance Test 35 3.3.4 Bioassay Test 37 3.3.5 Stability Test 40 3.3.5.1 Temperature 40 3.3.5.2 pH 44 3.3.5.3 Light Condition 47 3.4 Conclusion 50 4 Isolation of Bioactive Compound Produced by Bacillus subtilis 51 4.1 Introduction 51 4.1.1 Objective 52 4.2 Materials and Method 53 4.2.1 Microorganisms 53 4.2.2 Extraction and Thin Layer Chromatography (TLC) 53 4.2.3 Bioautography 54 4.2.4 Profiling of B. subtilis Bioactive Compound by HPLC 55 4.3 Results and Discussion 58 4.3.1 Extraction and Thin Layer Chromatography (TLC) 58 4.3.2 Profiling of B. subtilis Bioactive Compound by HPLC 62 4.4 Conclusion 67 5 Carbon Source Amendment and Its Effect on Bacillus subtilis

Hydrolytic Enzyme Production 68

5.1 Introduction 68 5.1.1 Objective 71 5.2 Materials and Method 72 5.2.1 Microorganism 72 5.2.2 Carbon Source Preparation 72 5.2.3 Minimal Media Preparation 72 5.2.4 Effect of Different Carbon Source Amendment on B. subtilis

Activity 73

5.2.5 Hydrolytic Enzyme Assay 73 5.2.5.1 Protein Assay 73 5.2.5.2 Glucanase Assay 74 5.2.5.3 Chitinase Assay 74 5.2.5.4 Protease Assay 75 5.2.6 Statistical Analysis 76 5.3 Results and Discussion 77 5.3.1 Effect of Different Carbon Source Amendment on B. subtilis

Activity 77

5.3.2 Hydrolytic Enzyme Assay 84 5.3.2.1 Glucanase Assay 84 5.3.2.2 Chitinase Assay 88 5.3.2.3 Protease Assay 89 5.3.3 Correlation 94 5.4 Conclusion 97 6 GENERAL DISCUSSION AND SUGGESTION FOR FURTHER

RESEARCH 98

6.1 Bacillus subtilis; A Potential Biological Control Agent Against 98

Selected Plant Pathogen 6.2 Isolation of Bioactive Compound Produced by Bacillus subtilis 102 6.3 Carbon Source Amendment and Its Effects on Bacillus subtilis

Hydrolytic Enzyme Production 104

7 CONCLUSION 106 BIBLIOGRAPHY 108 APPENDICES 126

LIST OF TABLES

Table Page

2.1 List of characteristics of the Genus Bacillus (Clause and Berkeley, 1986)

9

2.2 Mode of action by chitinolytic enzyme (Gohel et al., 2006) 19

2.3 Microorganisms and carbon source used in enzyme production 21

3.1 Comparison of Phaseolus vulgaris growth when treated with B. subtilis

34

3.2 Inhibition of B. subtilis (%) after tested with Benozide® and Dancodil 2787® at different concentrations

36

3.3 Inhibition (%) of R. solani, P. ultimum and S. rolfsii in the presence of B. subtilis culture filtrate after treated with different temperature

42

3.4 Inhibition (%) of R. solani, P. ultimum and S. rolfsii in the presence of B. subtilis culture filtrate after treated with different pH

45

3.5 Inhibition (%) of R. solani, P. ultimum and S. rolfsii in the presence of B. subtilis after treated with different light condition

49

4.1 Summary of HPLC analysis conducted 57

4.2 Effect of different extraction solvents on B. subtilis antifungal activity against P. ultimum, R. solani, S. rolfsii and C. albicans

59

4.3 Summary of all extraction and bioautography test against P. ultimum, R. solani, S. rolfsii and C. albicans

61

5.1 Diameter of inhibition in the presence of B. subtilis culture filtrate after supplemented with 1% (w/v) oil palm root

78

5.2 Diameter of inhibition in the presence of B. subtilis culture filtrate after supplemented with G. lucidum

80

5.3 Diameter of inhibition in the presence of B. subtilis culture filtrate after supplemented with ball-milled chitin

82

5.4 Correlations between enzymatic activities in culture filtrates of B. subtilis and their ability to inhibit P. ultimum, R. solani and S. rolfsii

95

LIST OF FIGURES

Figure Page

2.1 Regulatory pathways of antibiotic biosynthesis in B. subtilis 16

3.1 Optical density (OD), biomass and pH of B. subtilis grown in nutrient broth (NB). OD was measured by absorbance at 560nm (OD560)

32

3.2 Radial growth of P. ultimum, R. solani and S. rolfsii grown on Potato Dextrose Agar (PDA) in the presence (with) and absence (without) of B. subtilis culture filtrate. (a) P. ultimum, (b) R. solani, (c) S. rolfsii

38

4.1 HPLC profiles of compounds produced by B. subtilis after (A) 12h, (B) 24h, (C) 36h and (D) 48h of incubation. P1-Peak 1 and P2-Peak 2

64

4.2 Inhibitory activity of peak 1 (P1) and peak 2 (P2) when tested against

Candida albicans

66

5.1 Histogram representing different inhibitory activity of B. subtilis when supplemented with palm oil root on P. ultimum, R. solani and S. rolfsii. Inhibition was measured according to diameter of clear zone formed on PDA seeded with 106 spore/ml pathogen

78

5.2 Inhibitory activity of B. subtilis supplemented with Ganoderma lucidum on P. ultimum, R. solani and S. rolfsii. Inhibition was measured according to diameter of clear zone formed on PDA seeded with 106 spore/ml pathogen

81

5.3 Inhibitory activity of B. subtilis supplemented with ball-milled chitin on P. ultimum, R. solani and S. rolfsii. Inhibition was measured according to diameter of clear zone formed on PDA seeded with 106 spore/ml pathogen

83

5.4 Influence of different carbon source amendment on β-1,3-glucanase activities produced by B. subtilis. OPR-Oil Palm Root; GL-Ganoderma lucidum; CHIT-Chitin

86

5.5 Influence of different carbon source amendment on β-1,6-glucanase activities produced by B. subtilis. OPR-Oil Palm Root; GL-Ganoderma lucidum; CHIT-Chitin

87

5.6 Influence of different carbon source amendment on chitinase activities produced by B. subtilis. OPR-Palm oil root; GL-Ganoderma lucidum; CHIT-Chitin

89

5.7 Influence of different carbon source amendment on protease activities produced by B. subtilis. OPR-Oil Palm root; GL-Ganoderma lucidum; CHIT-Chitin

90

5.8 Effect of incubation period on hydrolytic enzyme produced by B. subtilis + OPR

92

5.9 Effect of incubation period on hydrolytic enzyme produced by B.

subtilis + GL

93

5.10 Effect of incubation period on hydrolytic enzyme produced by B. subtilis + CHIT

94

LIST OF ABBREVIATIONS

BCA Biological control agent

NVAF Non volatile antifungal compound

PDA Potato Dextrose Agar

NB Nutrient Broth

UV Ultra Violet

GlcNac N-acetylglucosamine

mm milimeter

cm centimeter

CHAPTER 1

INTRODUCTION

During the past few years, extensive green house and field trials were conducted to

control seed or plant pathogens either by using cultural control or chemical control

measures. Crop rotation and cover crop, plowing and seedbed preparation, site

selection and planting date, herbicide effects, fertilizer effect, fungicide-treated seed

and fungicide soil treatment were examples of many other control measures that have

been taken to prevent seed and crop destruction by pathogenic fungi. Even though the

cultural control treatments have been an effective traditional procedure in the

agricultural practices for a long time, this type of control measure was also found to

increase population of the root rot fungi and increase disease severity (Knudsen et al.,

2002).

Biological control appears to constitute an alternative strategy for controlling

diseases, perhaps as part of an integrated control system, thus reducing the use of

chemical products and contributing to the preservation of the environment (Souto et

al., 2004). In biological control, microorganisms or their secretions are biologically

used to prevent plant diseases (Wang et al., 2002). Biological control of soil borne

pathogens by introduced microorganisms has been studied for over 65 years and it is

in part a response to public concern about hazards associated with chemical

pesticides. It is however, important to know the performance of a biological control

in the environment in which it is expected to act upon may lead to improved

performance (Collins and Jacobsen, 2003) and wider use of biological control method

(Fravel, 1988). However, biological control takes more intensive management and

planning. It is also very often that the results of using biological control are not as

dramatic or quick as the results of pesticides used.

Microorganisms are currently considered as biological control agent which offers an

environmentally friendly supplement as an alternative to chemical control (Souto et

al., 2004) with high specificity against the targeted plant pathogens, high

degradability after effective usage and low cost of mass production. It may also act as

a potent means of reducing the inocula density or disease-producing activities of a

pathogen or parasite in organisms (Baker, 1985). Products produced biologically or

the microbial cells themselves are called biological control agents (BCAs), which

stably inhibit the environment as non-dominant species but still maintain their

effectiveness (Shoda, 2000). There have been many studies conducted in order to

isolate antifungal agents from a variety of antagonistic microorganisms. The

antagonistic activity of these microorganisms may be based on changeable modes of

action, including acid and antibiotic production, stimulation of host defense, and

direct parasitism of the pathogens or nutrient competition (Whipps, 1997). Therefore,

biological control of fungal diseases by applied bacterial inocula has become an

important approach to facilitate sustainable agriculture (Lang et al., 2002).

Bacillus spp. which showed antagonism are ideal for biological control experiment as

they are often soil isolates capable of forming endospores, a characteristic which

facilitates long term storage and formulation (Knox et al., 2002). Bacillus spp. have

been used to control a number of leaf spot and post- harvest diseases. They also have

shown the capacity to control early leaf spot of peanut, yam leaf spot, post harvest

apple diseases, gray mould of strawberries, and post- bloom fruit drop of citrus

(Collins and Jacobsen, 2003).

Bacillus subtilis is a ubiquitous bacterium commonly recovered from water, soil, air,

and decomposing plant residue. The bacterium produces an endospore that allows it

to endure extreme conditions of heat and desiccation in the environment (Knox et al.,

2002). B. subtilis produces a variety of proteases and other enzymes that enable it to

effectively degrade a series of biopolymers (i.e proteins, starches, pectins and lipids),

thus B. subtilis is assumed to contribute to nutrient cycling. However, under most

conditions the organism is not biologically active but exists in the spore form (Yu et

al., 2002).

It was decided to select for B. subtilis, as it is particularly amenable to formulation

and long term storage (Powell et al., 1990), concerned over inocula viability is less

than with gram-negative bacteria (Weller, 1998) and large scale production of

bacterial inoculate (Rodgers and Hajjar, 1993). It has been established that specific

isolates of B. subtilis can operate under subtropical climatic conditions, where the

higher ambient temperature favor to bacterial growth and metabolism (Fiddaman and

Rossall, 1995). Beside, the potential risk associated with the use of B. subtilis in

fermentation facilities is low.

The use of B. subtilis as a biological control agent of fungal plant pathogens is being

investigated because of the effects of antifungal compounds on Monilinia fructicola

(McKeen et al., 1986), Aspergillus flavus and A. parasiticus (Kimura and Hirano,

1988), and Rhizoctonia (Loeffler et al., 1986). Lumsden and Locke (1989) also

conclude that effective biological control inhibition of fungal growth in green house

and field controls of fungal diseases have been achieved with the application of B.

subtilis. Yu et al. (2002) carried out in vitro trials of B. subtilis against variety of

plant pathogens. They have evaluated B. subtilis with several modified antagonistic

experiments and identified several compounds produced that inhibit pathogens

growth. Alboleutin, bacitracin, botrycidin, clorotetain, dificidin, fengycin, iturins,

mycobacilin, rhizocticins and subtilin have been identified as strong causes in

pathogens inhibition process (Zuber et al., 1993). These antifungal peptides inhibit

the growth of a large number of fungi, such as Aspergillus sp., Penicillium sp. and

Fusarium sp. (Munimbazi and Bullerman, 1998), as well as yeasts, i.e. Candida

albicans and Saccharomyces cerevisiae (Thimon et al., 1992).

Besides using B. subtilis solely in producing bioactive compounds, raw materials that

will act as carbon source to B. subtilis were also added. These were aimed for

obtaining microbial growth and its productions similar to or higher than that attained

by routine laboratory media. The raw materials used included agricultural products,

i.e. oil palm root and basidiocarp of Ganoderma lucidum, and agro-industrial material

such as chitin. These fine materials were easy to find and can be obtained at low cost.

The ease in propagating B. subtilis to obtain high level of bioactive compounds will

undoubtedly lead to rapid development of the capability for industrial-scale

production. Mahmood et al. (1998) have used several additional substances in their

study that would support the growth and probably induce the production of

extracellular enzymes and other bioactive compounds in B. subtilis.

This study was aimed at the potential application of B. subtilis as biological control

agent against plant pathogenic fungi. Thus the first objective of the study was to

determine the antifungal activities produced by B. subtilis in culture filtrate. To

achieve this, dual culture test were used to test B. subtilis antifungal ability against

Pythium ultimum, Rhizoctonia solani and Sclerotium rolfsii and the effectiveness of

the antifungal produced. Stability test was then carried out to determine the optimum

condition for the growth of B. subtilis and to evaluate the stability of the bioactive

compounds when tested on extreme temperature, pH and light condition.

After establishing the ability of B. subtilis to inhibit the growth of all plant pathogens

tested, next objective was to isolate the bioactive compound produced by B. subtilis.

As for this, thin layer chromatography (TLC) and high performance liquid

chromatography (HPLC) techniques were carried out. By performing TLC, the

compounds produced were visualized in terms of spots or bands formed and further

characterized using different staining techniques or detectors. Each band was then

subjected to bioautography to detect its antifungal antibiotic activity against plant

pathogens and Candida albicans. HPLC were used to profile and isolate fractions

with activity. This will further explain the existence of compounds that were formed

by B. subtilis in relation to its antifungal antibiotic activity. A comparison to previous

studies conducted by others was done in order to get a brief idea on the compound

produced.

Assessment on different carbon source amendments was done to evaluate the

potential of utilizing bioproduct to induce antifungal production. The hydrolytic

enzymes secreted by B. subtilis, cultured in the presence of additional carbon source,

were measured and the correlation between antifungal activity and enzyme production

was quantified. This is to tackle the third and final objective which was to determine

whether the inhibitory activities shown were also caused by enzyme production.