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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,