ABSTRACT
A major characteristic shared among Bacillus strains is the ability to produce a wide range of antimicrobial compounds that are active against bacteria and fungi. Bacillus species was isolated from soil samples collected from refuse dump site. The bacterial isolate exhibited antagonistic activity when cultured with fungi on Potato Dextrose agar plates. Antifungal metabolites produced by Bacillus species in Potato Dextrose Broth were extracted from fluid culture supernatant by evaporation with ethyl acetate giving a yield of 0.21g dry metabolites from 1 litre fluid culture on average. The antifungal metabolites inhibited mycelial growth of fungal species – Aspergillus, Rhizopus and Fusarium. The inhibition zones were observed for more than four days and there was no observed change in the size of the zones. The antifungal metabolites were subjected to different pH levels and were active and stable over a wide range of pH (2-10). They were exposed to very high temperatures and were stable and remained active after sterilization at 121oC for 15min. The metabolites were soluble in water and organic solvents like ethanol and were resistant to denaturation by enzymes. The effect of these antifungal metabolites against postharvest fungal pathogens under various circumstances suggest that they could be a potent source of antifungal not just for postharvest fungal pathogens but human and animal fungal pathogens alike.
TABLE OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgement iv
Table of Contents v
List of Tables ix
List of Figures x
Abstract xi
CHAPTER ONE
INTRODUCTION 1
1.1
Introduction 1
1.2
Objectives 2
CHAPTER TWO
LITERATURE REVIEW 3
2.1
Bacterial Metabolites 3
2.1.1
Antimicrobial properties of bacterial metabolites 5
2.1.2
Bioactivity of Microbial secondary metabolites 5
2.2
The Bacillus species 6
2.2.1
History of Bacillus species 7
2.2.2
Taxonomy of Bacillus species 12
2.2.3
Life cycle of Bacillus species 14
2.3
Catabolite Repression 17
2.4
Resistance of Spores 19
2.5
Production of Antibiotics 21
CHAPTER THREE
MATERIALS AND METHODS
3.1
Sample Collection 26
3.2
Sterilization of Materials 26
3.3
Media Preparation 26
3.4
Isolation of Organism 26
3.4.1
Cultural characteristics on various media 27
3.4.1.1
Nutrient agar 27
3.4.1.2
Potato dextrose agar 27
3.4.1.3
Gram staining 27
3.5
Test for Antifungal Activity 28
3.6
Further Identification Tests 28
3.6.1
Motility 28
3.6.2
Catalase Test 28
3.6.3
Indole Test 29
3.6.4
Hydrolysis of starch 29
3.6.5
Methyl Red Test 29
3.6.6
Voges Proskauer 30
3.6.7
Citrate utilization Test 30
3.6.8
Sugar fermentation test 31
3.7 Extraction of Antifungal
Metabolites 31
3.8 Characterization of Antifungal
Metabolites 32
3.8.1
Antifungal Activity 32
3.8.2
Thermal Stability 32
3.8.3
pH Stability 32
3.8.4
Susceptibility to denaturation by enzymes 32
3.8.5
Solubility in organic solvents 33
CHAPTER FOUR
RESULTS
4.1
Identification of the bacterial isolate 34
4.2
Identification of fungal species 34
4.3
Extraction of antifungal metabolites 34
4.4
Antifungal activity of the extracted metabolites 34
4.5
pH stability of the extracted metabolites 35
4.6
Thermal stability of the extracted metabolites 35
4.7
Susceptibility to denaturation by enzymes and solubility in organic
solvents 36
CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATION 44
5.1
Discussion 44
5.2
Conclusion 46
5.3
Recommendations 47
References 48
LIST OF TABLES
Table Title Page
2.1 Origins
of isolates of Bacillus species 10
2.2 Some
Bacillus antibiotics 24
4.1 Cultural and biochemical characteristics
of the isolated antifungal-producing
bacteria 37
2.3
Cultural and
morphological characteristics of the fungal species 38
2.4
Inhibition
of mycelial growth of some fungal species by antifungal
metabolites
produced by Bacillus species 39
2.5
Inhibition of mycelial growth of Rhizopus by antifungal metabolites of
Bacillus
species treated at different pH values 40
2.6
Inhibition of mycelial growth of Aspergillus by antifungal metabolites of
Bacillus
species treated at different pH values 41
2.7
Inhibition of mycelial growth of Fusarium by antifungal metabolites of
Bacillus
species treated at different pH values 42
2.8
Inhibition
of mycelial growth by heat treated (121oC/15min) antifungal
Metabolites of Bacillus species 43
LIST OF FIGURES
Figure Title Page
2.1
B. megaterium sporeforming cells as seen in the phase contrast or
in the interference contrast microscope 9
2.9 Cycle
of germination, outgrowth and sporulation of a typical spore-
forming bacterium
16
CHAPTER ONE
INTRODUCTION
1.1 Introduction
Bacteria that show inhibitory action
against fungi can contribute immensely to the prevention of plant diseases and
post-harvest spoilage hence serving as alternative to chemical pesticides and
their metabolites as preservatives (Walsh et
al., 2001). The discovery of novel, non-antibiotic small molecule
pharmaceuticals or product candidates through screening microbial secondary
metabolites is an expanding activity throughout the pharmaceutical industry and
one that is becoming increasingly fruitful. There is widespread acceptance that
microorganisms are a virtually unlimited source of novel structures with many
potential therapeutic applications. Soil-borne bacteria, especially Bacillus
species, have been recognized for their role in plant health and soil
fertility. Soil and rhizosphere have frequently been used as model environment
for screening putative agents for use in biological control of soil-borne plant
pathogens.
Bacillus
sp., form endospores and can tolerate extreme pH, temperature and osmotic
conditions; therefore they offer several advantages over other organisms. Bacillus sp. was found to colonize the
root surface, increase plant growth and cause lysis of fungal mycelia (Turner
and Backman, 1991). They are regarded as safe biological agents and their
potential is considered high. As bacteria their antagonistic effect is assumed
to be as a result of the production of antifungal antibiotics (Korzybski et al., 1978).
Biosynthesis of antibiotics from microorganisms
is often regulated by nutritional and environmental factors. It has been
reported that antimicrobial substances produced by bacterial species were
greatly influenced by variation of carbon sources. Several abiotic factors such
as pH and temperature have been identified as having an influence on antibiotic
production from bacterial (El Banna, 2006; Raaijmakers et al., 2002). Antifungal peptides produced by Bacillus species include mycobacillins, surfactins, mycosubtilins
and fungistatins. The Bacillusspecies
can produce a wide range of other metabolites including chitinases, and other
cell wall-degrading enzymes, volatiles and compounds that elicit plant
resistance mechanisms (Sadfi, 2001). Volatile metabolites produced from Bacillus species have been reported to
inhibit mycelia growth of Fusarium oxysporum
with the highest effect on reduction of Fusarium
wilt of onion (Sharifi and Ramezani, 2003). Ryu et al., (2004) reported on promotion of growth and induction of
systemic resistance (ISR) response in Arabidopsis
thaliana against Erwinia carotovora
sub sp. carotovora by volatile
substance (vs) (acetylbutanediol and acetoin, same as the present purified
compounds) from Bacillus amyloliquefaciens
isolate IN937a and Bacillus subtilis
isolate GB03. Therefore, volatile-substance – producing bacteria can used as
biocontrol agents for protection against microbial plant diseases.
The present study was aimed at isolation
of potential antagonistic soil microorganisms and extraction of antifungal
components. Analysis of their characteristics, as well as their effects on
growth against tested pathogenic fungi was conducted.
1.2 OBJECTIVES
The objectives of this work are
·
To isolate Bacillus species capable of producing antifungal metabolites
·
To identify the characteristics of these metabolites
·
To check the antifungal activity of the
metabolites
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