ABSTRACT
Bacillus species isolated from five rhizospheric soil of turmeric plant root were evaluated for the potential antagonistic activity against fungi. Six isolates namely; Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus thuringinesis, Bacillus licheniformis and Bacillus pumilus were aseptically isolated and characterized respectively. These bacterial isolates were screened with the aim of producing those with antifungal tendencies and one of the bacterial isolates possessing great antifungal activity was identified and characterized according to Bergey’s manual of determinative bacteriology as Bacillus megateruim. Bacillus megaterium has been recognized as an endophyte and is a potential biocontrol agent against plant and animal diseases. Bacillus megaterium showed an occurrence of 80% in five difference turmeric rhizospheric soil which proved that the organism occurs in the rhizosphere. Statistical analysis showed that Bacillus megaterium showed inhibition activity against Fusarium 9.67mm, Candida 15.33mm, Rhizopus 14.33mm, Penicillium 9.33mm and Aspergillus 11.33. The anti-fungal activity of Bacillus megaterium were all much lower than the activity of the commercial drug (mancozade) but the organism inhibited all the test fungi species. This research shows that B. megaterium is quite important and effective as biocontrol agent against the fungi Aspergillus, Candida, Fusarium, Penicillium, and Rhizopus. This inhibition action is probably due to the synergistic effects of the factors such as the production of anti-fungal metabolites.
TABLE
OF CONTENTS
Title page
i
Certification
ii
Dedication iii
Acknowledgements
iv
Table of contents v
List of figures
ix
List of tables
x
Abstract
xi
CHAPTER
ONE
1.1 Introduction
1
1.2 Main
Objective
3
1.3 Specific Objectives
3
CHAPTER TWO
2.0 Literature Review 4
2.1 Metabolites
4
2.2 Types of metabolites or bioactive
molecules 5
2.2.1 Primary metabolites 5
2.2.2 Secondary metabolites 5
2.4 Antibiotics
6
2.4.1 Antibiotic produced by bacteria
7
2.4.2 Antibiotics produced by Bacillus species 8
2.5 Effects of antibiotics produced by Bacillus megaterium 9
2.6 Application of antibiotics produced by Bacillus megaterium 9
2.6.1 Application of antibiotics produced by Bacillus megaterium in medicine 10
2.6.2 Application of antibiotics produced by Bacillus megaterium in industries 10
2.6.3 Application of antibiotics produced by Bacillus megaterium in agriculture 10
2.7 Rhizosphere Environment
11
2.8 Natural Occurrence of Endospore Forming, Bacillus
species 12
2.9 Antagonistic
activity of Bacillus megaterium on
fungi pathogens 13
CHAPTER THREE
3.0
Materials and Methods
15
3.1 Materials
15
3.2 Methods
15
3.2.1 Sample
collection
15
3.2.2 Sterilization of materials
15
3.2.3 Sample preparation 15
3.2.4 Media
preparation
16
3.3 Isolation
of Bacillus species from the
rhizospheric soil 16
3.3.1 Pour
plate methods 16
3.3.2 Spread
plate method
17
3.4 Isolation
of Bacillus species 17
3.5 Characterization
of Isolates
17
3.5.1 Colony
Features/Macroscopic Features 17
3.5.2 Microscopic
features
17
3.5.3 Endospore
stain 18
3.5.4 Flagella
stain 18
3.6 Biochemical
Tests
18
3.6.1 Methyl
red
19
3.6.1.1 MR
positive
19
3.6.1.2 MR
negative
19
3.6.2 Citrate
Utilization Test
19
3.6.3 Coagulase
test
20
3.6.4 Carbohydrate
utilization 21
3.6.5 Catalase
test
21
3.6.6 Urease test
22
3.6.7 Indole Test
22
3.6.8 Oxidase Test
22
3.7 Identification
of Bacillus megaterium
22
3.8 Extraction
of antifungal molecules
23
3.8.1 Test
for antifungal activities
23
CHAPTER
FOUR
4.0 Results
25
CHAPTER
FIVE
5.0 Discussion, conclusion and
recommendation 32
5.1 Discussion
32
5.2 Conclusion 33
5.3 Recommendation
33
References 34
LIST
OF FIGURE
4.1 Occurrence of Bacillus species in the rhizosphere of turmeric 30
LIST
OF TABLES
TABLES
1 Showing the phenotypic characteristics of isolates 27
2 Showing the biochemical features of isolates 28
3 Showing the occurrence of Bacillus isolated from the rhizosphere of
turmeric plant 29 4 Showing
the antifungal activities of Bacillus
megaterium isolated
from
the rhizosphere of turmeric plant 31
CHAPTER ONE
1.1 INTRODUCTION
Controlling
disease has been an apparent priority, yet this has proved to be a difficult
task. Over the past years, chemical control methods have been the primary means
of combating diseases. However, many of the chemicals that are used to control
fungal and bacterial disease are detrimental to animal and human alike. The
truth is that chemicals persists and eventually accumulate in the natural ecosystem.
Consequently, chemical control may not be the most suitable means due to its
high cost and environmental risks associated with its application. As an
alternative, several examples of controlling foliar fungal pathogens with
bacteria have been reported. It is therefore desirable to replace this chemical
agent with biological approaches that are more environmental friendly. Thus,
biological control would be highly preferred for disease control in the future
(Mardanora, et al., 2017).
The
intensive use and misuse of antibiotics have generated a strong selective
pressure for the emergence of resistant strains. Actually, the constant
increase of multi-drug resistant pathogenic micro-organisms stimulates more
than ever effort to identify and develop new antibacterial compounds. In that field,
the recent advances in genome sequencing have highlighted the genus Bacillus as an unexpected source of
anti-biotic like compounds. Indeed, for some of them, such as Bacillus megaterium more than 4% of
the genome has been found potentially
devoted to the synthesis of polyketides, non-ribosomal peptide, bateriocins as
well as other unusual antibiotics (Patrick, 2012).
Antibiotics
have been recognized as the only means of effective microbial growth control
after the discovery of penicillin and other antimicrobial agents by Alexander
Fleming in1928. Since that time to date, there has been continues search for
more effective that can stand the emerging menace of drug resistance among
micro-organisms worldwide.
The
increase in antibiotic resistant have been attributed to inappropriate use,
inadequacies on the part of the
manufacturers and leads to the decline of effective antibiotics annually worldwide
(Kuta, 2008).
Bacillus
species are gram positive, aerobic or facultative anaerobic, sporulating rod
shaped bacteria that are widely spread in nature, being implicated in food poisoning.
Bacillus species exhibit a wide range
of physiological abilities that allow the organism to flourish in every
environment and compete favourably with other organisms within the environment
due to its ability to form spores, produce metabolites that are heat stable,
cold, radiation and desecration dis-infectant and have antagonistic effect on
other micro-organism (Faruk, et al,
2009).
The
rhizosphere is a habitat in which several biologically important processes and
interactions take place. It is the zone of intense activity of various groups
of micro-organisms. The rhizosphere is the 1mm zone of soil surrounding a plant
root where the biology and chemistry of the soil are influenced by the root.
This zone is where the majority of soil micro-organism (bacteria and fungi)
resides (Chakraborty, et al, 2015).
The
use of antibiotics along with communization and sterile technique in hospitals
has significantly decreased the number of lethal bacterial infections. However
antibiotics once seen as miracle drugs are now becoming useless in treating
various bacterial diseases. The rapid emergence of resist once to antibiotics
among pathogen generates visions of the potential post antibiotics era
threating present and future medical advances. The wide spread use of
antibiotics in medicine and in intensive animal husbandry is indicative of
selection pressure exerted on bacteria to enter the environment directly from
liquid manure and muck (Pushpendra, et
al., 2018).
1.2 Main
Objective
Ø To
isolate Bacillus megaterium from the
rhizosphere of turmeric and determine its antifungal activities against fungal
pathogens.
1.3 Specific
Objectives
i.
To isolate Bacillus species from the rhizosphere of turmeric plants.
ii.
To characterize and identify Bacillus megaterium from the rhizosphere
of turmeric plants.
iii.
To test for the antifungal activity of Bacillus megaterium isolated from the
rhizosphere of turmeric plant against fungi.
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