ABSTRACT
Chemical pesticides have disadvantages such as high production cost, short persistence, comparative low efficiency, development of resistance to toxin and causing ecological damage. In other to obtain better bio pesticides, Rhizopheric soils of turmeric plants were screened for the presence of Bacillus species which have antifungal tendencies against known fungal pathogens such as Aspergillus niger Rhizopus stolonifera, Penicillium species and Candida species. Four Bacillus isolates were identified as Bacillus subtilis, Bacillus cereus, Bacillus megaterium and Bacillus licheniformis. All the Bacillus isolates have inhibitory potentials with Bacillus subtilis exhibiting the highest potentials. Therefore, Bacillus subtilis was chosen as the ideal organism for optimization studies on the conditions for antifungal activities. Its optimal pH was found to be at 6.0 and temperature at 40 0C, incubation time at 96 hours and the best carbon source was glucose. Hence, it is therefore obvious that a good manipulation of the growth conditions for B. subtilis culture maybe an avenue for sourcing adequate quantity of these antifungal agents for the control of fungal phytopathogens. It is therefore recommended that optimization studies be carried out on the growth of Bacillus specie itself.
TABLE OF CONTENTS
Title page i
Certification ii
Dedication iii
Acknowledgments iv
Table of contents v
List of tables viii
List of figures ix
Abstract x
CHAPTER 1
1.1 Introduction 1
1.2 Objectives 6
1.2.1 General Objective 6
1.2.2 Specific Objectives 6
CHAPTER TWO
2.0 LITERATURE REVIEW 8
2.1 General characteristics
of Bacillus subtilis 8
2.2 Sporulation 8
2.3 Antifungal Tendency 11
2.4 The factors affecting
sporulation, antifungal and insecticidal activity of
Bacillus subtilis 12
2.4.1 Medium composition 12
2.4.2 Energy and carbon
sources 13
2.4.3 Cultural conditions 14
2.4.3.1 Aeration 14
2.4.3.2 Culture medium; pH 14
2.4.3.3 Temperature 15
2.4.3.4 Incubation time 15
CHAPTER
THREE
3.0 MATERIALS AND METHODS 16
3.1 Materials 16
3.2 Methods 16
3.2.1 Source of organisms 16
3.2.2
Collection of samples 16
3.2.3
Sample preparation 17
3.2.4.
Preparation of Media 17
3.2.5
Isolation of Bacillus species 18
3.2.6
Characterization of isolates 18
3.2.6.1
Colony features 18
3.2.6.2
Gram staining 18
3.2.6.3
Microscopic features 19
3.2.6.4
Biochemical reaction tests 19
3.3
Extraction of antifungal molecules 28
3.4
Antifungal activity tests 29
3.5
Optimization of the Bacillus isolate
against antifungal activity 29
3.5.1
Effects of pH on the production of antifungal compound 29
3.5.2 Effect of temperature on the production of antifungal
compounds 30
3.5.3
Effect of incubation period on the production of antifungal compound 30
3.5.4
Effects of carbon sources on production of antifungal compound 30
3.6 Data analysis 31
CHAPTER FOUR
4.0 RESULTS 32
CHAPTER 5
DISCUSSION AND CONCLUSION
5.1 Discussion 45
5.2 Conclusion 48
5.3 Recommendation 49
REFERENCES
APPENDICES
LIST OF TABLES
Table 1: colony
and microscopic features of suspected isolates 33
Table
2: Occurrence of Bacillus spp in biochemical test 34
Table 3: Cultural and
morphological characteristics of fungi and their
tentative identification 35
LIST OF FIGURES
Fig 1: Bacillus in a microscopic view 10
Fig 2: Antifungal
activity of Bacillus cereus 36
Fig 3: Antifungal
activity of Bacillus subtilis 37
Fig 4: Antifungal
activity of Bacillus licheniformis 38
Fig 5: Antifungal
activity of Bacillus licheniformis 39
Fig
6: Activities of B. subtilis isolates
against some named fungi 40
Fig 7: Effects of pH on antifungal activity of B.subtilis 41
Fig 8: Effects of temperature on antifungal activity 42
Fig.9: Effects
of Incubation time on Antifungal activity 43
Fig 10: A chart showing the effects of carbon source on antifungal
activity 44
CHAPTER 1
1.1 Introduction
Human’s progress depends largely on man’s ability to
control the forces of nature and on his efficiency to utilize them or his
benefits. In the process of understanding and harnessing the natural forces, several
advances in science and technology have taken place even though that man has
inadvertely interfered with balances of nature creating a feeling of fear that
biological species/microorganisms may become extinct on this planet in the near
future. The use of biological control in the management of agriculture pest and
disease I an effective alternative to the use of pesticides which are often
accumulated in plants and are lethal to beneficial microorganisms present in
the soil (Nogorska et al., 2017).
Antifungal agents produced by microorganism may be
used as bio control agents. Some soil borne fungi, bacteria and actinomycetes
has been identified and used as antagonistic microbes (Sivanantham et al., 2013). Bacillus strains with broad antifungal activity has been reported
by Awais et al (2007) also reported
the activities of Bacillus subtilis
Insects
are the most abundant groups of organisms on earth. They often negatively
affect humans in a variety of ways. They cause massive crop damage and act as vectors
of both humans and animal diseases, such as malaria and yellow fever.
Therefore, humans have desired to control insects. As being parallel to development
of chemistry, chemical substances have been used to control pests from the mid-1800s
(Yasutake et al., 2007). The use of inorganic chemicals and organic
arsenic compounds were followed by organochlorine compounds, organophosphates,
carbornates, pyrethroids and formamidines. These chemicals were very effective
in killing and controlling many species of pests. However, they have many
direct and indirect adverse effects on ecosystem including accumulation of
toxic residues in nature, leading health problems in mammals and development of
insect resistance (Fadel and Sabour, 2002; Sarfraz et al., 2005). The
problems related with chemical pesticides oriented humans to find out safer and
natural alternative ways of pest control.
In
developing countries like Kenya and Nigeria, where agriculture is the main
income generating activity, the excessive and widespread use of pesticides has
attained immense momentum during the recent past. Pesticides are the largest
group of possible hazardous chemicals that are introduced purposefully into the
environment (Ghribi et al., 2007). These toxic pesticides produce residues
which persist in the environment, causing pollution and resistance in many
target organisms against these chemicals (Brar et al., 2006). The residues persist in the soil and may contaminate
ground water and following absorption, get accumulated in plants, fruits and
vegetables. Pesticides affect wildlife, biological control agents and above all
are dangerous to fish, man and mammals (Bhattacharya, 2000).
Presently,
major concerns including insect resistance to chemicals, non-target effects of
pesticides and the cost of production of new compounds have renewed interest in
alternative forms of pest control, among which disease causing
micro-organisms hold out particular promise (Fadel and Sabour, 2002).
Increased concerns about the potential effects of pesticides on health,
the reduction in arable land per capita, and the evolution of pest
complexes likely to be accelerated by climate changes also contribute to change
in plant protection practices (WHO, 1985). Other drawbacks of synthetic insecticides
include resurgence and outbreaks of secondary pests and harmful effects on non-target
organisms (El-Bendary, 2006).
The
widespread use of a single chemical compound confers a selective evolutionary advantage
on the progeny of pests, since they acquire resistance to such chemicals.
Another problem is that some pesticides affect non-target species with
disastrous results. Unexpected elimination of desirable predator insects has
caused explosive multiplication of secondary pests. Other concerns include
environmental persistence, toxicity of many pesticides and increased cost of
developing new and safer 3 ones (Sayyed et al., 2000; Devi et al., 2005).
This calls for a demand for alternative control methods, including physical
controls (Sayyed et al., 2004; Sarfraz et al., 2005). The
drawbacks of using synthetic pesticides provide strong desire to find
alternative approaches, formulations and cost-effective bio pesticides
production for pest control.
Human
population is estimated to increase to 8.5 billion by the year 2020 (United
Nations, 2000). This increased population will cause an increase in the demand
for agricultural production. However, the land suitable for agricultural
production is limited due to restricted water availability, depletion of land
sources and already cultivated highly productive soils. Under these
limitations, it is important to develop the yield of agricultural production
(Luna et al., 2004). It has been estimated that up to 15% of crops
worldwide are lost annually due to insect damage only (Devi et al.,
2005). Therefore, the need to exterminate insects that are destroying crops
becomes urgent. Wheat, rice, maize and barley are the primary source for human
nutrition worldwide and cover more than 40% of global croplands (Tyagi et al.,
2002). Most of the pests giving damage to these grains belong to Coleoptera and
Lepidoptera orders. In addition, some species of Arachnida, Orthoptera,
Hymenoptera, Diptera and Psocoptera can also cause damage in stored grain products.
Early
pesticides were largely composed of chemical constituents. Certain properties
made them useful, such as long residual action and effective toxicity to a wide
variety of insects. However, their use led to many negative outcomes. The
chemical insecticides used today are considered as presumably safer to those
used in the past, but there are still some concerns. Long-term exposure to these
chemicals can cause cancer, liver damage, immunotoxicity, birth defects and
reproductive problems in humans and animals (Bhattacharya, 2000; Zouari et
al., 2002). Also, they can cause accumulation and persistence of toxic
residues in soil, water and food; toxicity against beneficial insects and development
of pest resistance (Luna et al., 2004). Nevertheless, chemical
insecticides have a large market volume, and global sales of them are about $5
billion a year (Luna et al., 2004)
By
contrast, microbial pesticides are safe for ecosystems. They are non-toxic and
nonpathogenic to wildlife and humans. Their toxic action is often specific to a
single group or species of insects, so they do not affect the other insect population
in treated area. Because they have no hazardous residues to humans or animals,
they can also be applied when crop is almost ready for harvest (Ouoba et al.,
2008). In spite of these attractive features, microbial pesticides represent
about 2% of global insecticide sales.
Bacillus
subtilis based pesticides account major share of
the bio insecticide market with 80-90%. For several reasons, the use of bio pesticides
as insecticide has grown slowly when compared to chemical ones. Microbial
pesticides are generally more expensive to produce than many chemicals. Large
quantities of toxins have to be applied to the field to ensure that each larva
will ingest a lethal dose. However, the cost can be decreased by increasing demands.
Many chemical pesticides have broad spectrum of toxicity, so pesticide users may
consider microbial pesticides with a narrower range to be less convenient
(Chang et al., 2008). The use of biological pest control agents has been
considered to be much safer than chemical ones for the ecosystem. Moreover, the
future prospects of them seem to be positive. It is estimated that, the growth
rate of usage of bio pesticides use over the next 10 years up to 2015 will be
10-15% compared with 2% for chemical pesticides (Crickmore, 2005). Also, the
cost of development of Bacillus thuringiensis insecticides is predicted
to be $3-5 million, compared with $50-80 million for chemical insecticides. In addition, the use of chemical insecticides
seems likely to decline in the future; restrictions for their registration will
increase resulting in a smaller chemical pesticide market (Zouari et al.,
2002).
In
nature, some microorganisms have the potential to produce some biological
agents capable of infecting other living organisms including insects. Many of
these infectious agents have a narrow host range and, are not toxic to
beneficial insects or vertebrates. Therefore, the use of these non-pathogenic microorganisms
has been developed as the biological way of pest control. Insect viruses (Baculoviruses),
some fungi, protozoa and bacteria have been used as biological pest control
agents. Among all, Bacillus subtilis is one of the most important
microorganism with entamopathogenic activity against certain insect orders. It
is ubiquitous, gram-positive and spore-forming bacterium which produces
insecticidal crystal proteins during sporulation. The toxic activity due to
proteins produced by plasmid encoded Cry genes varies with insect type.
The native strains of this bacterium have been used nearly for 50 years safely,
as an alternative to chemical pesticides. The discovery of the larvacidal
action of some bacterial strains from the genus Bacillus caused the
introduction of biopesticides. Bacillus thurugensis has been used for pest
control since 1920s and still accounts for over 90% of the biological
insecticide market (Crickmore, 2005). Bacillus subtilis was next
identified as a new biological insecticidal agent and its products appeared in
the market (Crickmore 2005).
Chemical
pesticides have certain disadvantages such as high production costs, short persistence,
comparative low efficacy and development of resistance to toxin. The ecological
damage occasionally caused by the lack of specificity in the toxic effects of insecticides
has provided the impetus to seek alternative methods of insect control. This observation
led to the development of bio insecticides based on the insecticidal action of Bacillus
subtilis. The discovery of bio larvicidal actions of Bacillus subtilis opened
a new perspective for insect control
The
use of isolated Bacillus subtilis strains in Nigeria for pest protection
will be a milestone in the reduction of chemical pesticides and hence reduce
negative impacts of chemical pesticides in the environment. It is also
necessary to understand the best temperature, pH, carbon, incubation time to
mass produce these antifungal substances. The goal then becomes how to find alternative
approaches for pest control at an optimized standard. The present study was
aimed at isolation of potential antagonistic Bacillus subtilis of antifungal components. Analysis on their
effects against tested pathogenic fungi, was also conducted.
1.2
Objectives
1.2.1
General Objective
To determine the optimal conditions for the antifungal
potential of Bacillus subtilis
1.2.2
Specific Objectives
1.
To isolate
Bacillus subtilis isolates from the rhizosphere of turmeric plant.
2.
To extract and characterize the antifungal
substances from the Bacillus subtilis isolates.
3.
To
carry out in vivo studies on the sensitivity of fungi species to the Bacillus
subtilis from the rhizosphere of the turmeric plant
4.
To optimize conditions for low cost production of antifungal substances from Bacillus
subtilis
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