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- Name: BIOCONTROL POTENTIAL OF BACILLUS THURINGIENSIS ISOLATED FROM SOIL SAMPLES AGAINST LARVA OF MOSQUITO
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A major challenge for achieving successful mosquito control is overcoming insecticide resistance. Bacillus thuringiensis which is one of the most effective biolarvacide for control of species of mosquitoes and monitoring of larval susceptibility is essential to avoid resistance development. Mosquito larvacidal activity of Bacillus thuringiensis was assessed by isolating them from ecologically different soil habitats in and around Enugu metropolis. The isolate organisms were confirmed as Bacillus thuringiensis based on biochemical characterization and microscopic observation. The larvacidal activity of Bacillus thuringiensis isolates was tested against the larval of mosquito by using the standard cup bioassay. The isolates of Bacillus thuringiensis showed a significant level of variation in their larvacidal activity.
TABLE OF CONTENTS
Title page: – – – – – – – – – i
Certification: – – – – – – – – ii
Dedication: – – – – – – – – iii
Acknowledgement: – – – – – – – iv
Abstract: – – – – – – – – – v
Table of contents: – – – – – – – vi
List of tables: – – – – – – – – viii
1.0 Introduction: – – – – – – – 1
1.1 Crystal composition and morphology: – – – – 3
1.2 General characteristics of Bacillus thuringlensis: – – 4
1.3 Classification of Bacillus thuringiensis subspecies: – – 5
1.4 Ecology and prevalence of Bacillus thuringrensis: – – 5
1.5 Other pathogenic factors of Bacillus thuringiensis: – – 7
1.6 Morphological properties of Bacillus thuringiensis: – – 8
2.0 LITERATURE REVIEW- – – – – – – 10
2.1 Mode of action on target organism: – – – – 10
2.2 Mechanism of action of Bacillus thuringensis formulation: 12
2.3 General application of Bacillus thuringiensis: – – 13
3.0 Material and method: – – – – – – 15
3.1 Soil sample collection: – – – – – – 15
3.2 Isolation of Bacillus thuringiensis: – – – – 16
3.3 Isolation of Bacillus thuringiensis from soil: – – – 16
3.4 Sample staining: – – – – – – – 17
3.5 Biochemical identification: – – – – – 18
3.6 Materials and method of Bacillus theringiensis against
mosquito lava: – – – – – – – 19
3.7 Catalase test: – – – – – – – 20
3.8 Oxidase enzyme activity: – – – – – – 20
3.9 Sugar test: – – – – – – – – 20
3.10 Methyl red test – – – – – – – 21
3.11 Indole test – – – – – – – – 22
4.0 Result of sample collection and isolation: – – – 23
4.1 Colony morphology of Bacillus isolates: – – – 24
4.2 Biochemical test: – – – – – – – 25
4.3 Bioassay: – – – – – – – – 27
Discussion: – – – – – – – – – 28
Conclusion: – – – – – – – – – 28
Recommendations: – – – – – – – 29
References: – – – – – – – – – 31
Appendix: 1 – – – – – – – – – 35
Appendix: 2 – – – – – – – – 38
Bacillus thuringrensis (Bt) is a well known and widely studied bacterium
which is known for its use in pest management. Today it is the most
successful commercial xenobiotic with its worldwide application when
compared with the chemical pesticides; Bacillus thuringiensis has the
advantages of being biologically degradable, selectively active on pests and
less likely to cause resistance. Safety of Bacillus thuringiensis formulations
for humans, beneficial animals and plants explains the replacement of
chemical pesticides in many countries with these environmentally friendly
pest control agents.
Bacillus thuringiensis was first isolated by the Japanese Scientist Ishiwata
(1901) from skilkworm larvae, bombyxmori, exhibiting sotto disease. After
10 years, Berliner (1911) isolated the square gram (+) positive, spore
forming, rod shaped soil bacterium from disease flour moth larvae, Anngasta
Kachmiccalla, in the Thuringia region of the Germany and named it as
In the early 1930s Bacillus thuringiensis was used against Ostrinianubilis, the
European corn borer. The first commercial product was available in 1938 in
France, with the trade name sporeine (Weiser, 1986). It was Bacillus
thuringiensis subspecies Kurstaki that was used for the control of the insect
(Lepidopteran) pests in agriculture and forestry (Luthy & Ebersold, 1981).
New commercial products arrived in 1980s after the discovering of
subspecies thuringiensis opened the gate for black fly and mosquito larvae
Like all organisms, insect are susceptible to infection by pathogenic
microorganisms, many of these infections agents have a narrow host range
and therefore, do not cause uncontrolled destruction of beneficial insects and
are not toxic to vertebrates. Bacillus thuringiensis is a major microorganism,
which shows entamopathogenic activity (Glazer & Nikaido, 1995, Schnepf,
et al. 1998) which forms parasporal crystals during the stationary phase of its
Most Bacillus thuringiensis preparations available on the market contain
spores with parasporal inclusion bodies composed of δ – endotoxins. In
commercial production, the crystals and spores obtained from fermentation
are concentrated and formulated for spray on application according to
conventional Agriculture practices (Baum, Kakefuda, & Gawron-Burke,
1996). There are many strains of Bacillus thuringiensis having insecticidal
activity against insect order (eg Lepidoptera, Diptera, Homoptera,
Mollaphage, Coloptera). Only a few of them have been commercially
Bacillus thuringiensis insecticides are divided into three groups, group one
has been used for the control of lepidopterans. These groups of insecticides
are formulated with Bacillus thuringiensis Subspecies. Kurstaki, group two
contains thesandiego and tenebrionis strains of Bacillus thuringiensisand has
been applied for the control of certain celopterans and their larvae. Group
three contains the Israelensis strains of Bacillus thuringiensis which has been
used to control black flies and mosquitoes.
CRYSTAL COMPOSITION AND MORPHOLOGY
The existence of parasporal inclusions in Bt was first noted I 1915 (Berliner
1915) but their protein composition was not delineated until the 1950s
(Angus 1954). Hannay (1953) detected the crystalline fine structure that is a
property of most of the parasporal inclusion. Bacillus thuringiensis
subspecies can synthesize more than one inclusion, which may contain
different ICPs. ICPs have been called data endotoxins; however since the
term endotoxin usually refers to toxin associated with the other membranes of
gram-negative bacteria, comprising a core lipopoly saccharide. Depending on
their ICP composition, the crystals have various forms (bipyramidal,
cuboidal, flat rhomboid, or a composition with two or more crystal types. A
partial correlation between crystal morphology, ICP composition, and
bioactivity against target insects has been established (Bulla et al.1977).
Hofte and Whitely, 1989, Lynch and Baumman, 1985).
GENERAL CHARACTERISTICS OF BACILLUS THURINGLENSIS
Bacillus thuringiensis is a member of the genes Bacillus and like the other
members of the taxon, has the ability to form endospores that are resistant to
inactivation by heat, desiccation and organic solvent. The spore formation of
the organism varies from terminal to subterminal in sporangia that are not
swollen, therefore, Bacillus thuringiensis resembles other members of
Bacillus species in morphology and shape (Stahly, Andrews, & Yousten,
1991). The organism is gram-positive and facultitative anaerobes. The shape
of the cells of the organism is rod. The size when grown in standard liquid
media varies 3 – 5um.
The most distinguishing features of Bacillus thuringiensis from other closely
related Bacillus species. (eg Bacillus anthracis, Bacillus. cereus) is the
presence of the parasporal crystal body that is near to the spore outside the
exosporangium during the endospore formation, which is shown in figure 1:1
(Andrews, Bibilops, & Bulla, 1985; Andrews, Faust, Wabiko, Raymond, &
Bulla, 1987; Bulla, Faust, Andrews, & Goodma, 1995). Bacillus thuringiensis
is an insecticide producing variant of Bacillus cereus (Gordon, Haynes, &
Pang, 1973) several Bt species also produce Bacillus cereus type
enterotooxin (Carlson, & Kolsto, 1993) plasmids coding for the insecticidal
toxin of Bacillus thuringiensis have been transferred into B. cereus to make it
a crystal producing variant of Bacillus thuringiensis(Gonzalez, Brown,
Carlton, 1982) molecular methods including genomic restriction digestion
analysis and 16 rRNA sequence comparison support that Bacillus
thuringiensis, Bacillus anthracis and Bacillus cereus are closely relocated
species and they should be considered as a single species (Carlson, Caugant,
& Kolstra, 1994; Ash , Farrow, Dorsch, Stackebrandt, & Collins. 1991;
Helgason et al.2000).
CLASSIFICATION OF BACILLUS THURINGIENSIS SUBSPECIES
The classification of Bacillus thuringiensis based on the serological analysis
of the flagella antigens was introduced in the early 1960s (de Barjac &
Bonnefoi, 1962). This classification by serotype has been supplemented by
morphological and biochemical criteria (de Barjac, 1981). Clutill (1977),
explains that only 13 Bacillus thuringiensis subspecies were toxic to
lepidopteran Larva only. And apparently Nematode (Narva et; al., 1991)
enlarged the host range and markedly increased the number of subspecies up
to the end of 1998, over 67 subspecies based on flagella H – Serovars had
ECOLOGY AND PREVALENCE OF BACILLUS THURINGRENSIS
Although our knowledge about Bacillus thuringiensis occurs naturally and
it can also be added to an ecosystem artificially to control pest, prevalence of
Bacillus thuringiensis in nature can be said as “natural” and can be isolated
when there is no previous record of application of the organism for pest
The Bacillus thuringiensis which belong to artificial habitat areas are sprayed
based insecticides (mixture of spores and crystal). (Stahly et al. 1991). Thus,
it is obvious that Bacillus thuringiensis is widespread in nature. However, the
normal habitat of the organism is soil. The organism grows naturally as
asaprophyle, feeding on dead. Organic matter, therefore, the spores of
Bacillus thuringiensis persist in soil and its vegetative growth occurs when
there is nutrient available. Moreover Bacillus thuringiensis has recently been
isolated from marine environments (Maeda et al. 2000) and from soil of
Antarctica also (Foresty & Logan 2000).
However the true role of the bacteria is not clear. Although it produces
parasporal crystal inclusions that are toxic to many orders of insects, some
species of Bacillus thuringiensis from diverse environments show no
insecticidal activity. The insecticidal activities of Bacillus thuringiensis are
rare in nature. For example, Iriarte et al.(2000) reported that there is no
relationship between mosquito breeding sites and pathogenic action level of
Bacillus thuringiensis in the surveyed aquatic habitats. While another study
suggested that habitat with a high density of insect were originated by the
pathogenic action of this bacterium (Itoqou Apoyolo et al.1995).
OTHER PATHOGENIC FACTORS OF BACILLUS THURINGIENSIS
At the period of the active growth cycle, the strains of Bacillus
thuringiensis produce extracellular compounds; this compound might yield to
virulence. These extracellular compounds include proteases, chitinases
phospholipases, and vegetative conseticidal protein (Zhang et al. 1993;
Sohneff et al. 1998).
Bacillus thuringrensis also produces antibiotics compounds having antifungal
activity (stab et al. 1994). However the crystal toxins are more effective then
these extracellular compounds and allow the development of the bacteria in
dead insect larvae.
Bacillus thuringiensis strains also produce a protease, which is called
inhibitor. This protein attacks and selectively destroys cecropiris and attacisis
which are antibacterial proteins in insects, as a result of this, the defence
response of the insect collapses. This protease activity is specific, it attacks an
open hydrophobic region near C – terminus of the cecropin and it does not
attack the globular proteins (Duthambar & Steiner, 1984).
Other important insecticidal proteins which are unrelated to crustal proteins
are vegetative insecticidal protein. These proteins are produce by some
strains of Bacillus thuringiensis during vegetative growth.
MORPHOLOGICAL PROPERTIES OF BACILLUS THURINGIENSIS
Colony forms can help to distinguish Bacillus thuringiensis colonies from
other Bacillus species. The organism forms white, rough colonies, which
spread out and can expand over the plate very quickly. Bacillus thuringiensis
strains have unswallon and ellipsoidal spores that lie in the subterminal
position. The presence of parasporal crystals that are adjacent to the spore in
another cell is the best criteria to distinguish Bacillus thuringiensis from other
closely related Bacillus species. The size number, of parasporal inclusion and
morphology may vary among Bacillus thuringiensis strains. However, four
distinct crystal morphologies are apparently the typical bipyramidal crystal,
related to crystal proteins (Aronson et al. 1976). Cuboidal usually associated
with bipyramidal crystal (Ohba&Aizawi 1986), amorphous and composite
crystals related to cry4 and cry proteins (federicet al. 1990), and flat, square
crystal related to cry3 proteins (Hernstadet al. 1986, Lopezmeza & Ibarra,
The classification was based in part on the possession of parasporal bodies.
Bernard et al.(1997) isolated 5303 Bacillus thuringiensis from 80 different
countries and 2793 of them were classified according to their crystal shape.
Bacillus thuringiensis vary’s based on geographical or environmental
location. Each habitat may contain novel Bacillus thuringiensis isolated that
have more toxic effects on target insects. Intensive screening programs have
been identified Bacillus thuringiensis strain from soil, plant surfaces and
stored product dust samples. Therefore many strain collections have been
described in the literature, such as Assian (Chak et al. 1994, Ben – Dov et al.
1997, 1999) and Maxican (Bravo et al. 1998).
Therefore the aim of this study is to isolate Bacillus thuringiensis from soil
sample and to isolate Bacillus thuringiensis against larva of mosquito or to
determine Bacillus thuringiensis against larva of mosquito.
Commercial Bacillus thuringiensis (Bt) products are microbial pest control
agent (MPCAs) containing specific insecticidal crystalline proteins (ICPs)
and most often living spores as well as formulating agent. They processed
fermentation products. (Baumann et al., 1993; and Hansen et al. 1998).
Bacillus thuringiensis is a facultative anaerobic motile, gram – positive, spore
forming bacterium. The formation of parasporal crystals adjacent to the
endospore during sporulation stage three to four distinguished Bt from other
Bacillus species. Bacillus thuringiensis like other Bacillus species, has been
classified on the basis of its cellular, cultural, biochemical and genetic
characteristics (Baumannet al. 1984; Barkley 1986; Slepecky; 1992;
Hansenet al. (1998). In 1958, Heimpel and Augus1958, introduce a
classification scheme to identify these crstalliferous bacteria base on their
morphological and biochemical characteristics but that specific biochemical
characteristics do not always refer to a specific serotype (Helyasonet al. 1998;
Hansen et al.1998).
MODE OF ACTION ON TARGET ORGANISM
The sporulation bacillus thuringiensis with insecticidal crystalline proteins or
spore – ICP complexes must be ingested by a susceptible insect larva. The
efficacy of the ICP depends on the solubilizaiton, in the midgut
(Heimpel&Anges 1993) resulting in the conversion of the protoxin to the
biologically active toxin by proteolytic enzymes, specific membrane receptor
binding by the C-termminal domain of the active toxin, and pore formation
by the N-terminal domain with subsequent lysis of the epithelia cell.
Proliferation of the vegetative cells into the haemocoel may result in a
septicuemia contributing to the cause of death.
Receptor binding by the ICP is the major determinant of host specificity by
the different Bacillus thuringiensis (ICPs) insecticidal crystalline proteins
(Barkley et al. 1996). The mode of action of Bacillus thuringiensishas been
reviewed by Schneptet al.(1998) and can be summarized in the following
1. Ingestion of sporulation Bt and ICP by an insect harva.
2. Solubilization of the crustalline ICP in the midgut.
3. Activation of the ICP by protease.
4. Binding of the activated ICP to specific receptors in the midgut cell
5. Insection of the toxin in the cell membrane and formation, followed by
destruction of the epithelia cells (Cooksey 1971 &Smodley 1996).
6. Subsequent Bacillus thuringiensis spore germination and septicaemia
may enhance motality.
The specific bioactivity of Bt is dominated by the ICPs that are encoded by
the cry gene and are active against subsceptible species (Feitelson 1993;
Zukowski 1995) the ICP must be effective against the target organism
Bacillus theringiensis is effective against the early stages of mosquito larvae
and does not affect mosquito to eggs, mature larvae, pupae or adults. (Murray
& Daniels, 2007). Mosquito larvae must eat the Bt formulated product
containing dormant spores. Crystals which are known as insecticidal crystals
proteins (ICPs) or delta – endotoxinal produced during Bacillus thuringiensis
sporulation. The mosquito larvae stop feeding and die when these proteins are
converted into toxins that work by damaging the gut wall of mosquitoes.
(Lacey, & Merrit, 2003).
MECHANISM OF ACTION OF BACILLUS THURINGENSIS
The ICP spore complexes of Bacillus thuringiensis are ingested by
susceptible insect larva. In the midgut the parasporal crystalline ICP is
dissociated to the protoxinform, and the protoxin is then activated to a
holotoxin by gut proteases (Warrenet al. 1984; Jaquetet al. 1987; Aronson et
al. 1991; Honee 1993). Shortly the gut becomes paralysed and the larun cease
The ICP structure and function have been reviewed in detail by Schneptet
al.(1998) binding of the ICP to pultative receptors is a major determinant of
ICPs specificity and the formation of pores in midgut epithelia cell is a major
mechanisms of toxicity (Van Frankenhuzen, 1993).
Binding to specific receptors has been demonstrated to be closely related to
the insecticidal spectrum of the ICPs (Denolfet al. 1997). Van Rieet al.
(1998) found the effinity of these toxins similar for the tobacco budworm
(Heliothisvirescen) and tomato hornworm (manducasexta) brush bord or
membrane vesicles, but the number of binding sites differed and reflected
varied bioactivity. However, the toxin affinity for binding sites does not
appear constant for all insects.
GENERAL APPLICATION OF BACILLUS THURINGIENSIS
Application Agricultural and Forest: Commercial use of Bt on Agricultural
and forest crops dates back nearly 30 years, when it became available in
France – use of B+ has increased greatly in recent years and the number of
companies with a commercial interest in Bt products has increased from four
in 1980 to at least 18 (Van Frankenhuyzen, 1993), several commercial
Bacillus thuringiensisproducts have been supplied to crops using
conventional spraying technology. Various formulation have been used on
major crops such as cotton, maize, soybeans, potatoes, tomatoes, various
crops trees and stored grains (Carlton et al. 1990).
In the main naturally occurring Bt strain have been used but transgenic
microorganism expressing and by genetic manipulation and in some cases,
these have reached the commercial market. These modified organisms have
been developed in order to increase host range, prolong field activity or
improve delivery of toxin to target organisms, for examples, the coleopteran
active crygene has been transferred to a hepidopteran – active Bt (Cartonet al.
1990). A plasmid bearing an ICP gene has been transferred from Bacillus
thuringiensis to a non-pathogenic leaf colonizing isolate of pseudomonas
fluorescens, fixation of the transgenic cells produces ICP contained within a
membrane which prolongs persistence (Gelenter, 1990). The delivery of ICP
effectiveness has been shown in European corn borer, feeding within plant
stems (Beach 1990). Improvement in performance arising from such
modification are such that transgenic organisms and their products are likely
to be used much more widely in the future.
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