BIOCONTROL POTENTIAL OF BACILLUS THURINGIENSIS ISOLATED FROM SOIL SAMPLES AGAINST LARVA OF MOSQUITO

ABSTRACT

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 CONTENT

Title Page 
Certification 
Dedication 
Acknowledgement 
Abstract 
Table Of Contents 
List Of Tables

CHAPTER ONE: 
1.0 INTRODUCTION 
1.1 Crystal Composition And Morphology 
1.2 General Characteristics Of Bacillus Thuringlensis 
1.3 Classification Of Bacillus Thuringiensis Subspecies 
1.4 Ecology And Prevalence Of Bacillus Thuringrensis 
1.5 Other Pathogenic Factors Of Bacillus Thuringiensis 
1.6 Morphological Properties Of Bacillus Thuringiensis

CHAPTER TWO: 
2.0 LITERATURE REVIEW
2.1 Mode Of Action On Target Organism 
2.2 Mechanism Of Action Of Bacillus Thuringensis Formulation 
2.3 General Application Of Bacillus Thuringiensis

CHAPTER THREE: 
3.0 MATERIAL AND METHOD 
3.1 Soil Sample Collection 
3.2 Isolation Of Bacillus Thuringiensis 
3.3 Isolation Of Bacillus Thuringiensis From Soil 
3.4 Sample Staining 
3.5 Biochemical Identification
3.6 Materials And Method Of Bacillus Theringiensis Against Mosquito Lava 
3.7 Catalase Test 
3.8 Oxidase Enzyme Activity 
3.9 Sugar Test 
3.10 Methyl Red Test 
3.11 Indole Test

CHAPTER FOUR: 
4.0 RESULT OF SAMPLE COLLECTION AND ISOLATION 
4.1 Colony Morphology Of Bacillus Isolates 
4.2 Biochemical Test 
4.3 Bioassay

CHAPTER FIVE: 
5.0 DISCUSSION 
5.1 Conclusion 
5.2 Recommendations 
5.3 References 
Appendix 1
Appendix 2

CHAPTER ONE
INTRODUCTION 
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 Bacillus thuringiensis. 
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 control. 
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 growth cycle. 
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 developed. 
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 been identified.

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 control. 
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, 1996 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.