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

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(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.

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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).

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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

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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

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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

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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

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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

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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.

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