The Largest Sector In The Economy Of Pakistan Biology Essay

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The largest sector in the economy of Pakistan is agriculture which is contributing 25 to the gross domestic products. Controlling agriculture pests, which cause damage to crops is necessary to increase in yield, quality and productivity. An integrated pest management program which comprises different practices including the use of pesticide, crop rotation, use of insecticide and use of biopesticide (Krattiger, 1997).Triggered by the discovery of DDT 1939, agriculture has changed markedly over the last forty years as a result of the use of synthetic chemicals to control pests on different crops (Prokopy, 1988). The insecticidal activity of this chemical was discovered after few years. From 1943 to 1985, the use of pesticide worldwide jumped from less than 30 thousand metric tons to

3 million metric tons in 1985 (Prokopy, 1988).

Due to over-dependence on chemical control, modern agriculture became extremely vulnerable with unwanted side effects to human health and the environment. It is establish in study that approximately 25 million people working as agricultural workers residing in developing countries are poisoned by pesticide (Jeyaratnam, 1990). Also the effect of pesticides on the environment is still lacking a global assessment. Among the pesticides, insecticides stand out as the most important group which effect human health and the environment (Balid et al., 1998).

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Synthetic agrochemicals are converting minor pests to major ones (Bull et al., 1979). Most important group of all the agricultural pests are insect pests of order Lepidoptera and most of them were pests created by the overuse pesticides. The Lepidopterans also have the leadership of the number of species with insecticide resistance among the agricultural arthropodal pests with 26076 of the total (Georghiou & Lagunes, 1991). Biological control is an environmentally friendly tool for maintaining crop production which involves the use of living organism as pest control agent. Fortunately enough,

B. thuringiensis exists and has shown from its discovery a marked efficiency against this group of agricultural pests. B. thuringiensis is fermentation friendly and therefore commercially exploitable and it is host specific or has narrow host range. These advantages favored development of about 100 formulations (Federici, 1993).

B. thuringiensis is a versatile pathogen capable of infecting protozoa, nematodes, flatworms, mites and insects that are either plant pests or human and animal health hazards (Feitelson, 1993). B. thuringiensis were globally recovered from soil (Martin & Travers, 1989). B. thuringiensis has been obtained from soil, phyllosphere, diseased insects, stored products, dumping pits, excreta of vegetarian animals etc. and about 30- 100% spore formers of phyllosphere were found to be B. thuringiensis (Martin & Travers, 1989; Boucias & Pendland, 1998). Numerous B. thuringiensis subspecies have been extracted from coniferous & deciduous trees and vegetables, as well as from other aromatic plants (Smith & Couche, 1991; Damgaard et al., 1997). B. thuringiensis could be isolated everywhere, including desert, beach and tundra habitats (Martin & Travers 1989; Attathom et al., 1995). It has also been discovered in dust particles in the form of suspension and also in marine sediments (Meadows et al., 1992; Damagaad, 1996; Bernhard et al., 1997; Akhurst et al., 1997; Smith & Barry, 1998; Maeda et al., 2000; Maduell et al., 2002). B. thuringiensis was also recovered from river and public water after an aerial application of Thuricide (Ohana et al., 1987; Hoti & Balaraman, 1991). They can also persist in air after application of B. thuringiensis pesticide and can withstand in air foe 17 days after application (Barry et al., 1993).

B. thuringiensis accounts for about 5-8% of Bacillus spp. population in the environment (Hastowo et al., 1992). Steinhaus was the first worker to demonstrate potential for commercial exploitation (Heimpel & Angus, 1963). Since this time interest in the use of B. thuringiensis has steadily increased and as long ago as 1971, B. thuringiensis was registered for use against 23 insects in the USA on some 20 agricultural crops

(Falcon, 1971).

B. thuringiensis is a rod-shaped which is positive for Gram test, spore-forming and soil-resident bacteria. This bacteria is approximately 5 (five) µm in length and 1 (one) µm in width (Sakai et al., 2007). These bacteria can grow up at body temperature and diamond shaped crystal are produced from its protein called crystal proteins. These cry proteins can be used to kill predators, insects, and other pathogens (Jimenez -Jaurez et al., 2007).

In 1901 B. thuringiensis was first isolated in Japan from contaminated silkworm larvae. Later on B. thuringiensis were then isolated from Mediterranean flour moths in 1911(Lambert & Peferoen, 1992). It was found historically in soil, sick or dead insects and stored products such as tobacco, flour, and grains (Bernhard et al., 1997; Bravo et al., 1998; Helgason et al., 1998; Hongue et al., 2000). The subspecies B. thuringiensis represents a group of organisms that can use to obtain insect control naturally or can be achieved by adding to ecosystem artificially (Andrews et al., 1987; Stahly et al., 1991).

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B. thuringiensis is closely related to B. cereus and B. anthracis. B. cereus causes food poisoning whereas B. anthracis causes a disease known as anthrax. Various researches have been done on the persistent health effects, carcinogenicity, or mutagenicity of B. thuringiensis. Exposure of people to B .thuringiensis resulted in eye, respiratory and skin irritation. Direct contact with a B. thuringiensis formulation can cause corneal ulcer. (Swadener, 1994).

B. thuringiensis forms toxin proteins near endospore (Schneph et al., 1998). More than one parasporal inclusions can be formed by them which may contains different ICPs (Hannay, 1953). These crystalline inclusions are toxic for some invertebrates, specially species of insect larvae belonging to the orders Coleoptera, Diptera and Lepidoptera (de Berjac, 1981; Andrews et al., 1987). There are more recent reports of B. thuringiensis isolates active against other insect orders (Hymenoptera, Homoptera, Orthoptera, and Mallophaga) and against nematodes, mites, and protozoa (Feitelson, 1993; Feitelson et al., 1992). The Cry proteins produced at the time of spotulation include alpha (α), beta (β), gamma (γ) and delta (δ) endotoxins. The δ endotoxins are frequently used to control agronomically important pests (Dulmage, 1981; Guillet et al., 1990; Mulla, 1990). B. thuringiensis is already a useful alternative to synthetic chemical pesticide application in commercial agriculture, forest management, and mosquito control. Two isolates of this genus are highly active against insects of great economic importance; (B. thuringiensis subsp. kurstaki, active vs. lepidopterous insects and B. thuringiensis subsp. israelensis, active vs. mosquitoes and blackflies) (Tomlin, 1997). B. thuringiensis is an important ingredient of pesticides now a days because more than 90% of the pesticides contains these insecticides, furthermore, it does not harm environment because it falls in the category of environment friendly family of insecticides (Cherif et al., 2007).

Many studies point toward and consider B. thuringiensis and B. cereus as one species. However, there is a significant difference between both of them because crystal proteins can be produced from B. thuringiensis. The synthesis of crystal protein is controlled by plasmid genes, which is prone to loss (Cherif et al., 2007). Another response is that the strains of B. thuringiensis produce enterotoxins which are released in the lower intestine through micro-organism. These enterotoxins are involved in the pathogenesis of B. cereus. This analysis indicates a fine-line between the two species (Cherif et al., 2007). A close resemblance has been characterized among bacteria which further establish that all varieties of such bacteria correspond to same species. (Swadener, 1994). B. thuringiensis's crystal proteins can be produced from B. cereus if it is cultured with B. thuringiensis cells and transfer of genetic material takes place (Swadener, 1994). Transfers of genetic material between B. cereus and B. thuringiensis made the receptor of B. cereus indistinguishable from B. thuringiensis (Gonzalez, et al., 1981).

Large-scale applications of B. thuringiensis can have far reaching ecological impacts.

B. thuringiensis is very effective and can eradicate significantly number and variety of those butterfly and moth species which has adverse impacts on caterpillars that are used to feed birds and mammals. Furthermore, it has bad impacts on various helpful insects (Swadener, 1994).

The extensive use of B. thuringiensis as a pest control technique shows some critical concerns that it can affect the animals which does not requires these type of pest control techniques (Salama et al., 1991). B. thuringiensis can also root-out those organisms which are indispensable for the existence of those animals that are dependent on these types of organisms for their food and other requirements (Salama et al., 1991).

Aquatic insects are also affected by B. thuringiensis treatments. It was also observed in Canadian studies that various kinds of insects (Simulium vittatum and Taeniopteryx nivalis) were badly affected by B. thuringiensis applications (Eidt, 1985; Kreutzueiser et al., 1992). Another study reveals that applications of B. thuringiensis israelensis have repeatedly killed Midges (chironomids) (Sinegre et al., 1980). It is shown that applications of B. thuringiensis harmfully impacts on life of those birds whose life is dependent on caterpillars; it is also worth-mentioning that impacts of B. thuringiensis in respect of spraying on birds have been documented. In Pakistan there are only six B. thuringiensis based products have been approved from Plant Protection Department, Govt. of Pakistan (Karim & Riazuddin, 1999). Because of lack of awareness and high costs, manufacture of biopesticide in Pakistan does not exist practically.

The aim of the present research was to isolate and preserve different isolates of

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B. thuringiensis which may be further used for biological control of wide variety of insect pests and disease vectors. The principal objectives include sampling from different ecological habitats of Jhelum i-e sample of cow dungs, dust, bird dropping and wheat dust and screening of isolates with high protein profile thorough biochemical tests and spore staining procedure. After isolation the isolates were preserved for future study.

CHAPTER 2

LITERATURE REVIEW

The formulations of Bacillus thuringiensis (B. t.) have been used for several decades since its discovery as an effective means of controlling Lepidoteran pests in agriculture because of the its many properties that are different from the synthetic chemical formulations. It is not toxic to mammals; it is environmental friendly, earlier it was believed to have immunity towards the pesticide resistance phenomenon, good combination with other pest control methods and the possibility of being mass produced at low cost at farm level, are the factors that make B. thuringiensis the much-needed means for IPM programmes in developing countries. It has been revealed by almost 85 years of research that Bacillus spp., especially B. thuringiensis and Bacillus sphaericus are the most effective biopesticides (Boucias & Pendland, 1998). B. thuringiensis is a bacterial species having insecticidal attributes and a specific range of insect orders are affected by it. B. thuringiensis has atleas 34 subspecies also called

serotypes or varieties. It has over 800 strain isolates (Swadener, 1994). B. thuringiensis comprises about 5-8% of Bacillus spp. population in the environment (Hastowo et al., 1992).it has been found that more than 130 species of lepidopteran, dipteran and coleopteran insects are controlled by B. thuringiensis till date (Dean, 1984).

Historical Background of B. thuringiensis

B. thuringiensis play an important role in the biological control of insect pest which form toxic crystal proteins at the time of sporulation. B. thuringiensis, a bacterium that produces insecticidal proteins during its sporulation is the source of well known and generally used biopesticide. This widespread soil bacterium, was discovered first in Japan in 1901 by Ishawata and then in 1911 in Germany by Berliner. It is found in grain dust from soil in abundance and other grain storage facilities, (Baum et al., 1999). Later it was found that thousands of strains of B. thuringiensis are present (Lereclus, 1993). The isolation of this bacterium from diseased larvae of Anagasta kuehniella, led to the establishment of B. thuringiensis as microbial insecticide.

The first record of its use to control insects can be traced back to Hungary at the end of 1920, and in Yugoslavia at the beginning of 1930s; it was used to control the European corn borer (Lords, 2005). The first commercial product of B. thuringiensis was Sporine it was available in 1938 in France (Waiser, 1986) for the controling flour moth (Jacobs, 1951). World War II resulted in the use of the product for very short time (Nester et al., 2002). Development of transgenic plant was also noticed. In 1987 first reports of insertion of genes encoding for B. thuringiensis delta-endotoxins into plants came. Tobacco and tomato plants were first transgenic plants to express B. thuringiensis toxins (van Frankenhuyzen, 1993). "Thuricide" was first commercialized strain on B. thuringiensis. It was commercialized in 1957 because of the increasing concern of biopesticide over the use of chemical insecticides.

During its stationary phase of growth B. thuringiensis, a gram-positive spore-forming bacterium produces crystalline proteins called deltaendotoxins (Schnepf et al., 1998). The crystal is released to the environment after analysis of the cell wall at the end of sporulation, and it comprises 20 to 30% of the dry weight of the sporulated cells (Schnepf et al., 1998)

Distribution & Habitat of B. thuringiensis

This bacterium is universal distribution (Martin & Travers, 1989). Its main habitat is believed to be soil; however it has been isolated from foliage, water, storage grains, and dead insects as well (Iriarte & Caballero, 2001). Strains isolated from dead insects has been the main source for commercially used varieties, including kurstaki, isolated from A. kuehniella; israelensis, isolated from mosquitoes, and tenebrionis, isolated from Tenebrio monitor larvae (Ninfa & Rosas, 2009; Iriarte & Caballero, 2001).. The spores of B. thuringiensis persist in soil, and vegetative growth being subjected to the availability of nutrients (Akiba, 1986; Ohba & Aizawa, 1986; Travers et al., 1987; Martin & Travers, 1989; DeLucca et al., 1981).

In USA, soil sample contains between 0.5% and 0.005% B. thuringiensis of all Bacillus species when isolated (DeLucca et al., 1981). B. thuringiensis were globally isolated from soils (Martin & Travers, 1989). Meadows (1993) has isolated 1115 soil samples and recovered 785 B. thuringiensis isolates, furthermore, he found that samples that were obtained from New Zealand contains 56% of B. thuringiensis and samples from central and southern Africa and Asia contains 94% of B. thuringiensis. Ohba & Aizawa (1986) have isolated 189 soil samples in Japan and recovered 136 B. thuringiensis isolates.

There are several theories regarding the ecological niche filled by B. thuringiensis. B. thuringiensis generally recycle poorly and rarely unlike most insect pathogenic microbes. The reason being natural epizootics in insects, leading to assumption that B. thuringiensis is essentially a soil micro-organism having incidental insecticidal activity (Martin & Travers 1989) are commonly reported in the environmental occurrence of B. thuringiensis is independent of insects and the lack of association between occurrence and insect activity are the evidence supporting this idea (van Frankenhuyzen 1993). Meadows (1993) suggested four possibilities for the presence of B. thuringiensis in soil: 1) seldom grows in soil but is deposited there by insects; 2) may be infective to soil-inhabiting insects (as yet undiscovered); 3) may grow in soil when nutrients are accessible; and 4) a resemblance with B. cereus.

B. thuringiensis has been found widely in the phylloplane. Various B. thuringiensis subspecies have been isolated from deciduous trees, vegetables, coniferous trees, further it can be isolated from other types of herbs (Damgaard et al., 1997; Smith & Couche, 1991). B. thuringiensis activeness is affected by sunshine, it means when B. thuringiensis placed upper most part of the leaves which is exposed to sunshine it may remains active for only 1-2 days, but if it is deposited underneath of the leaves that are protected from sunshine, it may remain active for 7-10 days (Swadner, 1994). After an aerial application of Thuricide 16B B. thuringiensis kurstaki has been isolated from rivers and public water distribution systems (Ohana, 1987). B. thuringiensis usually resides only for a short time in soil. The half life of the insecticidal activity (the time in which half of the insecticidal activity is lost) of the crystal is about 9 days (West & Burges, 1985). However, small amounts can be quite persistent.

Crystal Composition and Morphology

In 1915 for the first time the existence of parasporal inclusions in B. thuringiensis was noted (Berliner, 1915), but it was not until 1950s that their protein composition was delineated (Angus, 1954). Crystalline fine structure hasby Hannay (1953) and this structure is the property of most of parasporal inclusions. B. thuringiensis subspecies can produce more than one inclusion, which may have different ICPs (Hannay, 1953). crystalline inclusions of B. thuringiensis ranging from 27 to 140 KDa are protoxins that are proteolytically transformed into smaller toxic polypeptides in themed gut of larva (Peferoen, 1997). Crystal protein genes are present as satellite chromosome (Kronstad et al., 1983).

On the basis of their ICP composition, the crystals have different types and forms (flat rhomboid, cuboidal, bipyramidal, or a combination of two or more types of crystal) (Höfte & Whiteley, 1989; Bulla et al., 1977). A limited association between morphology of crystal, composition of ICP, and bioactivity in opposition to target insects has been recognized (Bulla et al., 1977; Höfte & Whiteley, 1989; Lynch & Baumann, 1985).

Classification of B. thuringiensis subspecies

In early 1960s, B. thuringiensis subspecies was classified on the basis of serological analysis of the flagella (H) antigens (de Barjac & Bonnefoi, 1962). Morphological and biochemical criteria further add-on to this classification by serotype (de Barjac, 1981). Only 13 subspecies of B. thuringiensis had been described until 1977, and all that subspecies were venomous to larvae of order Lepidopteran larvae at that time. The finding of other subspecies toxic to Diptera (Goldberg & Margalit, 1977) and Coleoptera (Krieg et al., 1983) expanded the host range and astonishingly amplified the number of subspecies. Greater than 67 subspecies based on flagellar H-serovars had been recognized up to the end of 1998,

Genetics of ICP

In the early 1980s, it was recognized that most genes coding for the ICPs exist on large plasmids which can be transmitted to other organism. The majority of which are readily replaced between strains by the process of conjugation (González & Carlton, 1980; González et al., 1981). After these early studies, most of the ICP genes were cloned and arranged in an order to create the strains of those B. thuringiensis who were having novel insecticidal spectra (Höfte & Whiteley, 1989).

Currently known ICPs that are encoded by crystal (cry) gene types are specific. Furthermore, that encoded ICPs are particular to either Diptera and Lepidoptera (cryII), Lepidoptera (cryI), Diptera (cryIV), Coleoptera (cryIII), or Coleoptera and Lepidoptera (cryV) (Höfte & Whiteley, 1989). All ICPs illustrated so far do violence to the insect gut when these ICPs are ingested. Each one of the proteolytically stimulated ICP molecules has a variable C-terminal domain with insecticidal activity. This C-teminal domain is responsible for receptrecognition (host susceptibility), and a conserved N-terminal domain, which encourages formation of pore (toxicity) (Li et al., 1991).

Naturally occurring B. thuringiensis strains generally contain ICPs which are active against a single order of insects. However, new strains with various plasmid contents can be formed by conjugative transfer between B. thuringiensis strains or related species (González & Carlton, 1980). Thus the complex and diverse activity spectra observed in B. thuringiensis can be explained by cry gene's mobility and plasmid switch over (Jarrett & Stephenson, 1990; González & Carlton, 1980; Reddy et al., 1987; González et al., 1982; González et al., 1981). By conjugation new B. thuringiensis strains have been developed that is toxic to two insect orders.

Nutritional status of B. thuringiensis

Availability of nutrients is the factor affecting sporulation and germination in bacilli (Hardwick & Foster, 1952), in order to delineate the mechanisms regulating spore and parasporal crystal formation it is important to study nutritional requirement of

B. thuringiensis var. thuringiensis. Growth, sporulation and crystal formation of B. thuringiensis var. thuringiensis are supported by certain amino acids, while others restrain the growth (Singer et al., 1966; Singer & Rogoff, 1968; Bulla et al., 1975; Nickerson & Bulla, 1975; Rajalakshmi & Shethna, 1977). Growth, sporulation and crystal formation in Î’. thuringiensis is promoted by a lower concentration of cystine (Nickerson & Bulla, 1975) or cysteine (Rajalakshmi & Shethna, 1977), while only vegetative growth was observed at a elevated concentration of cys/cysSH (Rajalakshmi & Shethna, 1977).

Classification of B. thuringiensis

In early 1960s, B. thuringiensis subspecies' classification, which was based on flagella (H) antigens' serological analysis, was introduced (de Barjac & Bonnefoi, 1962). Biochemical and morphological criteria has further supplemented this classification (de Barjac, 1981). By serological techniques, many strains of B. thuringiensis have been isolated and classified within more than 20 different varieties. These varieties have been further categorized into five pathotypes on the basis of their effectiveness or potency for insects (Hoftey & Whiteley, 1989):

Lepidopteran-Specific (e.g. B. thuringiensis .var Kurstaki)

Dipteran-Specific (e.g. B. thuringiensis . var israelensis)

Coleopteran-Specific (e.g. B. thuringiensis .var. tenebrionis)

Those active against Lepidoptera and Dipter(e.g. B. thuringiensis . var. aizawai)

Those with no toxicity recorded in insects (e.g. B. thuringiensis . var. Dakota)

Mode of Action

Schnepf et al., (1998) have reviewed the ICP structure and function in detail. Most important determinant of ICP specificity is the binding of the ICP to recognized receptors. And the development of pores in the epithelial cells of the midgut is a foremost mechanism of toxicity (Van Frankenhuyzen, 1993). The crystal is dissolved in the alkaline gut of the insect after ingestion of B. thuringiensis by insect. After that the digestive enzymes being present in insect's body disintegrate the crystal structure and trigger B. thuringiensis's insecticidal component, namely the delta-endotoxin (Swadner, 1994). The delta-endotoxin is attached to the cells of the midgut membrane and pores are created in the membrane. Formation of pores disturbs the ion balance of the gut. The insect soon stops feeding and is starved to death (Gill et al., 1992).

If the insect is not inclined to the direct action of the delta-endotoxin, death occurs after B. thuringiensis starts vegetative growth inside the gut of insect. When the gut membrane is broken, spore starts to germinate and reproduce and makes more spores. This body-wide infection ultimately kills the insect (Entwistle, 1993).

Target Organisms

The classification of B. thuringiensis Cry toxins was done in the past decade on the basis of the targeted pest attacked by them (Hofte & Whiteley, 1998); however, as a result of dual toxic activity shown by some cry genes and the inconsistencies in the original proposed classification by Höfte and Whiteley (1989), Crickmore et al., (1998) suggested a modification of the nomenclature for insecticidal crystal proteins, based on the capability of a crystal protein to show some experimentally demonstrable toxic effect in a target organism (Crickmore et al., 1998; Höfte & Whiteley, 1989). Almost 70 serotypes and the 92 subspecies described to date describe the diversity of B. thuringiensis (Galan-Wong et al., 2006).

It is an established fact that many insects are vulnerable to the toxic activity of

B. thuringiensis; they include, lepidopterans which have been remarkably well studied, and activity is shown against them by many toxins (Jarret & Stephens., 1990; Sefinejad et al., 2008). A bulk of susceptible species belonging to agriculturally important families such as Cossidae, Gelechiidae, Lymantriidae, Noctuidae, Pieridae, Pyralidae, Thaumetopoetidae, Tortricidae, and Yponomeutidae belong to order Lepidoptera (Iriarte & Caballero, 2001).

Genome of B. thuringiensis

B. thuringiensis has a circular chromosome and it has GC-content of approximately 32%~35%. Genome size of B. thuringiensis is between 5.2-5.8 Megabases. It has many plasmids and B. thuringiensis strains shelter a miscellaneous variety of plasmids that differ in number and in size (2-200kb) (Thomas et al., 2000). Plasmids which do self-replication are important to the organism's heredity and lifestyle. The largest class of genes consists of the "δ-endotoxins" (from the Cry proteins), these genes are produced in the insect gut under high pH as 130-140 kDa protein precursors to yield a 60-65 kDa biologically active core toxin (Cherif et al., 2007).

General patterns of use:

The major commercial uses of B. thuringiensis generally aims against pest of agricultural and forest crops belonging to order lepidoptera; however, in current years strains active against pests belonging to order Coleoptera have also been marketed (Tomlin, 1997). In public health programmes strains of B. thuringiensis kurstaki that are active against Dipteran insects which are vectors of parasitic disease organisms have been employed (Tomlin, 1997).

Applications in agriculture and forestry

After B. thuringiensis became available in France for about 30 years it is used commercially on agricultural and forest crops (Van Frankenhuyzen, 1993). The commercial use of B. thuringiensis has been increased to a large extent in recent year and it is now commercially produced by the companies. The number of these companies which produces B. thuringiensis products has been increased from four to at least eighteen since 1980 (Van Frankenhuyzen, 1993). Employing conventional spraying technology several commercial B. thuringiensis products including

B. thuringiensis aizawai, B. thuringiensis kuehniella or B. thuringiensis tenebrionise have been applied to crops. Different types of formulations have been used on major crops such as maize, cotton, soybeans, tomatoes, potatoes, various crop trees and stored grains. The range of these formulations is from ultralow-volume oil to high-volume, wettable powder and aqueous suspensions (Tomlin, 1997). Naturally occurring B. thuringiensis strains have been used mainly, but B. thuringiensis toxins expressed by transgenic microorganism using the technique of conjugation and genetic manipulation have reached the commercial market in some cases (Carlton et al., 1990). The reason for developing these modified organisms is to increase host range, lengthen field activity or improve toxin's deliverance to the required organisms e.g., the transfer of coleopteran-active cryIIIA gene to a lepidopteran-active B. thuringiensis kuehniella (Carlton et al., 1990). A plasmid bearing an ICP gene has been transferred from B. thuringiensis to isolate of Pseudomonas fluorescens which is leaf-colonizing non pathogenic organism. Fixation of the transgenic cells manufactures ICP limited within a membrane which prolongs persistence (Gelernter, 1990).

Applications in vector control

B. thuringiensis Kurstaki are very beneficial for the prevention and control of both blackfiles and mosquitoes and for this reason it has become the integral part of large scale programmes (Chilcott et al., 1983; Lacey et al., 1982; Bernhard & Utz, 1993; Cibulsky & Fusco, 1987; Becker & Margalit, 1993; Car, 1984; Car & de Moor, 1984). B. thuringiensis Kurstaki is very effective in control and prevention of mosquitoes. Its practical application can be observed in different countries like in Germany, between 1981 and 1991 in the Upper Rhine Valley. B. thuringiensis Kurstaki was used to control mosquitoes (Culex and Anopheles species) with the composition of 23 tons in form of wettable powder and 19,000 litters of liquid concentrate (Becker & Margalit, 1993). Further, China has used around 10 tons of B. thuringiensis Kurstaki in recent to control the material vector, Anopheles sinensis.

Resistance of Insect Populations

During the last 15 years several insect populations belonging to a number of different species with different heights of resistance to B. thuringiensis have been obtained by laboratory selection experiments (Schnepf et al., 1998). The species comprise of Cadra cautella, Culex quinquefasciatus, Plodia interpunctella, Chrysomela scripta, Tricholplusia ni, Spodoptera littoralis, Spodoptera exigua, Leptinotarsa decemlineata, Ostrinia nubilalis and Heliothis virescens (Schnepf et al., 1998). The first insect that showed resistance to B. thuringiensis Kurstaki was Indian meal moth, a pest of grain storage areas (Swadner, 1994).

The progression of resistance is quicker in laboratory experiments than under field conditions because of higher selection pressure in the laboratory (Tabashnik, 1991). In the field no indications of insect resistance to B. thuringiensis were noticed, until it was observed in diamondback moth. B. thuringiensis had been frequently used against diamondback moth in crops, as a result resistance to B. thuringiensis was developed in them. Since then, it was observed in the laboratory that resistance has been developed in the Colorado potato beetle, tobacco budworm and other insect species (McGaughey, 1992).

Transgenic Bacillus thuringiensis plants

Transgenic plants are the plants which have their DNA being modified by genetic engineering. B. thuringiensis transgenic plants possess gene from B. thuringiensis. This gene helps these plants to produce insecticide within their cells. Agriculture has been revolutionized by transgenic plants expressing insecticidal proteins from the bacterium, Bacillus thuringiensis. B. thuringiensis has become a key insecticide because genes producing B. thuringiensis toxins have been engineered into major crops grown on millions of ha worldwide in 2000 (Shelton et al., 2002).

Transgenic Bacillus thuringiensis plants were intended to control the European corn borer (Ostrinia nubilalis) and other Lepidoptera that feed on corn tissue. The B. thuringiensis toxins produced by transgenic corn are specific to Lepidoptera and kill specifically insects that consume the plant tissue. Owing to this specificity, the impact of B. thuringiensis corn on no target organisms was believed to be insignificant (Orr & Landis, 1997; Pilcher & Rice, 1998). The majority of commercial B. thuringiensis hybrids express the endotoxin in their pollen to varying degrees and thus may pose a risks to non target Lepidoptera consuming pollen deposited on their host plants (Losey et al., 1999; Jesse & Obrycki, 2000).

Ecological Impacts of B. thuringiensis

It has also been keenly observed that the use of B. thuringiensis at a very large scale as a pest control technique also affects some pests those are beneficial for animal in addition to those for which these techniques are to be applied. Almost all types of B. thuringiensis products can have adverse impacts on organisms other than those for which these products are applied for and in turn, it has bad impacts on those animals that are dependent on such organisms (Swadner, 1994).

Effect on Beneficial insects:

Most of the insects does not falls in the category of pests, therefore, pest management techniques are required to be carefully designed because it can harm those insets that are beneficial, as these insects prey and feed on pest species (Swadner, 1994). Various numbers of beneficial species can be affected by the applications of B. thuringiensis. For Example, wasp, a meal moth's parasite (Plodia interpunctella), generated less numbers of eggs when treated with B. thuringiensis (Salama, 1993). Lessening in the hatchability and Production of eggs has also been monitored in a predatory bug (Salama, 1991).

Other insects:

There are numbers of insects that are very important and beneficial for ecosystem's proper functioning and its structure but these insects are not directly beneficially important to agriculture. Furthermore, numbers of studies have shown that many applications of B. thuringiensis can adversely impacts on communities of insect (Swadner, 1994). Large scale application of B. thuringiensis to kill larvae of gypsy moth in Lane county, Oregon resulted in the reduction of number of caterpillars which feed on oak. The spraying of B. thuringiensis was done for three years and the numbers of caterpillars were continued to decline for two years (Miller, 1990).

Birds:

Many birds feed on caterpillars and insects that are affected by the application of B. thuringiensis. So unsurprisingly different impacts on birds have also been recognized (Swadner, 1994). It was observed in the New Hampshire that when the number of caterpillar was reduced due to B. thuringiensis treatment, nesting attempts of black-throated blue warbler were also fewer and they bought smaller numbers of caterpillars to their nestlings. (Rodenhouse, 1992).

Effects on Humans

Different effects on humans were observed in different researches. In one research, 1 gram (3 Ã- 109 spores/g of powder) of a B. thuringiensis kuehniella formulation was ingested by eight human volunteers daily for 5 days. Five out of these eight volunteers also inhaled 100 mg of the B. thuringiensis kuehniella powder daily for five days. Comprehensive medical examinations immediately before, after and 4 to 5 weeks later, were unsuccessful to reveal any undesirable health effects, and all the blood chemistry and urinalysis tests also showed negative result (Fisher & Rosner, 1959).

It was reported by Pivovarov et al., (1977) that intake of foods contaminated with

different concentrations of B. thuringiensis gastroenteitis caused vomiting, nausea, tenesmus, colic-like pains in the abdomen, and fever in most of the volunteers studied. The lethality of the B. thuringiensis gastroenteritis strain may have been caused by production of beta-exotoxin (Ray, 1990).

B. thuringiensis produces proteins and some of these proteins in their purified form are intensely poisonous to mammals. However, such toxicity of commonly-used B. thuringiensis varieties, in its natural form, is limited to mosquito larvae, caterpillars and beetle larvae (Swadner, 1994).

Special Concerns about B. thuringiensis Toxicity

The earliest tests done regarding B. thuringiensis's toxicity were conducted using B. thuringiensis var. thuringiensis, a B. thuringiensis strain that is known for having a second toxin called beta-exotoxin (Swadner, 1994). The beta-exotoxin is toxic to vertebrates, with an LD 50 (median lethal dose; the dose that kills 50 percent of a population of test animals) of 13-18 milligrams per kilogram of body weight (mg/kg) in mice when injected into the abdomen. When an oral dose of 200 mg/kg per day was given to the mice, it killed that mice after eight days (Swadner, 1994). Genetic damage to human blood cell is also caused by Beta-exotoxin (Meretoja, 1977).

CHAPTER 5

DISCUSSION

Bacteria are unicellular prokaryotes that are few millimeter in length. These bacteria are present in extensive range of shapes from spirals to spheres and rods. Bacteria are present in all kinds of habitats including soil, air, hot springs, deepest earth's crust and also in water. There are so many types of bacteria present on earth. Some of them are pathogens, some are mutualists and some are predators. Two types of bacteria are present, Gram positive bacteria and Gram negative bacteria. B. thuringiensis is one of the Gram positive spore forming bacteria which is present in every locality. This bacterium does not cause any harms to human and other animals except some insects. The spores formed by these bacteria are known as Crystal Proteins. These proteins are toxic for some insect which are pests of different crops. Due to this unique characteristic this bacterium is used as bioinsecticide. An extensive research work has been performed on the bacterium B. thuringiensis, involving aspects ranging from its molecular biology to its activity in a bioinsecticide. Many scientists are working on the isolation of new strains of B. thuringiensis with the aim of finding strains with new host range and increased toxicity against a specific pest or pests. The interesting arrangement of its genetic content and the high diversity of toxins derived from it, makes this bacterium a unique organism.

Insect pests are major limiting factors in successful crop production (Boulter et al., 1989). uncontrolled use of chemical pesticides has resulted in irreparable damage to environment. Continuous use of chemical insecticides has led to the emergence and spread of resistance in agricultural pests and vectors of human diseases (Georghiou, 1990). Of all the microbial agents, Bacillus thuringiensis has been successfully used as a biocontrol agent. Because of its ability to produce environmentally friendly crystal proteins (ICPs), B. thuringiensis has been used extensively as microbial insecticide. B. thuringiensis have different types of Cry toxins which are specific for specific order of insects, such as Lepidoptera, Dipteral, Coleoptera. Ingestion of these Cry toxins causes death of insect larvae. These crystal proteins are formed at the time of sporulation. B. thuringiensis has diverse kinds of habitats. B. thuringiensis isolates can be isolated from these diverse habitats and can be preserved for further formulation. These isolate can be used further to observe their toxicity against different insects.

Therefore the present research work is based on the search for the presence of B. thuringiensis and isolation and preservation of different isolates of B. thuringiensis from City Jhelum. Different media were applied for the isolation of different strains of B. thuringiensis from variety of habitats. The result of isolation showed that the B. thuringiensis is present in almost every habitat e.g., cow dung, soil, wheat dust, dust and bird droppings. B. thuringiensis is widely distributed in environment, since samples of soil, stored product material, insect and their habitat and the leaves of certain deciduous and coniferous trees have been found rich in B. thuringiensis (Theunis et al., 1998; Smith & Couche, 1991).

Hundred different samples from dust, cow dung, bird droppings soil and wheat dust were processed for the isolation of B. thuringiensis. LB medium is enrich medium for the growth of B. thuringiensis. One hundred µl volumes of the heat treated samples were spread on nutrient agar plates and incubated (Lee et al., 1995). Subsequent heat shock of 1 ml aliquot of the broth at 80°C for 10 min (Akiba and Katoh, 1986) eliminates all vegetative forms. Bacillus isolates were selected on the basis of their close resemblance and likeness with B. thuringiensis. Colony shapes including margins, surface, color and elevation confirmed the presence of B. thuringiensis. For further verification, gram staining and Spore staining was performed. All the Bacilli strains appeared purple with Gram staining process and green with Spore staining, hence confirmed the presence of B. thuringiensis.

The SDS-PAGE protein components of B. thuringiensis isolates were examined using SDS-PAGE. Six best isolates were selected on the basis of production of Crystal Protein. From these isolates Crystal Protein were extracted and loaded on 10% SDS-PAGE. High molecular weight marker in 1st well contained proteins band of the range 68KDa- 53KDa and compared the protein profile of isolates with HMW Marker (Figure: 27). Samples from same source showed similar protein profile while protein profiles of various B. thuringiensis isolates showed its diversity in natural populations.

One important and rapid method for identifying B. thuringiensis isolates is biochemical tests (Martin et al., 1985). Two types of biochemical tests were performed for the confirmation of B. thuringiensis in the isolated bacilli. LB media mixed with starch was used for the starch hydrolysis test. Clearing zone around the growth of all 77 isolated Bacilli confirmed their potential to release enzymes required for starch hydrolysis. Broth media which was supplemented with 7% Nacl was inoculated with isolated strains of B. thuringiensis. Turbidity in the broth confirmed the growth of bacteria. To divide the B. thuringiensis isolates into biochemical types, starch utilization, lecithinase production and acid formation from salicin_and sucrose was performed (Martin et al., 1985).

Highest numbers of samples which show the growth of B. thuringiensis were samples of cow dung. Also the highest number of isolates was found in cow dung. 95% of samples of bird droppings showed the growth of B. thuringiensis. Wheat dust, soil and dust showed 70%, 65% and 55% respectively (Table 2). According to their colony morphology, 5 types of isolates (A, B, C, D, and E) were observed (Table 6). All the samples of cowdung showed growth of B. thuringiensis colonies. 95% of sample of bird droppings showed the growth. While wheat dust, soil, and dust showed 70%, 65% and 55% respectively (Table 2). Soil and dust are the primary source of B. thuringiensis (Martin & Travers, 1989). Theunis et al., (1993) have reported the grain dust to be the richest source of B. thuringiensis.

Percentage of isolates that were present in different samples was, 25.71% in cow dungs, 15.24% in dust, 18.10% in soil 23.81%in bird droppings and 17.14% in wheat dust. (Table 7) Isolate 'B' & 'C' was -ive in cow dung. Isolate 'E' was -ive in bird droppings and soil, 'A' & 'C' were -ive in dust while 'B' was -ive in wheat dust (Table 6). This result showed that samples of cow dung showed greater number of isolates. B. thuringiensis bacteria were abundant in soils contaminated with such animal by-products (Obiedat et al., 2000)

B. thuringiensis subspecies are used to control insect pests and these subspecies can occur naturally or the can be introduced to ecosystem. (Andrews et al., 1987; Stahly, D et al., 1991). When different samples from various locations of Pakistan were studied for relative abundance of B. thuringiensis, the soil samples were found to be the richest source (Khan et al., 1995). However, the isolation of B. thuringiensis from soil is variably successful with rates ranging from 3-85% (Martin & Traverse, 1982) and from 22-50% (Chilcott et al., 1991). According to Ohba and Aizawa, (1986) certain

B. thuringiensis strains appear to accumulate in the environments where insects are abundant and/or breeding, as higher concentrations of B. thuringiensis were found in samples taken from grain dusts (DeLucca et al., 1981), animal feed mill residues (Meadows et al., 1992) as compared to the soil samples (Ohba & Aizawa 1986). Numerous B. thuringiensis subspecies have been recovered from coniferous trees, deciduous trees and vegetables, as well as from other herbs (Smith & Couche 1991; Damgaard et al., 1997).

The obtained results confirmed that different isolates of B. thuringiensis are present in different localities of City Jhelum and the soil contaminated with cow dung showed greater number of isolates (Obiedat et al., 2000). Unlike this study, Hongyu et al. (2000) and Bernhard et al. (1997) reported that B. thuringiensis is more abundant in stored product environments than soil. Isolates of B. thuringiensis can be isolated from all areas of City Jhelum. Soil and Cow dung are the main sources of B. thuringiensis (Martin & Travers 1989; Obiedat et al., 2007). This study also showed that isolates could be changed in different areas and different types of samples.

CONCLUSION

The present research was done on isolation of B. thuringiensis from different localities of City Jhelum. B. thuringiensis was isolated in greater number from different habitats such as soil, cow dung, bird dropping and dust. It was clearly shown in the result that all the samples from different areas contain different isolates of B. thuringiensis. Overall five types of isolates were observed and their percentages in different samples deviate from each other. This shows that different isolates of B. thuringiensis are present in different localities of City Jhelum. These isolates could be utilized for production of bioinsecticides, aiming to reduce the use of chemical insecticides.

RECOMMENDATIONS

B. thuringiensis may be very useful for the control of insect pests of agricultural crops and forests.

It may be used against different insect species to check against which insect it is more active.

Isolated strains of B. thuringiensis and their toxins can be used to make effective formulations against agriculturally important insect pests.

B. thuringiensis can be used in the development of transgenic insect resistance plants.

It can be used for genomic studies in order to obtain new strains.

Further studies can be done on cloning and characterization of different cry genes from these new isolates of B. thuringiensis that will be useful to use in integrated pest management for sustainable agriculture.