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Bacteria are unicellular prokaryotes that are few millimeter in length. They are present in wide range of shapes from spheres to rods and spirals. 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. There is a great amount of scientific research 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 Âµl 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 80Â°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)
The B. thuringiensis subspecies represents a group of organisms that occur naturally and can be added to an ecosystem to achieve insect control (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.
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.
B. thuringiensis may be safely used 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.