Abstract
Bacillus thuringiensis (Berliner) (Bt) based pesticides has become the greatest successes among the microbial pesticides. This research was focused on the isolation and characterization of Bt from chicken manure samples as a comprehensive effort to contribute to the establishment of native Bt strains from various types of habitats in Malaysia. Initially, selection was done mainly based on colony morphology, Gram staining and phase contrast microscopy. Total of 85 colonies were identified and isolated based on colony morphology, in which off-white, matte and creamy appearance showed the characteristics of the genus Bacillus. The number reduced to 35 colonies after Gram staining was done. Subsequently, total of 8 isolates were confirmed to be B. thuringiensisafter Coomassie Brilliant Blue staining was carried out. The presence of B. thuringiensisstrain was confirmed when a blue cap-like structure and a darkly stained oval structure, insecticidal crystal proteins (ICP) appeared in the sporulation phase. An isolate was chosen out of the 8 isolates in order to generate the growth curve of B. thuringiensisbacteria. The similarity of the aspects above confirms that the selected isolate was a Bt strain. Isolates were further characterized by SDS-PAGE analysis to determine the molecular masses of the Bt proteins. These isolates produced two major ranges of polypeptides, the Cry protein which corresponds to a range of 70-150kDa and the Cyt protein profile which corresponds to the range of 18-40kDa. The protein profile obtained correlates the strains to the typical Bt strain. A total of 6 isolate yielded SDS-PAGE products that demonstrated major protein bands of approximately 135kDa and 140kDa, possibly encoded by the genes cryI and cryIV, respectively. Key words: Bacillus thuringiensis, chicken manure, protein analysis.
Highlights
Bacillus thuringiensis (Bt) is a naturally occurring bacterium common in soils throughout the world and belongs to the group Bacillus cereus sensu lato (Huang et al, 2001)
This research was focused on the isolation and characterization of Bt from chicken manure samples as a comprehensive effort to contribute to the establishment of native Bt strains from various types of habitats in Malaysia
All the 8 isolates were subjected to Coomassie Brilliant Blue staining technique in which the technique allows high-throughput evaluation of the presence of crystals (Rampersad et al, 2002)
Summary
Bacillus thuringiensis (Bt) is a naturally occurring bacterium common in soils throughout the world and belongs to the group Bacillus cereus sensu lato (Huang et al, 2001). Cry proteins have been classified according to their insect specificity and nucleotide sequence (Konecka et al, 2006; Ghelardi et al, 2007). Most of the insecticidal activity of Bt is due to intracellular crystal inclusions produced during the process of sporulation. These parasporal crystals are comprised of one or more related ICPs encoded by cry (crystal) and
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