Molecular characterization of Bacilllus thuringiensis strains with differential toxicity to Chilo partellus

Abstract

Bacillus thuringiensis (Bt) is one of more than 20 species of soil growing Bacilli, it is an ubiquitous gram positive, aerobic, spore- forming organism that forms parasporal crystals during the stationary phase of its growth cycle. Bt was initially characterized as an insect pathogen, and its insecticidal activity was attributed largely or completely depending on the insect and the parasporal crystals. This study addressed the need to characterize three Bt isolates with varying levels of toxicity to C. partellus using molecular characterization techniques. The intention was to establish the basis for differential toxicity to C. partellus and to determine if the unique bands and other properties can be used to screen other isolates. Growth rate of Bt isolates were determined by inoculating the isolates in separate flasks containing (Luria Bertani) LB broth then incubated at 37 ºC while shaking at 200 rpm. The absorbance at 600nm was then read at intervals of every 6 hrs from 0 hrs to 72 hrs. 1M isolates had a slightly elevated growth rate at the lag phase and exponential phase of growth than both K10-2 and V24-M isolates, although at the plateau phase the growth seems to be equal for all the three isolates. Smirnoff staining protocol was used to detect the level of crystal and spore formation over a period of 72 hrs. All crystals were bi-pyrimidal in shape and stained with an almost black luster with lilac blue tint. The spores stained pink, whereas bacterial cells and their fragments assumed a light lilac tint. In the process of Bt isolate growth; secreted protein concentrations were determined using the Lowry assay and absorbance taken at 750nm. The three Bt isolates showed varying levels of protein concentration with 1M isolate showing a significantly higher concentration than both V24-M and K10-2. Analysis of protein profiles using SDS- PAGE for the Bt isolates at time intervals of 54 hrs, 60 hrs and 72 hrs were performed respectively. The major protein bands in this study were of molecular weights, 28kDA, 65kDA and 130kDA in all the isolates except isolates 1M that showed two protein bands of molecular weight 28kDA and 65kDA. Restrictions of plasmid DNA of the Bt isolates reveal unique bands in all the different isolates when digested with EcoRI, BamHI and HindIII. One particular unique band that was characteristic to all the digests were one of molecular weight ranging between 7,000 to 7,200bp. This was a unique band that was characteristic of all the toxic Bt strains and hence a crucial indicator of effective toxicity among Bt isolates in this study. In conclusion growth rates, protein determination and restriction digestions on their own did not reveal much information on which Bt isolates that are most toxic. However when the techniques are used collectively they have proven to be effective tools in the characterization of different Bt isolates hence should be incorporated instead of lengthy and expensive bioassays