Role of chitinases in bacterial insect pathogens
Chitinases have been proposed to play a key role in the virulence of microbial insect pathogens. The chitinolytic properties of the biopesticides Bacillus thuringiensis and Bacillus sphaericus were investigated using in vitro enzyme assays and in vivo bioassay studies. It was observed that when a suspension of 'colloidal' chitin was added to a solution of Calcofluor White M2R there was a significant enhancement of fluorescence observed. Using this observation a plate-reader chitinase assay was developed and the activities of a range of B.thuringiensis strains were measured. A similar fluorescent effect was observed with a glycol chitosan suspension and a chitosanase assay was developed. The chitosanase enzyme properties of B.thuringiensis strains were also characterised. More detailed analysis of the chitin and chitosan degrading properties of three selected strains of B. thuringiensis were performed using enzyme assays and SDS-PAGE activity gels. To investigate the role of chitinases in B.thuringiensis, bioassay studies were developed using 4th instar larvae of the biting midge C.nubeculosus and newly hatched caterpillars of the cotton leaf worm S.littoralis. Samples of B.thuringiensis were added at a range of concentrations along with additions of either an exogenous chitinase preparation or the specific chitinase inhibitor allosamidin. The results showed the addition of chitinase to enhance the insecticidal activity and addition of allosamidin to inhibit insecticidal activity of B.thuringiensis. Collectively, the data shows chitinase and chitosanase production to be widespread among different subspecies and pathotypes of B.thuringiensis and also, shows chitinases to play an important role in the virulence of B.thuringiensis.