Purification and characterization of a bacillus thuringiensis crystal & endotoxin active against chilo partellus and spodoptera exempta

Abstract

Microbial insecticides based on Bacillus thuringiensis (B.t.) strain berliner are valuable for the control of many lepidopteran pests. Li ttle work has been done on the potential of B. t. to control either Chilo partellus or Spodoptera exempta. Many of the identified exotic B. t. strains have been isolated in temperate zones and may not be well suited for use in a tropical country environment. Therefore, there is need to identify and carry out studies on local B.t. strains adapted to the tropical environment. In this study, purification and biochemical characterization of strain MF4B/2 crystal delta endotoxin shown to be active against c. partellusj S. exempta larvae, was carried out. The B. t. strain MF4Bj 2 crystal delta endotoxin was purified by centrifugation in a 40-70% linear sucrose gradient. On separation by SDS-PAGE, the crystal yielded a protein band (Mr -130 KD). Upon solubilization under alkaline pH and reducing conditions, the crystalline toxin yielded a protoxin (Mr -63 KD). The solubilization process did not result in loss of toxicity. The in vitro activation of the protoxin by s. exempta midgut homogenates, bovine pancreas trypsin and Glossina trypsin in each case, did not result in any apparent cleavage of the protoxin. However, in vitro activation by c. partellus midgut homogenate yielded a similar toxin (Mr -60 KO) as chymotrypsin activation. staining of the protoxin separated by SOS-PAGE (4-15%) with periodic acid schiff revealed that the protoxin was glycosylated. The carbohydrate moiety was of the high mannose type as shown by (Li ) staining with fluorescein isothiocyanate conjugated concanavalin-A. Antibodies raised against MF4Bj2 protoxin showed no crossreactivity with B.t. protoxins against Glossina species and Aedes aegypti.