Bacillus thuringiensis (B. t.) strains are important microorganism because they produced a large amount of δ-endotoxin protein per bacterial cell and their toxins are highly toxic to Lepidoptera, Coleoptera, and Diptera depending on B. t. To date, more than a hundred Cry proteins have been identified and classified into 195 holotypes, based on the amino acid sequence identity. The Cry proteins or cry genes from the Korean native B. t. isolates in this study were not identified yet. The electrospray ionization of quadrupole time of flight mass spectrometry (ESI Q-TOF MS) was used to get the internal amino acid sequences of the endotoxin-spore culture mixtures of B. t. isolates, for which polymerase chain reaction (PCR) techniques were unable to detect the cognate genes. Most of Cry proteins seperated, excized, and extracted from the one dimensional - polyacrylamide gel electrophoresis (1D-PAGE), instead of 2D-PAGE, were matched with protein databases using MS-MASCOT search program. The internal amino acid sequences which were submitted to protein BLAST (basic local alignment search tool) had partially homology with the Cry protein databases. Hence, present data strongly suggest that the de novo amino acid sequencing and ESI Q-TOF/MS analysis along with MASCOT search could be used as a simple and rapid method for detection of novel Cry toxins from B. t. isolates and identification of B. t. isolates.

A B. thuringiensis kurstaki was first discovered by H. Dulmage in 1970 and commercialized as a DipelTM due to powerful toxicity to various Lepidoptera. Previously we isolated B. thuringiensis kurstaki on the basis of plasmid DNA profiling and H-antigen serotyping. The aims of this study were to screen larvicidal activities and select the highly active B. thuringiensis isolates against the important polyphagous pests of mandarine oranges and vegetables. The colony forming unit (CFU/ml) of each of culture mixtures was determined to estimate the δ-endotoxin concentration. The bioassay against artificial diet-rearing insects was conducted by surface contamination methods using the Petri dishes specially designed and manufactured by SPL Lifesciences. The insecticidal activities to the natural diet-rearing insect were measured by the application of spore and crystal mixtures to the leaf discs of the chinese cabbage with Potter spray tower. The following insects were used for the larvicidal activities of B. thuringiensis isolates: beet armyworm Spodoptera exigua, diamondback moth Plutella xylostella, giant looper Ascotis selenaria, tobacco cutworm Spodoptera litura, and variegated cutworm Peridroma sucia.