Molecular characterization of a new dual Bacillus thuringiensis strain.

BERÓN, C.M.; GROSSI DE SÁ, M. F.; SALERNO, G. L.

Goiânia, Goiás, Brasil

Congreso; IV Latin - American Meeting on Plant Biotechnology; 2001

Bacillus thuringiensis is a spore forming bacterium well-known for its insecticidal properties and its ability to produce a crystal inclusion during sporulation. The specific activity of B. thuringiensis against insect larvae is due to the crystal proteins (Cry proteins). The boll weevil, Anthonomus grandis, is a major pest of cotton grown in USA, and in some countries from South America. The damage is important because adults feed on fruiting forms, leaf petioles and terminal growth. Females oviposit in squares and bolls, and the squares containing eggs are aborted. Infested bolls often fail to open properly and may be subject to boll rot. Adult feeding on leaf petioles in young, presquaring cotton causes "black flags", wilted leaves on the damaged petioles. Chemical control for this pest is expensive and harmful for the environment. A novel strain of B. thuringiensis, isolated from Córdoba (Argentina) a soil sample was found to be toxic to the larvae of lepidoptera (Anticarsia gemmatallis and Spodoptera frugiperda) and coleoptera (Diabrotica speciosa, Tenebrio mollitor and Anthonomus grandis). This strain (named FBt 7) has a rounded crystal protein inclusion and its SDS-PAGE polypeptide pattern showed the presence of two major bands at 50 and 135 kDa. In immunoblotting analysis, the polypeptides of FBt 7 were not recognized by anti-Btt (antibodies against the 66-KDa polypeptide of B. thuringiensis serovar tenebrionis) or anti-Btk (antibodies agaisnt the130-KDa polypeptide of B. thuringiensis serovar. kurstaki). A strategy based on PCR amplification of DNA fragments from FBt 7 homologous to cry genes with degenerated oligonucleotides, followed by Tail-PCR methodology was used to clone the genes. BLASTp analysis using the deduced amino-acid sequence of one of the Cry protein from the FBt 7 strain, indicated that it is related to proteins of the Cry 8AA type. This result was confirmed by a dendogram generated with the Clustal W method with the PAM 250 residue weight table and representative members of Cry proteins present in public data bases. The dual novel strain FBt 7 constitutes a very promising tool in the control of cotton pests.