Abstract

Bacillus thuringiensis strains were isolated from sugarcane farming soil in three provinces of northern Vietnam. Of these, 24 strains were identified as serotype H5a, 5b, B. thuringiensis serovar galleriae. A strain, namely MHB11.3 (deposited in GenBank: FN796428) belonging to the serovar galleriae, was highly toxic to larvae of the scarab beetles, Anomala cuprea and Cotinis nitida (Coleoptera: Scarabaeidae). A 2-kb DNA fragment, obtained by PCR from the strain MHB11.3 using cry8 specific primers, namelycry8Dat (truncated cry8Da gene), was cloned and expressed in Escherichia coli. The sequence of the cry8Dat from B. thuringiensis serovar galleriae MHB11.3 showed almost complete identity (99%) to that of B. thuringiensis serovar galleriae SDS502 (GenBank: AB089299), differs only by two nucleotides (positions A26G; A1493T), leading to 2 amino acid changes (Y9C and N498I). The recombinant protein pET32a(+)-Cry8Dat of 96 kDa molecular weight was purified by Ni⁽²⁺⁾ affinity chromatography. This polypeptide was tested to be toxic to A. cuprea and C. nitida. The amino acid change (Y9C) is located in a region responsible for the host specificity of the Cry8Da toxin. The strain B. thuringiensisserovar galleriae MHB11.3 harboring cry8Da gene as characterized in this study is a promising candidate as a biopesticide and suggested as an important genetic material for gene transfer into sugarcane for scarab beetle control.

Key words: Bacillus thuringiensis serovar galleriae, Coleoptera, cry8Da gene, Anomala cuprea, Cotinis nitida.