Seleção de estirpes de Bacillus thuringiensis e determinação da atividade de proteína cry tóxicas para importantes lepdopteros da cultura do milho no Brasil

Abstract

This work aimed to select and characterize through bioassays and biochemical and molecular methods, strains of B. thuringiensis which are toxic for Agrotis ipsilon. Selective bioassays were done with 71 strains, selected according to toxic activity against Lepdopteran insects. Some Bt strains caused 100% of mortality to A. ipsilon larvae. These strains belonged to serotypes galleriae (S597), sotto (S615), aizawiai (S616), kurstaki (S1450) described as toxic against Lepdopteran insects. The strains named S906, S234 and S844 caused mortality of 75% to A. ipsilon larvae. Molecular analysis were conduced using the strains S597, S615 and S844, which have no characterization described, while all others strains had biochemical and molecular characterization described. The results from molecular tests obtained with strain S615 identified the presence of genes cry1Ab, cry1Ac and cry2. The strain S844 showed the presence of genes cry2 and cry11. On strain S597 wasn’t identified any gene cry, according to methodology employed. Aiming the identification of individual toxic activity of Cry proteins were conduced bioassays for determination of lethal dosis necessary to causes mortality in 50% of population (LC50) of S. frugiperda larvae. Were used the proteins Cry1Aa, Cry1Ab, Cry1Ac and Cry2A present in B. thuringiensis subspecies kurstaki HD- 1 (Btk). Additionally, experiments to verify the binding of these proteins to membrane cell receptors of S. frugiperda larvae were conduced. Three recombinant strains of B thuringiensis and one wild type strain were utilized at this work to produce of proteins Cry1Aa, Cry1Ab, Cry1Ac and Cry2A. Bioassays with S. frugiperda larvae of second instar showed LC50 of 12.416 ng/cm2, 13.434 ng/cm2, 108.773 ng/cm2 and 164.350 ng/cm2, respectively, evidencing different toxic activity. The results of binding assays done with BBMV (brush border membrane vesicles) of S. frugiperda showed binding with membrane cell receptors of proteins Cry1Aa, Cry1Ab, Cry1Ac and Cry2A marked with biotin.