Characterization of Bacillus thuringiensis isolates by their insecticidal activity and their production of Cry and Vip3 proteins

Bacillus thuringiensis (Bt) constitutes the active ingredient of many successful bioinsecticides used in agriculture. In the present study, the genetic diversity and toxicity of Bt isolates was investigated by characterization of native isolates originating from soil, fig leaves and fruits from a Turkish collection. Among a total of 80 Bt isolates, 18 of them were found carrying a vip3 gene (in 23% of total), which were further selected. Insecticidal activity of spore/crystal mixtures and their supernatants showed that some of the Bt isolates had significantly more toxicity against some lepidopteran species than the HD1 reference strain. Five isolates were analyzed by LC-MS/MS to determine the Cry protein composition of their crystals. The results identified the Cry1Ac protein and a Cry2A-type protein in all isolates, Cry1Ea in 3 of them and Cry1Aa in one. The sequence analysis of the new vip3 genes showed that they had a high similarity to either vip3Aa, vip3Af or vip3Ag (94-100%). The vip3Aa gene of the 6A Bt isolate was cloned and sequenced. The protein was named Vip3Aa65 by the Bacillus thuringiensis Nomenclature Committee. The expressed and purified Vip3Aa65 protein was tested against five lepidopteran species and its toxicity compared to that of a reference protein (Vip3Aa16). Both proteins had similar toxicity against Grapholita molesta and Helicoverpa armigera, whereas Vip3Aa65 was less active than Vip3Aa16 against three species from the Spodoptera genus. A tetrameric structure of the Vip3Aa65 protein was detected by gel filtration chromatography. The study revealed some isolates with high insecticidal activity which can be considered promising candidates to be used in pest control.