A potent Vip3Aa94 protein from Thai Bacillus thuringiensis: molecular characterization and insecticidal potential

Vegetative insecticidal proteins (Vip) are produced by Bacillus thuringiensis during its vegetative growth stage. Vip3 proteins play a significant role in the insecticidal activity of B. thuringiensis against a wide spectrum of economically important crop pests. In this study, 163 B. thuringiensis strains were screened for the presence of vip3 using a polymerase chain reaction-based approach. Seventeen (10.43%) strains yielded an amplification product for vip3. Ten of these strains exhibited larvicidal activity against second-instar larvae of Spodoptera litura, Spodoptera exigua, and Spodoptera frugiperda, with corrected mortality rate ranging from 18.30% to 100%. JC20 exhibited the highest efficacy against the three Spodoptera species, achieving corrected mortality rates of 98.30% for S. litura and both 100% for S. exigua and S. frugiperda. Given its potency, JC20 was further characterized to identify its unique features and to assess its potential as a bioinsecticide. Scanning electron microscopy revealed the presence of bipyramidal crystal proteins in JC20, correlating with this strain harboring cry1D and cry2A. The vip3 gene of JC20 was cloned and sequenced. The complete 2,370 bp sequence was subsequently named Vip3Aa94 by the Bacterial Pesticidal Protein Database. The size of Vip3Aa94, determined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 90 kDa. Broad-spectrum efficacy for Vip3Aa94 was established against the second-instar of S. frugiperda, S. exigua, and S. litura, with low LC₅₀ values of 67.38, 81.97, and 147.92 ng/cm², respectively. Phylogenetic analysis based on the Vip3Aa94 sequence indicated that geographic separation led to the divergence of vip3, consistent with its distinct features compared with other B. thuringiensis strains globally. This study highlights JC20, with its high insecticidal activity, as a promising candidate for controlling Spodoptera pests.