Screening of vip genes from a Spanish Bacillus thuringiensis collection and characterization of two Vip3 proteins highly toxic to five lepidopteran crop pests

- Five hundred Bacillus thuringiensis strains from Spain were screened for vip genes.
- Seven and 14.5% potentially novel vip1 and 2, and vip3 were detected, respectively.
- The novel Vip3Aa45 and Vip3Ag4 differed from Vip3Aa1 in specific sequence features.
- Vip3Ag4 was highly toxic to Lobesia botrana, Chrysodeixis chalcites and Spodoptera littoralis.
- Vip3Aa45 was highly toxic to L. botrana, Mamestra brassicae and S. littoralis.

A Spanish Bacillus thuringiensis strain collection was screened for the presence of vip genes. One hundred strains from a Canary Island collection were screened for vip1 and vip2 genes and 7% contained potentially novel vip1 and vip2-like genes, as indicated by the low degree of similarity with previously known vip1 and vip2 genes. Four hundred strains from a collection originating from the Spanish mainland were screened for vip3 genes and 14.5% of them contained potentially novel vip3-like genes. Reconstruction of the full-length vip sequences could only be achieved for two vip3 gene variants encoding 789 and 787 amino acid proteins that were designated as Vip3Aa45 and Vip3Ag4, respectively. These proteins showed 82% pairwise identity between them and differed from Vip3Aa1 in the putative signal peptide, two specific proteolytic processing sites and the 66-kDa insecticidal fragment. The purified proteins were tested against nine lepidopteran pest species and displayed toxicity, expressed as mean lethal concentration, for five of them. The two toxins were highly toxic for Lobesia botrana (∼1-2 μg/ml) and Spodoptera littoralis (∼20 ng/cm2), moderately toxic for Spodoptera exigua (∼100-300 ng/cm2), and varied greatly in their toxicity for Mamestra brassicae or Chrysodeixis chalcites, with high toxicity for Vip3Aa45 in M. brassicae (∼40 ng/cm2) and for Vip3Ag4 in C. chalcites (∼45 ng/cm2).