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

PLoS One. 2018 Nov 1;13(11):e0206813. doi: 10.1371/journal.pone.0206813. eCollection 2018.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacillus thuringiensis / genetics
  • Bacillus thuringiensis / isolation & purification
  • Bacillus thuringiensis / pathogenicity*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / toxicity*
  • Bacterial Toxins / genetics
  • Bacterial Toxins / isolation & purification
  • Bacterial Toxins / toxicity*
  • Ficus / microbiology
  • Fruit / microbiology
  • Moths / microbiology*
  • Moths / pathogenicity
  • Pest Control, Biological / methods*
  • Plant Diseases / parasitology
  • Plant Diseases / prevention & control
  • Plant Leaves / microbiology
  • Soil Microbiology
  • Turkey

Substances

  • Bacterial Proteins
  • Bacterial Toxins

Grants and funding

This research was supported by the Spanish Ministry of Economy and Competitivity (AGL2015-70584-C02-1-R to JF), by grants from the Generalitat Valenciana (GVPROMETEOII-2015-001 to JF), and by European FEDER funds, and by a grant (15/082 to HG) from BAP of Muğla Sıtkı Koçman University, Turkey. JGC was recipient of a PhD grant from the Spanish Ministry of Economy and Competitivity (grant ref. BES-2013-065848). BS was supported 2214/A Scholarship Program of The Scientific and Technological Research Council of Turkey (Doctorate level). The proteomic analysis was performed in the proteomics facility of SCSIE University of Valencia that belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001 of the PE I+D+i 2013‐2016, funded by ISCIII and FEDER funds.