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.
Highlights
Bacillus thuringiensis (Bt) is a gram positive entomopathogenic bacterium widely used in biological control against various economically important pests
Eighty isolates in a Bt collection were screened for the presence of vip3 genes by PCR using screening primers (Table 1)
For the spore/crystal fraction, a preliminary screening at a single dose allowed us to select those isolates with apparently higher insecticidal activity than the HD1 strain, which were further tested in dose-response assays (Table 2)
Summary
Bacillus thuringiensis (Bt) is a gram positive entomopathogenic bacterium widely used in biological control against various economically important pests. Bt products are used in pest management because they are highly specific to the target organism and harmless to non-target organisms and the environment [1, 2]. Because of their effectiveness to control insect pests, they represent the 90% of the bioinsecticide market [3]. 001 to JF), and by European FEDER funds, and by a grant (15/082 to HG) from BAP of Muğla Sıtkı Kocman University, Turkey. 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
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