Isolation of Iranian Bacillus thuringiensis strains and characterization of lepidopteran-active cry genes

Maryam Rashki,Pariya Soltani Nezhad,Mahmood Maleki,Masoud Torkzadeh-Mahani,Shahryar Shakeri

doi:10.1186/s41938-021-00432-3

Maryam Rashki, Pariya Soltani Nezhad + Show 3 more

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https://doi.org/10.1186/s41938-021-00432-3

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Abstract

BackgroundInsecticidal crystal proteins (encoded by cry genes) produced by Bacillus thuringiensis (Bt) are fatal for insects of different orders such as Lepidoptera. The genes that encoded these crystal proteins can be detected on plasmids and chromosomal DNA and show different types in various strains. Therefore, the objective of this study was to determine molecular characteristics of Iranian Bt strains as well as their toxicity against Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae).ResultsThe collection sites included fields, gardens, and desert and semi-desert areas in 8 provinces of Iran. For crystal formation, each isolate was cultured in T3 medium. The results showed that the Bt isolates produced different types of crystals including spherical (73.33%), bipyramidal (53.33%), irregular (40%), cubical (33.33%), and elliptical (13.33%). Plasmid DNA extraction was performed and showed that most of the strains exhibited similar pattern in the number and the size of the plasmid bands to those of Bt kurstaki (Btk). Some specific primers were used for PCR amplification to distinguish different crystal genes including cry1 (A, C, and D) and cry2A (a and b). The primers related to cry1D and cry2Aa1 genes produced no amplicons. The results revealed that the most abundant gene was cry1-type. All strains analyzed for the cry2Ab2 gene presented unexpected bands. Electrophoretic profile of the protein crystals showed bands with different diversity in number, and size ranged from about 16 to 140 KDa. The bioassay result of some more toxic strains exhibited that the pathogenicity of 1019 was higher than the rest, even the reference strain, Btk. However, the toxicity of other strains was the same as Btk. According to the phylogenetic tree, 1019 was located in the same group with Bt subspecies coreanensis, Bt subspecies indiana and Bt subspecies tolworthi.ConclusionsThe investigated Iranian strains had the lepidopteran-active cry genes. The strains with the same toxicity to E. kuehniella had various cry genes, plasmid, and crystal protein profiles and vice versa. Therefore, characterization of cry genes of native strains could lead to access potent isolates as biocontrol agents against native insect pests.

Highlights

IntroductionInsecticidal crystal proteins (encoded by cry genes) produced by Bacillus thuringiensis (Bt) are fatal for insects of different orders such as Lepidoptera
Insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are fatal for insects of different orders such as Lepidoptera
Cry7A and cry1B were found in Bt subspecies galleriae (Btg) (Nazarian et al, 2009)

Summary

Introduction

Insecticidal crystal proteins (encoded by cry genes) produced by Bacillus thuringiensis (Bt) are fatal for insects of different orders such as Lepidoptera. The genes that encoded these crystal proteins can be detected on plasmids and chromosomal DNA and show different types in various strains. Insecticidal crystal proteins (encoded by cry genes) produced by a gram-positive bacterium, Bacillus thuringiensis (Bt) (Nazarian et al, 2009) are fatal for insects of different orders such as Lepidoptera, Coleoptera, and Diptera (Aronson and Shai, 2001). The genes that encoded these crystal proteins can be detected on plasmids and chromosomal DNA (Höfte and Whiteley 1989) and show different types in various strains. Cry7A and cry1B were found in Bt subspecies galleriae (Btg) (Nazarian et al, 2009)

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