Characterisation of lepidopteran-active Bacillus thuringiensis isolates recovered from infected larvae

Abstract

As a part of an ongoing nationwide programme focused on finding novel strains of Bacillus thuringiensis (Bt) that are toxic to some of the major pests that impact economically important crops, we initiated a search for Bt isolates native to Syria. We succeeded in assembling a collection of 40 Bt isolates recovered from infected larvae of Galleria mellonella, Helicoverpa armigera and Ephestia kuehniella. Light microscopy showed that all isolates produce bipyramidal and cuboidal crystal proteins. The 50% lethal concentration of the spore-crystal mixture of the 40 isolates against E. kuehniella larvae varied from 3 to more than 200 µg g⁻¹. A comparison of the LC₅₀ values of the tested isolates with the reference strain Bt kurstaki HD-1 (20.55 µg g⁻¹), showed that some of these isolates have a similar or up to six times higher toxicity potential. PCR screening revealed that all obtained isolates contain cry1 and cry2 genes, whereas only four contain cry9. Moreover, the proteins of 130 and 65/70 Kda encoded by these genes were detected in the SDS-PAGE of the purified parasporal bodies. Flagellar serotyping classified 30 as serovar kurstaki, six isolates serovar aizawai, one isolate cross-reacted with more than one H3 antisera and three were not typeable. Assays of toxicity of the aizawai isolates against third instar of G. mellonella showed that four, which contain cry9, have almost similar toxicity to the commercial strain Bt aizawai B401. Therefore, these isolates could be adopted for future applications to control G. mellonella. Moreover, this study contributes to our knowledge of Bt diversity in Syria where to date very few collections have been described.

Keywords:

• Bacillus thuringiensis
• cry gene
• Galleria mellonella
• Syria

Acknowledgements

The authors would like to thank the Director General of the Atomic Energy Commission of Syria and the head of the Molecular Biology and Biotechnology department for their continuous support throughout this work. A scientific visit for H. Ammouneh to the Lab of biological control of Arbovirus vectors, Wuhan Institute of Virology, China was supported by TWAS-CAS fellowships programme. We also thank Prof. J.W. Mansfield from the Imperial College London, UK for critical reading of the manuscript.