Chromosome-Directed PCR-Based Detection and Quantification of Bacillus cereus Group Members with Focus on B. thuringiensis Serovar israelensis Active against Nematoceran Larvae

Abstract

ABSTRACTBacillus thuringiensisserovar israelensis is a wide-spread soil bacterium affiliated with theB. cereusgroup (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of appliedB. thuringiensisserovar israelensis and its effect on indigenousB. thuringiensisserovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties ofB. thuringiensisserovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity betweenB. cereusandB. thuringiensis. Currently available PCR-based detection and quantification tools target markers located on plasmids. In this study, a new cultivation-independent PCR-based method for efficient and specific quantification ofB. thuringiensisserovar israelensis and Bcg is presented, utilizing two sets of PCR primers targeting the bacterial chromosome. Sequence database searches and empirical tests performed on target and nontarget species, as well as on bulk soil DNA samples, demonstrated that this diagnostic tool is specific forB. thuringiensisserovar israelensis and Bcg. The method will be useful for comparisons of Bcg andB. thuringiensisserovar israelensis abundances in the same samples. Moreover, the effect ofB. thuringiensisserovar israelensis-based insecticide application on the total Bcg assemblages, including indigenous populations, can be investigated. This type of information is valuable in risk assessment and policy making for use ofB. thuringiensisserovar israelensis in the environment.