e Bacillus thuringiensis serovar israelensis is a wide-spread soil bacterium affiliated with the B. cereus group (Bcg) and is widely used in biocontrol products applied against mosquito and black fly larvae. For monitoring and quantification of applied B. thuringiensis serovar israelensis and its effect on indigenous B. thuringiensis serovar israelensis and Bcg assemblages, efficient and reliable tools are essential. The abundance and properties of B. thuringiensis serovar israelensis strains in the environment traditionally have been investigated with cultivation-dependent techniques, which are hampered by low sensitivity and the morphological similarity between B. cereus and B. 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 of B. thuringiensis serovar 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 for B. thuringiensis serovar israelensis and Bcg. The method will be useful for comparisons of Bcg and B. thuringiensis serovar israelensis abundances in the same samples. Moreover, the effect of B. thuringiensis serovar 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 of B. thuringiensis serovar israelensis in the environment.
The bacterium Bacillus thuringiensis (Bt) is one of the grand successes in microbial pest control (1). After its first discovery in 1901 and description by Berliner in 1911, the first biocontrol product based on Bt, Sporein, for control of the European corn borer (Ostrinia nubilalis), was commercially available already in 1938 (1). The crystalline, proteinaceous ␦-endotoxins of Bt are formed during sporulation, and their lethal effect is manifested only after larval ingestion of spores or free crystals (2, 3). Bt is a member of the Bacillus cereus group (Bcg), which consists of the Gram-positive rod-shaped spore-forming bacterial species B. cereus, Bt, B. anthracis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis (4,5). However, a multidatatype phylogenetic analysis of Bcg strains isolated from various sample types, including environmental, clinical, food, and diary sources, did not reflect current Bcg taxonomy well. This finding indicated that the phylogeny of this bacterial group is more complicated than reflected by the division into the current six species (6).The bacterium B. thuringiensis serovar israelensis produces toxins active against nematoceran larvae and has been widely used for biocontrol of mosquitoes and black flies (3). B. thuringiensis serovar israelensis was described in 1976 (7) and has been shown to have ...