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

Abstract

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.

FIG 1

Phylogenetic tree built on NCBI accession numbers with 100% similarity to sequence homology groups formed of soil clones obtained with Bcg1_for/rev primers from targets amplified from soil DNA. Representative sequences from each accession number at subspecies, species, and genus level for each of the sequence homology groups, as well as from different homology groups among clone sequences (SHG1 Bcg to SHG4 Bcg; KP863940 to KP863943), were included. The tree was constructed using maximum-likelihood analysis based on the Jukes-Cantor model of Bacillus cereus group 1 (Bcg) sequences from Bcg bacteria with 1,000 iterations of bootstrapping.

FIG 2

Quantitative real-time PCR analyses for determining potential PCR inhibition by soil DNA extracts. Extracts corresponding to 1, 5, 10, 15, or 20 ng of soil DNA were added to PCR mixtures containing 10⁶ copies of the recombinant plasmid pCR4-Topo (Invitrogen). The dashed line indicates the mean C_T of the control without addition of soil DNA. C_T values are the averages from three replicates, and error bars represent standard deviations. Asterisks indicate significant deviations from the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001).