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

Abstract

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 ...

Detection of pine needle diseases caused by Dothistroma septosporum, Dothistroma pini and Lecanosticta acicola using different methodologies

“…Weak amplification of the marker gene below the detection limit by qPCR further indicated the presence of the pathogens. The detection limit was set at 25 copy numbers per reaction, allowing for robust repeatability in ecological studies (Schneider, Hendriksen, Melin, Lundström, & Sundh, 2015;Schneider et al, 2017). In theory, one copy of the target gene present in the reaction volume can be detected by qPCR.…”

The abundance and mineral‐weathering effectiveness of Bacillus strains in the altered rocks and the soil

“…). Although species‐ or strain‐specific PCR primers were designed for the identification of Bacillus subtilis , B. amyloliquefaciens , B. cereus , and B. thuringiensis in various environments , this was the study where qPCR and culture‐based methods were applied to Bacillus members living on the altered rock surfaces and in the soil. The high abundance of Bacillus members on the altered rock surfaces and in the soil was confirmed by using qPCR and culture‐based methods (Fig.…”

Do Multi-year Applications of Bacillus thuringiensis subsp. israelensis for Control of Mosquito Larvae Affect the Abundance of B. cereus Group Populations in Riparian Wetland Soils?

“…Soil was collected once every month, in May (17)(18)(19)(20), June (18)(19), July (17)(18), August (16)(17), and October (22)(23)(24)(25) 2013. Five soil cores of 2.5 cm diameter and 10 cm depth were taken evenly distributed within 1 m 2 at each subsite.…”

“…We recently developed a cultivation-independent qPCR tool targeting chromosomal DNA that allows for detection and quantification of Bcg and Bti in soil samples [16]. In this study, we investigated whether long-term Bti applications affected overall Bti-and Bcg-abundances in riverine wet meadows and alder swamps, using these tools.…”