Molecular identification and characterization of Bacillus thuringiensis strains.

摘要

Bacillus thuringiensis Berliner ("Bt") is a species of Gram positive, spore-forming, ubiquitous soil bacteria. Its entomopathogenic property was first identified by Ishiwata in 1901 and it has been used as a biological pest control agent with several varieties targeting insects different orders. Bacillus thuringiensis subsp. kurstaki (Btk) particularly has been most commonly used in commercial formulations as it is effective against lepidopteran larvae. To examine differences among commercial strains and environmental isolates of Btk, molecular, biochemical and morphological methods were used to compare two commercial Btk products (Dipelo RTM and JavelinRTM WG), and two Btk type strains (HD-1 and HD-73) along with 17 Btk environmental isolates. The original goal was to develop molecular markers to discriminate JavelinRTM WG from the other Btk strains by comparing the genetic polymorphisms generated by RAPD primers and identify strain-specific amplicons that were excised, cloned, sequenced and primer sets were designed for those sequences. Three primers sets identified the Btk and as a field application they were used to confirm the presence of Btk from an agricultural farm with a history of commercial Btk usage. The primer sets detected Btk in 63% of samples tested. The subspecies-specific primer sets presented here, have several applications including their use in study of Bt persistence and dispersal through various living systems. Extensive testing revealed that one specific preparation, Javelin technical powder contained at least three different strains of Bt plus Bacillus sphaericus Neide. In an effort to determine the active ingredient(s) of the Javelin technical powder, we examined and compared cry genes using the general primers for cry1, cry2, cry3, cry4 and cry6 and compared them to cry genes exhibited by 197 environmental isolates collected from soils across 22 states in the U.S.A. The most abundant of the cry genes was found to be cry4 (30%) followed by cry1 (15%); these percentages may be higher if some or all of the PCR-negative reactions were possibly due to cell-lysis failure.;The following two manuscripts and an additional chapter summarize my thesis research project. The first manuscript, entitled 'Molecular markers to determine ecological fate of Bacillus thuringiensis subsp. kurstaki,' is written in the format for publication in Molecular Ecology Notes. Supplemental data relating to this first portion of the study are provided after the manuscript. The second manuscript, entitled 'A more complete characterization of Bacillus thuringiensis isolates from USA soils,' is formatted for publication in Applied and Environmental Microbiology. The chapter titled 'Characterization of Javelin technical powder,' provides a more complete understanding of the components of one particular commercial preparation, Javelin technical powder.