MONITORING THE INSECTICIDAL TOXINS FROM BACILLUS-THURINGIENSIS IN SOIL WITH FLOW-CYTOMETRY

The accumulation and persistance in soil and other natural habitats of the insecticidal toxins from Bacillus thuringiensis may result in env ironmental hazards, such as toxicity to nontarget species and the sele ction of toxin-resistant target species, We describe the use of flow c ytometry as a method for detecting and tracking the fate of these inse cticidal toxins in soil that does not require their extraction and pur ification. The toxins from B. thuringiensis subspp. tenebrionis and ku rstaki were bound on clay- or silt-sized particles separated from Kitc hawan soil that was unamended (naturally contains predominantly kaolin ite) or amended to 6% v/v with the clay minerals montmorillonite or ka olinite (as an internal control). The particle-toxin mixtures were sus pended in 0.1 M phosphate buffer (pH 7) containing 3% nonfat milk powd er to block nonspecific binding of antibody, resuspended in a solution of antibody to the toxin from B. thuringiensis subsp. tenebrionis, an d then resuspended in a solution of anti-rabbit antibody conjugated wi th fluorescein isothiocyanate (FITC-Ab), Controls consisted of the par ticles alone and bound complexes of the particles with the toxin from B. thuringiensis subsp. kurstaki. All particles that bound the toxin f rom B. thuringiensis subsp. tenebrionis showed a significant shift in the peak of fluorescence to the right on the x axis as compared with t he nonspecific fluorescence from the control FITC-Ab complexes with pa rticles in the absence of the toxin. There was also a slight shift in the peak to the right for some particles that bound the toxin from B. thuringiensis subsp. tenebrionis, as there is some cross-reactivity be tween the toxins from B. thuringiensis subspp. tenebrionis and kurstak i and the antibodies that they induce. This method is more sensitive a nd rapid than the dot-blot ELISA, and processing of many samples is ea sily accomplished.