Q-244. Recovery of Bacillus thuringiensis and Insect Toxic Related strains from Forest Soil

P. A. W. Martin, D. E. Gundersen-Rindal, M. B. Blackburn;
USDA, Beltsville, MD.

Bacillus thuringiensis has been applied for gypsy moth control in forests in the Northeastern US for many years. The subspecies of B. thuringiensis that is used (kurstaki) is not common in the US soil. We attempted to recover B. thuringiensis from soil that had been sprayed two years prior with B. thuringiensis for gypsy moth control. By amplifying the bacteria found in the soil on bacterial agar and feeding this diverse microbial population to tobacco hornworm larvae, an insect that is very sensitive to B. thuringiensis, we were able to further improve on traditional recovery methods. Out of 20 soil samples, 15 were found to have bacteria that killed these larvae. These strains were identified to species by their 16S rDNA sequences. Phylogenetic relationships among new strains compared to the B. thuringiensis strain applied will be presented. Most of the bacteria formed spores (14/15) and 12 belonged to the Bacillus cereus group including 2 B. thuringiensis. The other two sporeformers were identified as B. weihenstephanensis and Lysinobacillus fusiformis. All 15 strains were phenotypically distinguishable from one another, as well as from the B. thuringiensis strain that was applied, by both substrate utilization and antibiotic resistance. However, the phenotypic profiles of these 15 strains were similar to the B. thuringiensis strain that was applied in their ability to produce acid from glucose (14/15), but not from mannitol, (14/15), mannose (13/15) and sucrose (13/15). Phenotypic tests that were variable include the production of urease (9/15), the hydrolysis of esculin (11/15), and acid production from salicin (9/15) and cellobiose (2/15). The toxicity to gypsy moth larvae of the two B. thuringiensis strains isolated from the soil samples was also similar to the applied B. thuringiensis strain. Thus, amplification of bacteria present in soil, in combination with a sensitive insect, can recover insect toxic strains that are related to an applied strain.