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Title: AN EFFICIENT METHOD TO ISOLATE INSECT PATHOGENS: MICROBIAL COMBINATORICS

Author
item Martin, Phyllis
item MONGEON, ELIZABETH - ELENAOR ROOSEVELT HS

Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 3/10/2006
Publication Date: 5/20/2006
Citation: Martin, P.A., Mongeon, E.A. 2006. an efficient method to isolate insect pathogens: microbial combinatorics. American Society for Microbiology. Orlando, Florida May 24- 25, 2006 - 106th General Meeting pg. 259.

Interpretive Summary:

Technical Abstract: Very few bacteria have been successfully used to control pest insects. We developed an efficient and cost-effective approach to isolate insect pathogenic bacteria. Analogous to the simultaneous screening of multiple compounds used in combinatorial chemistry, microbes from a single soil were grown on laboratory media and tested as mixed cultures for the ability to kill Manduca sexta L., tobacco hornworm larvae. We call this method combinatorial microbiology or microbial combinatorics. While feeding soil suspensions to hornworm larvae directly killed some hornworms, amplifying the bacteria present in soil by growth on solid or in liquid media decreased the time to kill and increased total mortality to M. sexta larvae. The bacterial mixtures grown from 18 of 21 soils tested killed 100% of the 2nd instar larvae within 96 h. One bacterial mixture killed 100% of the M. sexta larvae in less than 16 h while only one larva died when fed the bacteria from the unamplified soil extract. Furthermore, the bacteria isolated from 80% of the larvae that died from all 18 samples consisted of a predominant colony type. When these bacteria were grown in pure culture and re-tested for toxicity to hornworm larvae, 16 of 18 pure cultures killed M. sexta larvae. Fourteen bacterial strains isolated from this second set of larvae were identical to the bacteria fed the larvae by 14 biochemical tests and 8 antibiotic sensitivities, thus fulfilling Koch’s postulates. This approach resulted in the discovery of 14 confirmed insect pathogens, all of which formed spores. Three spore forming bacteria also formed crystals. One was identical to the Bacillus thuringiensis (Bt) var. kurstaki, which had been sprayed three years earlier for control of gypsy moth in the location sampled. The other two crystal-forming bacteria differed from the Bt that was sprayed by a single biochemical test. Four other spore-forming strains had identical biochemical and antibiotic resistance profiles, to the B. thuringiensis strain that was sprayed, but did not form crystals. This approach to isolating insect pathogens can be used with other insects, as well as having a more general application for rapid screening of mixed cultures for other useful compounds produced by bacteria.