Submitted to: Journal of Applied & Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/13/1996
Publication Date: N/A
Interpretive Summary: The Colorado potato beetle is a major pest of potatoes, tomatoes, and eggplant. In North America, the beetle is often infected with host-specific, gut-inhabiting spiroplasma (a helical bacterium in the class Mollicutes). This spiroplasma may be useful in biocontrol if it can add and express an insect-lethal gene. Difficulty in cultivation of the organism, however, has hindered development of a suitable transformation system. In the current study, we focused primarily on improving methods for isolation and culture of the spiroplasma. Systems with low oxygen were found to be ideal, resulting in more effective isolation, at lowered cost. Use of these strategies permitted the first cultivation of the spiroplasma on solid media, which will facilitate selection of lethal spiroplasma. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America. This information should be of use to those scientists developing genetically engineered spiroplasmas for biocontrol of agricultural pests, or those isolating other mollicutes (such as mycoplasmas) associated with human, veterinary, and plant diseases.
Technical Abstract: In North America, the Colorado potato beetle (CPB), Leptinotarsa decemlineata, is often infected with a host-specific, gut inhabiting spiroplasma (CPBS). CPBS is apparently a commensal, but may be useful in biocontrol if it can be transformed to express an insect-lethal gene. Difficulty in cultivation of the organism, however, has hindered development of a suitable transformation system. In this study, we focused primarily on eliminating the need for co-culturing CPBS with insect cells. CPBS was reliably isolated using BBL GasPak (Anaerobic) Jar systems (low redox, enhanced CO2), which are easier to use and less expensive than cell cultures. Use of anaerobiosis should also permit early passage screening of isolates for extrachromosomal elements, for use in gene vector constructs. The unique spiral (decreasing amplitude of coils) morphology of CPBS was preserved by anaerobiosis. Use of low pH media allowed aerobic adaptation of CPBS to M1D and SP-4 broth media. Use of these formulations permitted the first cultivation of CPBS on solid media, which will facilitate selection of molecular transformants. Potato beetles collected at four sites in Poland yielded CPBS strains similar to those previously obtained from populations in North America.