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Abstract:
A comparative study was made of all available milky-disease species and strains that have been isolated around the world from beetle larvae (family Scarabaeidae). Included in the study were Bacillus popilliae Dutky, B. lentimorbus Dutky, and B. lentimorbus var. maryland from the United States; B. euloomarahae Beard and B. lentimorbus var. australis Beard from Australia; B. fribourgensis Wille from Switzerland; and New Zealand milky disease (Dumbleton). The organisms were classified into three groups: (i) those containing parasporal bodies, including B. popilliae Dutky, B. fribourgensis Wille, and New Zealand milky disease (Dumbleton); (ii) those without a visible parasporal body and with spore morphology similar to B. lentimorbus Dutky, including B. lentimorbus var. australis Beard; and (iii) those with very tiny spores and no parasporal body, including B. euloomarahae Beard and B. lentimorbus var. maryland. All available milky-disease species and strains were cultivated in vitro on Brain Heart Infusion Agar plates. However, the most fastidious organisms-B. euloomarahae and B. lentimorbus var. maryland-could not be grown until they were passed through a life cycle in larvae of a large scarabaeid beetle infesting rotting wood. Then they remained stable for only one or two subcultures. All the milky-disease organisms produced larger cells in vitro than they did in vivo. The pattern of sugar fermentations was similar for all milky-disease species. It appears that there is a very low percentage of strains of B. popilliae, B. lentimorbus, and the other milky-disease organisms that have the inherent genetic makeup to permit them to sporulate on artificial media, if conditions are favorable. Among these conditions are a sufficiently high cell population and a reduced oxygen tension. Spores produced in vitro may have a low virulence via the normal ingestion pathway, even though they show apparent virulence when injected directly into the hemocoel.
Insights
This study compared global milky disease species from beetle larvae. Most strains struggled to sporulate in vitro, requiring specific conditions for successful cultivation and showing reduced virulence.
Area of Science:
- Microbiology
- Entomology
- Bacteriology
Background:
- Milky disease affects scarabaeid beetle larvae, caused by various Bacillus species.
- Previous research has isolated numerous strains globally, necessitating a comparative analysis.
Purpose of the Study:
- To comparatively analyze available milky-disease species and strains worldwide.
- To classify these organisms based on morphological and physiological characteristics.
- To assess their in vitro cultivation and sporulation capabilities.
Main Methods:
- Comparative analysis of Bacillus popilliae, B. lentimorbus, and related strains from diverse global locations.
- In vitro cultivation on Brain Heart Infusion Agar.
- Life cycle passage through beetle larvae for fastidious strains.
- Morphological classification based on parasporal bodies and spore size.
- Sugar fermentation pattern analysis.
Main Results:
- Organisms were grouped based on parasporal bodies and spore morphology.
- Fastidious strains (B. euloomarahae, B. lentimorbus var. maryland) required larval passage for in vitro growth and were unstable.
- All strains produced larger cells in vitro than in vivo.
- Sugar fermentation patterns were consistent across species.
- A low percentage of strains sporulated effectively in vitro under optimal conditions (high cell population, low oxygen tension).
- In vitro-produced spores exhibited reduced virulence via ingestion compared to injection.
Conclusions:
- Cultivation and sporulation of milky-disease bacteria in vitro present significant challenges.
- Specific environmental conditions are critical for successful in vitro sporulation.
- In vitro-derived spores may have diminished efficacy for biological control applications through natural infection routes.