Submitted to: Journal of Sugarbeet Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/14/2001
Publication Date: N/A
Interpretive Summary: Fungal pathogens cause the most serious disease problems of sugarbeet in the U.S. Use of molecular gene technologies has revolutionized the ability to determine the chemicals that fungi use in order to cause disease in plants. A fundamental aspect of these technologies is the ability to form "naked" cells of the fungus--that is, those that possess no cell walls. This enables the researcher to insert foreign DNA into the fungus, often in a way that creates defined mutants of the fungus. If the mutants are no longer able to infect the plant, the research often can readily find the gene that is mutated using these techniques. This report characterizes for the first time the ability to form wall- less cells of a major root and seedling pathogen of sugarbeet, the beet water mold fungus (Aphanomyces cochlioides). The wall-less cells were culture in a solution that then allowed them to re-form back into the fungus. A prerequisite for success of the technique also was met, as it was found that the regenerated fungus retained the ability to infect sugarbeet plants. The results pave the way for gene technologies to be applied to pathogenesis studies with this fungus.
Technical Abstract: Conditions were tested for the digestion of cultured Aphanomyces cochlioides and A. euteiches mycelia to promote the formation of protoplasts. Similar to protoplasting conditions for other oomycetes, 0.1% of cell wall-digesting enzymes in osmoticum were sufficient to convert the mycelia to protoplasts within 2 hr. Protoplast integrity was maintained upon embedding in molten agar containing 1M mannitol. Within 4 days post-plating on potato dextrose agar, 10-20% of the embedded protoplasts of both fungal species formed germ tubes that subsequently formed mycelial colonies. Fungal isolates derived from regenerated protoplasts of A. cochlioides and A. euteiches retained the ability to induce black root disease in sugarbeet seedlings and water soaking in pea seedlings, respectively. The generalized protocol for production and regeneration of protoplasts for Aphanomyces species may be of use in the development of a gene transfer protocol for this fungal genus.