Regeneration of pathogenic Aphanomyces cochlioides and A. euteiches from protoplasts

Enzyme mixtures were tested for the digestion of cultured Aphanomyces cochlioides and A. euteiches mycelia to promote the formation of protoplasts. Cell wall-digesting enzymes at 0.1% (w/v) in osmoticum were sufficient to convert the mycelia to protoplasts within 2 hr, similar to digestion conditions for other oomycetes. Protoplast integrity was maintained upon embedding in molten agar containing 1M mannitol. Within 4 days post-plating on potato dextrose agar, 10 to 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 important crop pathogen.