Protoplast preparation and regeneration from spores of the biocontrol fungus Pseudozyma flocculosa

This paper describes a specific protocol for yielding and regenerating prot oplasts from spores of the recently described powdery mildew biocontrol age nt Pseudozyma flocculosa. With this protocol, a large quantity of protoplas ts was obtained from beta-mercaptoethanol-pretreated spores in 3-day-old cu ltures of P. flocculosa grown in YMPD. Enzymatic digestion was optimal with 0.5% Novozym 234 and 5% Glucanex prepared in 0.6 M KCl in 0.1 M citrate bu ffer. All liberated protoplasts fluoresced in the presence of fluorescein d iacetate indicating that viability was nearly 100%. The regeneration rate w as equally outstanding reaching 75% when 0.8 M sucrose was used as osmotic stabilizer in the regeneration medium. This protocol will find useful appli cations in genetic studies of this poorly characterized and understood bioc ontrol agent. (C) 2000 Federation of European Microbiological Societies. Pu blished by Elsevier Science B.V. All rights reserved.