Studies on preparation and viability of Phytophthora parasitica spheroplasts

Spheroplasts were obtained from Phytophthora parasitica mycelium by digestion with Novozym 234 using 035 m CaCl 2 (pH 5.7) as initial osmotic stabilizer. Spheroplast yield decreased rapidly with increasing age of the mycelium. Young mycelium gave highest yields of 9.3±4.5 × 10 6 cells per Petri dish. Regeneration was investigated on synthetic and complex media in the presence of different osmotic stabilizers. Survival on solid and in liquid media was enhanced in the presence of CaCl 2 in addition to mannitol as osmotic stabilizer. When spheroplasts were spread on agar containing 0.1 M CaCl 2 plus 0.4 M mannitol as osmotica the regeneration rate was 12±4 %. After embedding in soft agarose stabilized with 0.8 M mannitol spheroplast regeneration was 19±6%. Incubation in osmotically stabilized liquid medium resulted in a rapid loss of viability. High concentrations of CaCl 2 as osmotic stabilizer caused abnormal growth of spheroplasts which eventually gave rise to normal mycelium.