An evaluation of methods for obtaining mycelium-free oospores of Pythium aphanidermatum and P. myriotylum

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Abstract
Sonication of aqueous suspensions containing mycelial fragments with or without oospores of Pythium aphanidermatum or P. myriotylum at 20% of the maximum intensity of a Biosonic III ultrasonic system for periods in excess of 100 s, or at 40,60, or 80% of maximum intensity for 20 s or longer resulted in suspensions free of viable mycelial fragments. Oospore germination was not affected after sonication at rates as high as 80% of maximum intensity for periods lasting as long as 60 s. In comparison with untreated controls, oospore germination of p. aphanidermatum or P. myriotylum was not affected by any of the following methods that were used for the preparation of oospore suspensions free of viable mycelial fragments: filtering or sonicating suspensions, treating suspensions with a cellulose–hemicellulase solution, or with a solution of commercial snail enzymes. Germination of P. aphanidermatum oospores was reduced by 78% and no germination of p. myriotylum oospores occurred after freezing the suspensions. Feeding mycelial mats to live water snails (Physa fontinalis) did not affect oospore germination of p. aphanider matum but increased germination of p. myriotylum oospores by 17% in vitro. At 25 oospores per gram of soil, only 7% of the tomato seedlings were infected after growth in soil infested with oospores of p. aphanidermatum that had been frozen, whereas 70–80% of the seedlings were infected after growth in soil infested at the same inoculum density with oospores exposed to the other treatments. No infection of rye seedlings occurred in soil infested with p. myriotylum oospores that had been frozen. A significantly higher level of infection of rye seedlings occurred with oospores of p. myriotylum that had been ingested by Physa fontinalis than with oospores that had been treated by the other methods. No infection occurred when tomato and rye seedlings were exposed to soil infested with mycelial fragments of 21-day-old cultures of p. aphanidermatum or P. myriotylum, respectively.

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