Induction of Phytophthora parasitica zoospores with V-8 broth

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Purpose

This procedure was developed specifically for Phytophthora parasitica; however, many isolates do not readily produce zoospores.

To clarify, filter broth through several layers of cheesecloth and dilute broth with an equal volume of distilled water.
Pipette 15 ml of the medium into 250 ml screwcap bottles. Autoclave. Adjust pH after autoclaving to 6.4 to 6.7.
Inoculate each bottle with a 7 mm mycelial disk taken near the edge of a colony of Phytophthora parasitica growing on V 8 juice agar. Incubate the bottles for 24 hrs. at 25°C.
Shake bottles vigorously by hand (20 to 30 short strokes) to fragment the mycelium. Incubate further in a horizontal position at 25°C for 5 days.

Aseptically pour the contents of each bottle into a sterile, 30 to 50 cc screw cap centrifuge tube.
Centrifuge for 10 min. at 3,200 rpm (1610 G).
Decant and add 20 ml sterile distilled water; centrifuge again. Repeat entire process 3 times.
Resuspend the mycelial mat in 10 ml sterile distilled water and pour the entire contents of each tube into a sterile, glass Petri dish.
Aseptically remove the inoculum disk and spread out the mycelial mat. Incubate for 5 days at 25°C. To avoid premature release of zoospores, do not disturb.

Decant water from the mycelial mat and resuspend mycelial mat in 10 ml distilled water; repeat 3 times.
Incubate at 20°C for 15 min. or longer to induce zoospore swarming.
Bring back to room temperature (25°C). Wait 30 min. after initial swarming begins, then pipette zoospore suspension to another vessel and pool zoospores prior to use.

Menyonga, J. M. and P. H. Tsao. 1966. Production of zoospore suspensions of Phytophthora parasitica. Phytopathology 56: 359 360.

From the Virginia Polytechnic Institute and State University Mediabook; Originally created by Robert Wick; contributed by Mary Ann Hansen