Pyrenopeziza brassicae conidiospore production protocol

You will need:

Sterile mortars and pestles

Sterile distilled water

Sterile 1 ml Gilson tips

9cm 3% MA (malt agar) plates: [30 g l -1 malt extract (Oxoid) 12 g l -1 Difco Bacto agar]

Procedure:

Scrape a small amount of mycelium from the edge of an actively growing P. brassicae culture and place in a sterile mortar.

Add 3 ml sterile distilled water and grind mycelium into a paste.

Aliquot 1 ml of the macerate onto 9 cm 3% MA plates and rotate to ensure that all the agar is covered by the macerated mycelium.

Incubate plates at 16 °C in the dark. After 2-3 weeks conidiospore production should be evident as creamy-white blobs on the surface of the culture (often easier to see using a binocular microscope).

Conidiospores can be collected by flooding the plates with 3-5 ml sterile distilled water and agitating the mycelium with a sterile glass spreader. The resulting spore suspension can be collected with a Gilson pipette.

NOTE – as an alternative to mortars & pestles, micro-homogenisers (from Anachem / Eppendorf) can be used to macerate mycelium in 1.5ml micro-tubes (with 500µl water). Make this up to 1ml with water before spreading out on a plate.