Bulk production of Pyrenopeziza brassicae inoculum from plant material

This protocol often produces a larger amount of inoculum than the plate method and in general, the inoculum is more infective.

Generate P. brassicae conidia using the plate protocol (from macerated mycelium) and collect the spores as detailed. Adjust the spore suspension to 10 6 spores ml -1 .

Spray oilseed rape plants at the 4-5 leaf stage with the spore suspension until liquid runs from the leaves. Cover the plants with a plastic bag to maintain humidity for 16-24 hours.

Remove the plastic bags for the plants and keep them at 16 °C preferably in a controlled environment facility.

When a large amount of sporulation is evident remove the leaves exhibiting the sporulation and place them in a flask with water. Shake / swirl the flask to allow the spores to go into suspension – leave the leaves in water for approx. 1 hour to achieve this.

Remove the leaves and measure the spore concentration – adjust this to 10 5 spores ml -1 .

The inoculum is now ready to be sprayed on further plants. Alternatively it may be necessary to use this inoculum to inoculate more plants to further increase the inoculum.