Bremia lactucae storage:

We store Bremia in two ways:

A. Long and Intermediate storage –80 C:

1: Maintain your inoculum in seven days old Cobham green seedlings

2: Separate the spores seven to eight days after inoculation by removing the filter paper or subtract

from the magenta box

3: Add 50 ml of cold dd water to seedling with spores and shake them to remove spore to water.

4: Filter suspension using cheesecloth. Transfer to 50 ml centrifuge tube.

5: Centrifuge suspension at 1000 RPM for 5 minutes (low temperature)

6: Discard supernatant to 5 to 10 ml. Resuspend spores in water by gently shaking tube.

7: Aliquot suspension in 1ml eppendorf tubes and centrifuge again to obtain a pellet.

8: Label tubes and store them at –20 C for a day or 2 and then move them to –80 C

B. Intermediate and short storage –20 C:

After seedlings show heavy sporulation pick filter paper with seedlings and spores and transfer to a tall petri dish. Seal plate with electric tape and store at –20 C.

To rescue isolates from any source from the freezer let the plate or the tube thaw for few minutes. Cut 7 to 8 days old cotyledons and plate them up side down in a petri dish with moisture (mist plates). Do about 20 per plate.

Micropipete small amounts of suspension on the top of each cotyledon.

In the case of plates with sporulating seedlings add 1 to 2 ml of water and micropipete after washing the spores from the tissue.