| Bremia lactucae storage:
We store Bremia in two ways:
- Maintain the isolate on universally susceptible lettuce seedlings e.g. cv. Cobham Green
Intermediate and short term storage -20 ºC:
- After seedlings show heavy sporulation, approximately 7 to 10 days after inoculation,
place filter paper with sporulating seedlings into a tall petri dish. Seal plate with tape and store at -20 ºC.
Long and Intermediate term storage -80 ºC:
- Harvest the spores seven to eight days after inoculation.
- Remove the filter paper or extract sporulating seedlings from Magenta box.
- Add 50 ml of cold dd water to seedlings and shake to suspend spores in water.
- Filter suspension using cheesecloth to remove plant debris.
- Transfer to 50 ml centrifuge tube.
- Centrifuge spore suspension at 1,000 RPM for 5 minutes at ~5 ºC.
- Discard supernatant leaving 5 to 10 ml.
- Resuspend spores by gently shaking tube.
- Aliquot suspension into 1 ml Eppendorf tubes and centrifuge again to obtain pellet.
- Label tubes and freeze them at -20 ºC for 1 to 2 days and then move to -80 ºC.
Store as pelleted spores; do not resuspend before freezing.
To rescue isolates:
- Thaw the plate or the tube for few minutes on ice.
- Cut 7 to 8 day old cotyledons and place them upside down in a petri dish (~20 per plate) that has been moistened by misting.
- In the case of plates with sporulating seedlings, add 1 to 2 ml of water and flush the spores from the tissue.
- Micropipete small amounts of suspension on the top of each cotyledon.