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  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.

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