Frozen stock of infected tissue (stored at -80°C or as transported on dry ice)
Spray gun (e.g., Preval paint sprayer available from Alltech Associates)
A flat containing -100 10-17-day-old seedlings with the first set of true leaves expanded are best. The seedlings must be well watered and placed in a container with drenched filter paper, or other absorbent material, underneath them.
Appropriate growth chamber (see Step 4) Procedure
1. Allow the infected tissue samples to thaw gradually on ice for 1-3 hours. Add 10 ml of distilled water to the tube, and vortex vigorously for 30-60 seconds. Remove any residual pieces of plant tissue that may later block the spray nozzle.
2. Store the resulting spore suspension on ice until plants are ready to be inoculated.
3. Immediately before spraying, vortex the suspension again. Inoculate by spraying the entire spore suspension onto the flat of seedlings.
Placing a dome behind the flat as a backdrop for the sprayer helps to keep the spore-mist from settling outside the flat. If the number of viable spores is low, several rounds of infection may be necessary to obtain sufficient spores for experiments.
4. Cover the seedlings with a clear dome that has been sprayed inside with a fine mist of water. Incubate in a growth chamber on a 12hour light cycle at 19°C and a minimum of 80% humidity.
Conidiophores should begin to appear on the leaves 5 days after inoculation. Conidiophore density peaks between 7 and 9 days after infection.
5. After 7 days, assess the level of infection (see below, Rating Per-onospora Infections). If the level of infection is low (i.e., a rating of <3), collect 15 ml of infected tissue and repeat Steps 3 and 4 on a fresh set of seedlings. If infection is sufficiently well established (i.e., a rating of 3 or more), collect batches of 15 ml of infected tissue, add 10 ml of chilled distilled water, and vortex well (1-2 minutes). These suspensions can be used immediately for further infections or kept at -80°C as frozen stock suspensions (see below, Preparation of Frozen Tissue Stocks).
Generally, one well-infected flat is enough to provide two to three 10-ml spore suspensions for maintenance and experimental infections with enough left over for frozen stock.
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