I. Introduction:

II. Materials:

• 4 small flower pots (4" diameter), paper or foam coffee cups, or disposable beakers filled with a potting soil or vermiculite
• 4 plastic bags in which the above containers will fit easily
• 2 forceps
• 1 dropper bottle containing 0.1 M glucose
• 1 dropper bottle containing 0.012 g of Ornalin® (active ingredient, vincolzolin, Ornalin 50WP; Grace-Sierra Hort. Products Co., Milpitas, CA) in 0.1 M glucose
• 24 sunflower seeds (Helianthus annuum cv. Sunspot; G.W. Park Seed Co, Greenwood, SC 29647)
• 2 plates of potato dextrose agar
• 1 strain of Botrytis cinerea known to be resistant to dicarboximides
• 1 strain of Botrytis cinerea known to be sensitive to dicarboximides

Cautionary Notes: Anyone wishing such strains of Botrytis cinerea can obtain them from the author at the address above. If outside Pennsylvania, an Application to Move Live Plant Pests must be filed with your state department of agriculture and a permit to move this fungus obtained from the U.S.D.A. (see attached). The permit must be sent to Moorman so it can be attached to the shipping container. This process generally requires 6-8 weeks.

III. Methods

• Pre-Class Preparation: At the time of the exercise, the plants should have cotyledons and first true leaves just emerging. Therefore, 7-10 days prior to the exercise:
  1. Moisten the potting mix in the containers enough so that seeds will germinate.
  2. Plant six sunflower seeds in each container about 1/2" deep and put the containers in a warm, bright location out of direct sunlight.
  3. Inoculate 1 potato dextrose agar plate with the Botrytis cinerea strain designated RESISTANT and a second plant with the SENSITIVE strain.
• The Test:
  1. Be certain the containers are well watered. Then, put them in separate plastic bags.
  2. Put 3-4 tablespoons full of water in the bottom of each of the plastic bags outside the containers. This will maintain the necessary high humidity in the bag and is REQUIRED.
  3. To each of the seedlings in 2 containers, apply a large drop of 0.1 M glucose from one dropper bottle to the top of the growing point of each seedling. The cotyledons and first true leaves will hold the drop there (E in the illustration). If the drop rolls off, put another one on.
  4. To each of the seedlings in the other 2 containers, apply a large drop of fungicide from the second dropper bottle to the top of the growing point of each seedling.
  5. Using the forceps, remove small pieces of the SENSITIVE strain from the petri plate and place on piece directly in the drop of liquid on each seedling treated with 0.1 M glucose (F in the illustration).
  6. Similarly, put a piece on each seedling in one of the containers of fungicide-treated seedlings.
  7. Repeat 5 and 6, inoculating the remaining seedlings with the RESISTANT strain.
  8. Pull the top of each plastic bag up and close them at the top with a twist-tie, leaving as much room for the plants as possible (F in the illustration). MAKE CERTAIN YOU PUT SOME WATER IN THE BOTTOM OF THE BAG TO MAINTAIN HIGH HUMIDITY DURING THE TEST.
  9. Place the beakers back in a warm, bright location, but out of direct sunlight. Periodically check the plants without opening the bags. The actual time required will depend on the temperature and light conditions where the plants are left, but usually take 5-10 days.
  10. Record how many of the plants browned or died as a result of Botrytis infection.

IV. Interpreting Results: If most of the seedlings in both bags inoculated with a given strain of Botrytis become infected and die, the strain is resistant to dicarboximides. If most of the glucose-treated seedlings become infected and died, but the fungicide-treated seedlings remained healthy, the strain's not resistant to dicarboximides.

V. References: