Anthracnose Leaf Blight Assay

Maize plants are typically grown the V-4 stage (about 3 weeks old) in Cone-tainers (small narrow cone shaped pots).

Plant Inoculation Assay

Potting the plants

1. Corn seeds are planted in soil that is 2:2:1 College Station field soil: Metro Mix soil: (soil from the bag)
2. Plants are grown for 21-25 days in the green house where they should be watered every other day with a solution of water and Peter’s fertilizer (20:20:20).

Preparation of inoculum

1. Colletotrichum graminicola is grown on Petri dishes of Potato Dextrose Agar (PDA) at room temperature under fluorescent lights. The cultures should be 14-21 days old.

2. Place 8-10 ml of sterile distilled water on the surface of the Petri dish.

3. Scrape the surface of the media with an inoculating loop to release the conidia from the hyphae. Be careful not lift too much mycelia by scraping too hard.

4. Filter the suspension (water + spores) into a 50 ml tube using a funnel and cheesecloth that have been sterilized. This should trap any mycelia that were accidentally lifted.

5. Rinse the spores from the dish one more time with an additional 8-10 ml of water and filter through the funnel again.
Fill the tube to approx 40 mL with sterile distilled water and mix.

6. Spin at 3000 rpm for 3 min to pellet the conidia in the eppendorf centrifuge in room 305. Adaptors for the centrifuge are in the lab. Be sure to balance the centrifuge.

7. After centrifuging the tube, pour off the supernatant and fill the tube again. Vortex or shake to resuspend the conidia.

8. Spin again for 3 min.

9. Pour the supernatant out and refill with water to approximately 30 ml.

10. Count with a hemacytometer. (see hemacytometer directions)

11. Make the spore dilutions (with ddH2O) and add tween 20. (We use a spore concentration of 1 X 105 spores/ml. For every 40 ml of conidia solution, add 10 μl of a 1:100 Tween 20 solution.

* From the moment that the conidia is collected off the PDA culture to the moment the plants are inoculated no more that 2 ½ hours should elapse. When the conidia are in solution too long and not on the plant, less conidia will germinate.

Inoculating plants

1. Cover the bottom of a 31″ tray with paper towels (we have them as kitchen rolls).

2. Lay the plants down so that the top of the 3rd and 4th leaves are available for inoculation.

3. Tape the leaves down onto the paper towels. Make sure the leaves are taped gently so it won´t  be difficult to lift the plants after inoculation.

4. Place 10 ul drops all along the top side of the leaves. Be careful not to place any drop along the mid vein of the leaf.

5. After the drops are on the leaves, draw a small arrow next to each drop for identification once the drop has dried.

6. Wet the bottom of the tray very careful so as not to disturb the drops on the leaves. Only add enough water to wet the towels, but water should not puddle because it may roll onto the leaves and carry away the drops of inoculum.

7. Seal the tray with GLAD Pressâ™n Seal â„¢ plastic. The objective is to create a sort of moisture chamber.

8. Allow the plants to remain sealed in the tray for at least 24 hours. Extra time may be needed depending on the purpose of the experiment. Open (take away the seal) and wait until inoculating drops dry (6-12 hrs) then un-tape the plants and put on trays. After 24 hours, remove the plastic and wait several hours for the droplets to dry before returning the plants to an upgright position. Symptoms are usually visible in 48-72 hours, depending on the corn line.

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