Spore germination assay

1. Colletotrichum graminicola is grown on Petri dishes of Potato Dextrose Agar (PDA) at room temperature under fluorescent lights. The cultures should be 12-15 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. 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 104 spores/mL for the slides assay and 1 x 105 for the in planta assay. For every 40 mL of conidia solution add 10 uL 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.


Assay in 100 uL drops in the botton of Petri dishes (60×15)

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