A Spray Inoculation Technique to Establish Fungal Infection in Rice Plant Leaves

Use sterile surgical scissors to cut three layers of filter paper into 8-centimeter circles. Place the filter paper circles onto 100-millimeter sterile plastic plates. Add just enough distilled deionized water to each dish to soak the filter paper. Then, place two sterile toothpicks 2 to 3 cm apart in each culture dish.

Two weeks after sowing, collect the leaves of the rice plants by cutting the lower part of the stem. Using a thermostatic incubator, culture the M. grisea strains on OTA plates at 25 degrees Celsius for four days. Then, use a pipette to add 2 milliliters of distilled deionized water to each plate. Using an inoculation loop, scrape the mycelia from the wild-type and mutant strains into the mycelia debris.

Collect the mycelia debris, and transfer it to a new OTA plate. Then, dry the mycelia debris on a clean bench. Add 2 milliliters of distilled deionized water to the cultured mycelium dish, and use a sterile cotton swab to gently scrape the conidia. Filter the conidia suspension through two layers of lens paper, and transfer the suspension to a new 50-milliliter tube. Then, centrifuge the tube for 5 minutes at 5,000 times g.

After centrifugation, remove the supernatant, and resuspend the pellet with Tween 20 solution, resulting in a concentration of 20,000 conidia per milliliter. Pour the suspension into a hand-held sprayer. Then, spray approximately 10 milliliters of the conidial suspension onto the leaves from the two-week-old rice plants. Spray the leaves of the control plants with Tween 20 solution.

After this, incubate the leaves in a dark humid box for 24 hours. Next, transfer the leaves to a moist chamber with fluorescent light for 12 hours.