This study investigates the infection process of the rice plant by the pathogenic fungus Magnaporthe grisea. The methodology involves the application of conidia to rice leaves and the subsequent observation of fungal growth and infection severity.
Take a Petri plate with sections of rice plant leaves positioned above a wet filter paper to prevent desiccation.
Take a suspension of conidia — reproductive spores — of the pathogenic fungus Magnaporthe grisea. Spray on the leaf sections and incubate under dark and humid conditions.
Moisture induces conidia to release mucilage, facilitating attachment to the leaf surface.
The conidia extend germ tubes, the tip of which differentiates into dome-shaped appressorium.
High solute concentration inside the appressorium generates turgor pressure, forming a penetration peg.
The small diameter of the peg focuses turgor-mediated force on a small leaf area, causing penetration into the leaf cell.
The peg differentiates into invasive hyphae to derive nutrition.
Incubate the leaf sections under fluorescent light for fungal growth.
The hyphae ramify within infected cells and invade neighboring cells through plasmodesmata.
The fungus secretes toxins to induce necrosis, resulting in leaf lesions. Score the lesions to assess infection severity.
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.
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