Establishment of Rice Plant Leaf Infection through Fungal Inoculation via Wounding

Begin with a culture dish containing a rice leaf with small wounds, exposing the underlying epidermis.

Apply a moistening solution containing Tween-20, creating a conducive environment for fungal growth.

Place a mycelial plug of Magnaporthe grisea, consisting of conidiophores with conidia  — reproductive fungal spores, over the wounded site.

Incubate the leaf in a humid chamber under photoperiod conditions to promote fungal growth.

During incubation, conidiophores release spores that adhere to the exposed epidermis, developing germ tubes.

The germ tube differentiates into a dome-shaped appressorium, which generates high turgor pressure, forming a sharp hypha — or penetration peg.

The penetration peg secretes cell wall-degrading enzymes, facilitating its entry into the epidermal cell and differentiation into invasive hyphae for nutrient acquisition.

These hyphae grow and spread within the epidermal cells through plasmodesmata  — connecting channels between cells, and establishing the infection.

The fungus secretes toxins, causing cell necrosis and lesion formation near the wounded site on the leaves, indicating fungal infection.

Using an anatomical needle, scrape three 2- to 3-centimeter-long wounds in the main veins of detached rice leaves. Place the scraped leaves on toothpicks, and spray them with Tween-20 solution. Next, cut a 1/2- by 1/2-centimeter mycelial plug from the OTA plates containing each M griseus strain. Place the plugs on the wounded leaves, and incubate them in a humid chamber at 25 degrees Celsius for 3 to 8 days.

After the incubation period, examine and evaluate the lesions according to the scoring system provided by the International Rice Research Institute. Finally, photograph the diseased leaves to evaluate the infectiousness of the tested strains.