Establishment of Rice Plant Leaf Infection through Fungal Inoculation via Wounding

1. Fungal culture for M.grisea

1. Prepare the oatmeal tomato agar (OTA) culture medium for fungal strains.
2. Weigh 30 – 50 g of oatmeal, add this to 800 mL of distilled/deionized water (ddH₂O), and boil the mixture for 30 min in the electric pot.
3. Filter the boiled oatmeal juice into the beaker through a piece of gauze.
4. Add 150 mL of tomato juice and 20 g of agar to the filtrate in the beaker and add ddH₂O up to 1,000 mL.

2. Preparation of experimental materials

1. Soak about 50 seeds of rice (Oryza sativa) cultivar Lijiangxintuan-heigu (LTH) in ddH₂O for 3 d or soak about 50 seeds of barley (Hordeum vulgare cv Golden Promise) in ddH₂O for about 1 d.
2. Wrap the seeds of rice or barley in moist gauze and germinate them on moist filter paper in about 30 Petri dishes with a diameter of 10 cm x 10 cm at 28 °C. The rice seed germination time is about 2 – 3 d, and the barley seed germination time is about 2 d. The relative humidity in the greenhouse is approximately 70%.
3. Plant the seedlings of the rice or barley in pots using autoclaved potting soil and watering and then cover them with a layer of vermiculite.
4. Place the plants in a suitable glasshouse or growth cabinet at 25 °C for about 1 ~ 2 weeks.
5. Cut 3 layers of filter paper in circles with sterile surgical scissors (each circle should have an 8 cm diameter) and place them onto 100 mm sterile plastic plates.
6. Add ddH₂O to each dish to soak the filter paper.
7. Make sure the filter paper is completely wet but add no extra water.
8. Remove any excess water with a vacuum pump.
9. Place 2 sterile toothpicks into the culture dish to support the rice/barley leaves; space the toothpicks ~2 – 3 cm apart.
10. Collect the leaves of the rice 2 weeks after sowing the seeds or take the leaves of the barley 7 days after sowing the seeds.
11. Using 4- to 6-leaf seedlings of rice/barley, cut the lower part of the stem at ~5 cm from the top and collect the leaves.

3. Wounding Inoculation with Mycelium Cubes or Droplets of Conidial Suspension of M. grisea

1. Using an anatomical needle, scrape three 2 – 3 cm long wounds in the main veins of detached rice/barley leaves; take care not to penetrate the leaves.
2. Put the scraped leaves on toothpicks and spray a 0.02% (v/v) Tween-20 solution on the leaves to form a layer of droplets.
3. Cut a 0.5 cm x 0.5 cm mycelial plug for each M. grisea strain (wild-type, mutant, complement strains, or other test strains) from an OTA plate.
4. Put the mycelial plugs or 25 µL droplets of conidial suspension onto the wounded leaves and incubate the leaves at 25 °C in a humid chamber for 3 – 8 d.
5. Examine the lesions at 5 – 7 d post-inoculation. The method of examination is the same as it was for the spray inoculation method (see step 1.3.13).
6. Examine the diseased rice/barley blades of ~6 cm in length and photograph them to evaluate the infection of the tested strains. The infection was assessed by the number of lesions per 3.6 cm².