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

1. Spray Inoculation with a Suspension of M. grisea Conidia

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. Spray inoculation protocol
   1. Culture the fungal strain on OTA plates in a thermostatic incubator (25 °C) for ~4 d.
   2. Add ~2 mL of ddH₂O using a 0.5 – 5 mL pipette to each 4-day-old plate.
   3. With an inoculation loop, scrape the mycelia of the M. grisea wild-type strain and the mutant strain into mycelia debris.
   4. Collect the mycelia debris and transfer it to a new OTA plate. Take the mycelia debris and blow dry on the clean bench.
   5. Cover the plate with 3 layers of gauze to ensure the humidity required for the growth of the conidia in a greenhouse at 25 °C during the day (14 h) and 23 °C at night (10 h) for 24 – 48 h.
   6. Add 2 mL of ddH₂O to each dish and scrape the conidia gently with sterile cotton swabs followed by a filtration through 2 layers of lens paper. Be careful not to scratch the surface of the culture medium.
   7. Transfer the conidia suspension into a new 50 mL tube with a 100 – 1,000 µL pipette.
   8. Centrifuge for 5 min at a minimum of 5,000 x g at 25 °C.
   9. Remove the supernatant and resuspend the pellet to give 2 x 10⁴ conidia per mL in a 0.025% (v/v) Tween-20 solution. The Tween-20 solution is usually about 10 – 20 mL.
   10. Pour the spore suspension into a hand-held sprayer.
   11. Spay about 10 mL of the conidial suspension onto the rice leaves of 2-week-old rice seedlings or barley leaves of 7-day-old barley seedlings and incubate them at 25 °C in a dark, humid chamber for ~24 h. Spray the control plants with the 0.025% (v/v) Tween-20 solution.
   12. Transfer the leaves into another moist chamber under fluorescent light at 25 °C for a photoperiod of 12 h.
   13. Record the disease symptoms at 5 d after the inoculation. Examine the diseased rice/barley blades of ~6 cm in length. The evaluation standard is according to the scoring system for blast resistance of the International Rice Research Institute (IRRI). The details of the scoring system for blast resistance are shown in Table 1.
   14. Photograph the leaves to evaluate the infection of the tested strains. The infection was assessed by the number of lesions per 3.6 cm².

Table 1: A scoring system for blast resistance. The table is cited from the International Rice Research Institute (IRRI).