a spray inoculation technique to establish fungal infection in rice plant leaves

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.

a-spray-inoculation-technique-to-establish-fungal-infection-rice

Research

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Host-Pathogen Interaction Models

EoE Sub Articles

eoe sub articles

1. Spray Inoculation with a Suspension of M. grisea Conidia
<ol>
	<li>Fungal culture for M.grisea
	<ol>
		<li>Prepare the oatmeal tomato agar (OTA) culture medium for fungal strains.</li>
		<li>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.</li>
		<li>Filter the boiled oatmeal juice into the beaker through a piece of gauze.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Preparation of experimental materials
	<ol>
		<li>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.</li>
		<li>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 &#176;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%.</li>
		<li>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.</li>
		<li>Place the plants in a suitable glasshouse or growth cabinet at 25 &#176;C for about 1 ~ 2 weeks.</li>
		<li>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.</li>
		<li>Add ddH2O to each dish to soak the filter paper.</li>
		<li>Make sure the filter paper is completely wet but add no extra water.</li>
		<li>Remove any excess water with a vacuum pump.</li>
		<li>Place 2 sterile toothpicks into the culture dish to support the rice/barley leaves; space the toothpicks ~2 - 3 cm apart.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Spray inoculation protocol
	<ol>
		<li>Culture the fungal strain on OTA plates in a thermostatic incubator (25 &#176;C) for ~4 d.</li>
		<li>Add ~2 mL of ddH2O using a 0.5 - 5 mL pipette to each 4-day-old plate.</li>
		<li>With an inoculation loop, scrape the mycelia of the M. grisea wild-type strain and the mutant strain into mycelia debris.</li>
		<li>Collect the mycelia debris and transfer it to a new OTA plate. Take the mycelia debris and blow dry on the clean bench.</li>
		<li>Cover the plate with 3 layers of gauze to ensure the humidity required for the growth of the conidia in a greenhouse at 25 &#176;C during the day (14 h) and 23 &#176;C at night (10 h) for 24 - 48 h.</li>
		<li>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.</li>
		<li>Transfer the conidia suspension into a new 50 mL tube with a 100 - 1,000 &#181;L pipette.</li>
		<li>Centrifuge for 5 min at a minimum of 5,000 x g at 25 &#176;C.</li>
		<li>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.</li>
		<li>Pour the spore suspension into a hand-held sprayer.</li>
		<li>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 &#176;C in a dark, humid chamber for ~24 h. Spray the control plants with the 0.025% (v/v) Tween-20 solution.</li>
		<li>Transfer the leaves into another moist chamber under fluorescent light at 25 &#176;C for a photoperiod of 12 h.</li>
		<li>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.</li>
		<li>Photograph the leaves to evaluate the infection of the tested strains. The infection was assessed by the number of lesions per 3.6 cm2.</li>
	</ol>
	</li>
</ol>
Table 1: A scoring system for blast resistance. The table is cited from the International Rice Research Institute (IRRI).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Scale</td>
			<td>Description</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Small brown specks of pin-point size</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Small roundish to slightly elongated, necrotic gray spots, about 1-2 mm in diameter, with a distinct brown margin. Lesions are mostly found on the upper leaves</td>
		</tr>
		<tr>
			<td>3</td>
			<td>The lesion type is the same as in 2, but a significant number of lesions are on the upper leaves</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 4% of the leaf area</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 4-10% of the leaf area</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 11-25% of the leaf area</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 26-50% of the leaf area</td>
		</tr>
		<tr>
			<td>8</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infecting less than 51-75% of the leaf area, many leaves dead</td>
		</tr>
		<tr>
			<td>9</td>
			<td>Typical susceptible blast lesions, 3 mm or longer, infect more than 75% of the leaf area</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>AOBOX Biotechnology (China)</td>
			<td>01-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>GE Healthcare brand(Sweden)</td>
			<td>10311387</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL tube</td>
			<td>CORNING(Amercia)</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf(Amercia)</td>
			<td>5804R</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Coolaber (China)</td>
			<td>CT11551-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish</td>
			<td>Thermofisher(Amercia)</td>
			<td>150326</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5-5 mL pipette</td>
			<td>Eppendorf</td>
			<td>4920000105</td>
			<td></td>
		</tr>
		<tr>
			<td>100-1000uL pipette</td>
			<td>Eppendorf</td>
			<td>4920000083</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Leybold</td>
			<td>D25B</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>MEMMERT</td>
			<td>PYX313</td>
			<td></td>
		</tr>
		<tr>
			<td>Inoculation ring</td>
			<td>Greiner Bio One</td>
			<td>731175</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Zhang, M., et al. <a href="https://app.jove.com/v/57675">The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea.</a> J. Vis. Exp. (2018).
This video demonstrates a technique to establish the pathogenic fungus Magnaporthe grisea infection in rice plant leaves. The fungal conidia suspension is sprayed on sections of rice leaf, and the disease progression is assessed by scoring the formation of necrotic lesions.

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

	Fungal culture for M.grisea
	
		Prepare the oatmeal tomato agar (OTA) culture medium for ...

Take a Petri plate with sections of rice plant leaves positioned above a wet filter paper to prevent desiccation.
Take a suspension of conidia&#160; &#8212; reproductive spores &#8212; 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.

79 조회수. 벼 식물 잎에 곰팡이 감염을 일으키기 위한 스프레이 접종 기술

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

내레이션 대본