The Plant Infection Test: Spray and Wound-Mediated Inoculation with the Plant Pathogen Magnaporthe Grisea

Summary

Here, we present a protocol to test plant virulence with the plant pathogen Magnaporthe grisea. This report will contribute to the large-scale screening of the pathotypes of fungi isolates and serve as an excellent starting point for understanding the resistant mechanisms of plants during molecular breeding.

Abstract

Plants possess a powerful system to defend themselves against potential threats by pathogenic fungi. For agriculturally important plants, however, current measures to combat such pathogens have proved too conservative and, thus, not sufficiently effective, and they can potentially pose environmental risks. Therefore, it is extremely necessary to identify host-resistance factors to assist in controlling plant diseases naturally through the identification of resistant germplasm, the isolation and characterization of resistance genes, and the molecular breeding of resistant cultivars. In this regard, there is need to establish an accurate, rapid, and large-scale inoculation method to breed and develop plant resistance genes. The rice blast fungal pathogen Magnaporthe grisea causes severe disease symptoms and yield losses. Recently, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions. Hence, we report the development of a plant virulence test method that is specific for M. grisea. This method provides for both spray inoculation with a conidial suspension and wounding inoculation with mycelium cubes or droplets of conidial suspension. The key step of the wounding inoculation method for detached rice leaves is to make wounds on plant leaves, which avoids any interference caused by host penetration resistance. This spray/wounding protocol contributes to the rapid, accurate, and large-scale screening of the pathotypes of M. grisea isolates. This integrated and systematic plant infection method will serve as an excellent starting point for gaining a broad perspective of issues in plant pathology.

Introduction

Rice blast, caused by M. grisea, is one of the most serious diseases for rice varieties worldwide^1,2. The process by which M. grisea infects host plants includes a conidia production and surface attachment, a conidia germination and appressorium formation, a formation of the penetration peg and infectious hypha differentiation, and a disease spread³. All these stages are common in many other plant pathogenic fungi, and, indeed, a blockade of any single stage prevents the infection of host plants. Owing to its economic importance and genetic tractability, M. grisea has emerged as a model organism for studying the mechanisms of plant-fungal pathogen interactions^1,4. Therefore, studying the molecular basis of these developmental stages in M. grisea will help to elucidate the molecular mechanisms underlying fungal pathogenicity and the identification of candidate target genes for screening and designing novel fungicides⁵.

Recent reports concerning M. grisea infection have focused on the molecular mechanisms of the pre-penetration stages, especially the conidiation, the appressorium formation, the penetration pegs, and the infectious growth^3,6. Therefore, it is essential to develop a detailed protocol to test M. grisea infection. Herein, we present a detailed method for an infection test that utilizes spray-mediated infection assays with a conidial suspension and the inoculation of wounds with mycelial plugs of M. grisea. In this report, the protocol focuses on the culture of strains, the preparation of the conidiation solution for spraying, and the mycelial plug-mediated inoculation of plants with M. grisea. These steps are described in detail below, and a schematic view showing the entire workflow of the method and a typical lesion are shown in Figures 1 and 2, respectively.

Protocol

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, 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 d 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 in 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 in the day (14 h) and 23 °C in the 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.

2. 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.

Results

The entire workflow for the technique is shown in Figure 1. The plant infection assays were performed on 14-day-old susceptible rice seedlings (O. sativa cv CO-39) or susceptible 7-day-old barley leaves (H. vulgare cv Golden Promise)^7,8,9. To test for an infection on the rice leaves, a conidial suspension (1.0 x 10⁵ spores/mL) of the ...

Discussion

Plant disease resistance genes play an essential role in preventing infections by pathogens, including fungal pathogens^1,12. Rice blast has been used as a model to understand the nature of pathogen population structures and to identify plant resistance genes⁴. Therefore, it is necessary to examine the disease resistance genotype and avirulence genotypes of the main varieties of agricultural plants on a large scale to identify disease-resis...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Filter paper	GE Healthcare brand(Sweden)	10311387
50-mL tube	CORNING(Amercia)	430290
Centrifuge	Eppendorf(Amercia)	5804R
Culture dish	Thermofisher(Amercia)	150326
0.5-5 mL pipette	Eppendorf	4920000105
100-1000uL pipette	Eppendorf	4920000083
Vacuum pump	Leybold	D25B
Dissection needle	FST	26000-35
Incubator	MEMMERT	PYX313
Inoculation ring	Greiner Bio One	731175