This video can help instruct key methods in the plant infection field such as free and wanted mediated inoculation with the plant pathogen Magnaporthe grisea. The main advantage of this technique is that the spraying, wounding protocol contributes to the rapid, accurate, and large scale screening of the pathotypes of fungi isolates. The implications of this technique relates to the study of physiology to combat plant pathogens because it is very important for the prevention and control of large scale plant diseases.
This method can provide insight into conservation pathogeny mechanism in fungi. It can also be applied to the main variety of agriculture plants such as rice or barley. Generally individuals new to this method will struggle because it is difficult to scrape conidia gently with the sterile cotton swabs.
The demonstration of this method is critical as a collection of scrape wounds without penetrating the leaves as the main means of detached rice or barley leaves is difficult. To begin weigh out 30 to 50 grams of oatmeal. Add the oatmeal to 800 milliliters of distilled deionized water and boil the mixture for 30 minutes in an electric pot.
Next filter the boiled oatmeal mixture through a piece of gauze into a beaker. Then add 150 milliliters of tomato juice and 20 grams of agar to the beaker. Add distilled deionized water to the beaker up to 1000 milliliters.
Soak about 50 rice seeds in distilled deionized water for 3 days then wrap the seeds in moistened gauze and germinate them on moist filter paper in Petri dishes. Next plant the seedlings in pots with autoclaved potting soil. After watering the seedlings cover them with a layer of vermiculite.
Place the seedlings in a glass house maintained at 25 degrees Celsius for one to two weeks. Next use sterile surgical scissors to cut three layers of filter paper into eight 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 tooth picks two to three centimeters 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 two 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 in a clean bench. Add two 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 millimeter tube. Then centrifuge the tube for five minutes at 5000 times g. After centrifugation remove the supernatant and re-suspend 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 the 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. Using an anatomical needle scrape three two-to-three-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 half-by-half-centimeter mycelial plug from the OTA plates containing each M.grisea strain. Place the plugs on the wounded leaves and incubate them in a humid chamber at 25 degrees Celsius for three to eight 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. In this protocol plant infection assays using M.grisea strains P131 and Com1 were performed on susceptible rice seedlings. The P131 inoculated wild type leaves displayed the typical robust lesions of rice blast.
However the Com1 inoculated leaves showed obvious infection defects and did not elicit a full infection. To determine if M.grisea could infect host cells through wounds abraded leaves of wild type plants were inoculated with mycelial plugs or conidial droplets. The spray inoculated leaves showed no infection defects.
In contrast the leaves inoculated via mycelial plugs displayed obvious defects. While attempting this procedure it is important to scrape three two-to-three-centimeter long wounds in the main veins of the detached rice leaves. Following this procedure other methods like pathogen inoculation can be performed in order to identify and analyze crop disease resistance.
After its development this technique paves the way for researchers in the field of plant pathogens to explore rice blast disease in modern organisms. Don't forget that working with Magnaporthe grisea can be hazardous and precautions such as wearing gloves should always be taken when performing these procedures.
