A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

This method can help answer key questions related to the biological control of soilborne plant pathogens. The gold standard for testing materials like compost is to have a plant bioassay to test whether it's toxic to the plants. However, that takes three weeks.

This method can be done in about two to three days. The main advantage of this technique is that it is simple, affordable, and effective. This assay is a rapid screening of specific compost to suppress the growth of Rhizoctonia solani.

However, this assay can also be used against other soilborne plant pathogens, such as Fusariums, Pythiums, and Phytophthoras. Visual demonstration of this method is critical because it takes care to isolate and maintain a pure culture of a fungus while avoiding contamination. Demonstrating the isolation procedure will be Taylor Readyhough, a graduate student from my laboratory.

Emma Wright, an undergraduate student from my laboratory, will demonstrate the competition assay. To begin, sow red radish seeds in soil known to have a history of disease caused by R.solani. After three to four weeks, remove the seedlings from the soil, and rinse them with tap water.

Using a razor blade, cut one-centimeter segments of the hypocotyl and root that have a brown color. Then, use flame-sterilized forceps to dip the segments into 10%bleach solution for one minute. After this, rinse the segments in sterile water.

Next, use flame-sterilized forceps to transfer the segments to a paper towel. Pat the segments dry, and transfer them to a Petri dish with water agar. Place the Petri dish inside of a container with a lid, and incubate the segments at room temperature.

To establish a daughter culture, transfer one of the segments to the center of a fresh plate of potato dextrose agar. After this, autoclave 25-milliliter test tubes containing 10 milliliters of water. Add two 0.5-gram samples of each test sample to a pair of autoclaved test tubes with 10 milliliters of sterile water, and shake the test tubes overnight.

After 24 hours, add 1.5 grams of plain agar and 90 milliliters of distilled water to a pair of conical flasks. After autoclaving the flasks, place them into a 45-degree Celsius water bath for 30 minutes. Next, pour the autoclaved reference sample and the living sample into the molten agar.

Pour the mixtures from each flask into five Petri dishes. To protect the indigenous microbes, it is important not to add the compost while the agar is too hot. Therefore, allow the agar to cool to 45 degrees Celsius before adding the compost.

Using aseptic technique, transfer pieces of R.solani to each pair of sample plates. Then, incubate the plates at room temperature for one to two days. Finally, use a clear, flat ruler and a stereo microscope to measure the radius of the mycelium in each plate to the nearest millimeter.

In this protocol, extracts from living composts suppressed R.solani growth significantly more than autoclaved samples, demonstrating that the effect was microbial and not nutrient-based. Suppressiveness is measured as a reduction in growth compared to the autoclaved control. Growth was not affected by the duration of maturation or curing of the compost.

Growth was significantly affected by compost method and recipe ingredients, specifically with vermicompost, windrow processes, and recipes with hardwood bark. After its development, this technique may be a candidate for commercial certification of compost for disease-suppressive properties.