Preparing Reagents and Poplar Material for In Vivo Leaf Inoculation

Begin by adding potato extraction, dextrose, and auger in water to prepare potato dextrose auger or PDA culture medium for fungal strains. Heat the solution at 100 degrees Celsius for 10 minutes to completely dissolve the components. Poor PDA medium into 25 milliliter tubes.

Sterilize all tubes at 121.1 degrees Celsius for 30 minutes. Then cool the sterilized tubes on a clean bench to 50 to 60 degrees Celsius. Pour the medium into 9.0 centimeter diameter culture plates and cool them at room temperature.

Next, cut the mycelium of the fungal pathogen cultured in PDA medium at 28 degrees Celsius for seven days into approximately 0.5 centimeter square cubes. Inoculate the fungal cubes at the center of the PDA plates, ensuring the mycelium sides face the medium. Then culture, the inoculated fungal pathogens in a thermostatic incubator for seven to 10 days.

After incubation, cut the PDA medium containing the fungal mycelium into square cubes with sides 1.0 to 1.2 centimeters long. Select the newly matured fully extended leaves from the top of the main branches of 1-year-old poplar clones, ensure the leaves are free from pests, diseases, and mechanical damage. For one year old branches of Perennial poplar hybrid clones, select the top fifth to seventh leaves of the selected branches as the inoculated materials.

Spray the selected poplar clones or branches with clean water. After drying, wipe the selected leaves with 75%alcohol, either one hour or one day before the inoculation manipulations.