In Vivo Inoculation of Stem Canker Pathogens in Poplar Leaves to Test the Canker Disease Resistance

To begin, prepare fungal culture for canker pathogen and poplar material for leaf inoculation. For small leaves, inoculate two previously prepared square fungal mycelium cubes and PDA cubes onto the upper surface of the top fifth to seventh leaves of poplar clones. Ensure that the mycelium faces the leaves and the inoculation sites are located at the center of the half leaves.

Inoculate four square fungal mycelium cubes and four PDA cubes onto the upper surface of the top fifth to seventh leaves of large poplar leaves. Then, wrap the inoculated leaves with transparent adhesive tape. Gently press the tape to make it adhere to the leaves, preventing the movement and water loss of the mycelium cubes during the experiment.

Next, using a five milliliter syringe needle, pierce the leaves and the mycelium cubes at five sites until the needle penetrates the cubes from the upper to the lower surface of the poplar leaves. Place one piercing site at the center and the other four one to two millimeters near the four vertices of the cubes. Inspect the inoculated leaves within three days for any location shifts or water loss of the mycelium inoculants.

Observe the onset of necrotic lesions around the piercing wound sites on the lower surface of the leaves. At the end of the experiment, pick off all inoculated leaves and place them in plastic sample bags.