In Vivo Infection Process of Pinus pinaster Seedlings with Pinewood Nematodes

To begin, set suspensions of mixed life-stage pinewood nematodes to 1000 nematodes per milliliter. Count the nematodes on a concave slide under the binocular microscope with up to 40x magnification. Before inoculating nematodes, manually remove needles from a section below the upper five centimeters of the pine stem, and make a superficial longitudinal incision using a sterilized scalpel.

At the wounding zone, place a piece of sterilized cotton to hold 0.5 milliliters of pinewood nematode suspension, and fix it with a transparent strip to maintain humidity. Maintain the greenhouse in humid conditions and water frequently. Follow pine wilt disease progression regularly, and score symptomatology by quantifying the percentage of discolored and wilted needles.

At 20 days after inoculation, cut the seedlings at the base of the stem for volatiles extraction. Place the seedlings in brown paper bags and freeze at minus 20 degrees Celsius. The successful infection of pine seedlings with the pinewood nematode resulted in symptoms of pine wilt disease at around 20 days after infection.