In Vitro Cultivation and Maintenance of Pinewood Nematodes Using Botrytis cinerea

To begin, transfer a culture plug of botrytis cinerea from the outermost border of the colony onto a sterile potato dextrose auger plate. Incubate the plate at 25 degrees Celsius for seven to 10 days, or until the fungal colony reaches the edge of the plate. Culture B cinerea in steam-sterilized, hydrated, certified organic barley grains for seven to 10 days.

Or until the surface of the cereal is fully colonized. Next, inoculate the B cinerea culture with a Pinewood nematode suspension. Incubate the flask in the dark for seven to 10 days, or until the fungal colony is completely consumed and the nematode climbs the flask walls.

To isolate Pinewood nematodes, rinse the walls of the flask with tap water. Then pour the contents onto a paper towel, placed in a Baermann funnel or tray, and immerse the nematodes in water. After 24 to 48 hours, recover the nematodes using a 38 micrometer mesh sieve.

Wash the accumulated nematodes into a container with tap water. Using this methodology, the Pinewood nematode population increased 100 fold within an eight day growth period.