Infection of In Vivo and In Vitro Pines with the Pinewood Nematode Bursaphelenchus xylophilus and Isolation of Induced Volatiles

We have established an in vitro co-culture system of pine shoots with the pinewood nematode to mark the study changes in host volatile response to these phytoparasites. Greenhouse bioassays allow control of environmental variability. However, the genetic diversity of pine seedlings still influences the resulting volatile profiles.

In vitro pine shoots infected with the pinewood nematodes allow sampling of easily obtainable plant clones, which reduces the influence of variability coming from pine genetic diversity. Plant volatiles are isolated by hydrodistillation and distillation extraction, but headspace techniques allow the profiling of volatiles emitted by the plant. Resin-packed columns will be used as a non-invasive technique to profile volatiles emitted by in vitro ground pine infected with pine nematode for direct analysis of the chemical interplay between the host and the phytoparasite.

To begin, transfer a culture plug of Botrytis cinerea from the outermost border of the colony onto a sterile potato dextrose agar plate. Incubate the plate at 25 degrees Celsius for 7 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 7 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 7 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.

To begin, set suspensions of mixed life stage pinewood nematodes to 1, 000 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.

To begin, wash certified Pinus pinaster seeds in running tap water to remove larger debris. Then wash the finer debris using a common detergent solution with vigorous agitation. In a flow hood, add a commercial bleach solution until the seeds are covered and mix vigorously for 15 minutes.

After discarding the bleach solution, rinse the seeds three times with sterilized tap water. Now immerse the seeds in 80%ethanol for 15 minutes with vigorous agitation. After disposing of ethanol, wash the seeds three times with sterilized ultrapure water.

For pine germination, break the sterilized seed coats with a sterile mechanical lathe. Isolate the pine nuts and set them in a closed container with sterilized wet filter paper to hydrate overnight. The next day, stratify hydrated pine nuts at four degrees Celsius for seven days to break dormancy and synchronize germination.

Then maintain the pine nuts in the dark at 25 degrees Celsius until germination. Transfer the aseptic germinated pines to controlled environmental conditions for two weeks or until the main stem begins developing. After two weeks, cut the aerial portion of the seedling above the root with a scalpel and transfer it to a sterilized Schenk and Hildebrandt culture medium.

Maintain the culture in controlled environmental conditions as described previously. Subculture periodically by cutting the callous tissue at the base of the shoot with a sterile scalpel. Transfer the shoot cluster using sterile tweezers to a fresh multiplication culture medium.

Next, inoculate the microshoots into the Schenk and Hildebrandt culture medium, containing three grams per liter of activated charcoal for microstem elongation. Now, place the elongated shoots in the Schenk and Hildebrandt culture medium without phytohormones or activated charcoal. Add 100 to 150 mixed life stage sterilized pinewood nematodes at the bottom of the microshoot.

Maintain the culture in controlled environmental conditions as described previously, the establishment and infection of in vitro pine shoots showed the appearance of chlorotic needles within a month of co-culture with pinewood nematodes. To begin, prepare a setup for trapping headspace volatiles with packed stainless steel traps. Place pine material set in covered glassware or inside PET bags to accumulate headspace volatiles.

For a closed loop system, connect the pump outlet to the PTFE tubing. Then attach the PTFE tubing to activated charcoal air filters. Connect the air filters to additional tubing leading to the glassware or PET bags with the pine material.

Next, attach the packed tube to the glassware or PET bag outlet and connect additional PTFE tubing from the stainless steel tube outlet to the mass flow pump inlet. Using a mass flow pump, pump 100 liters of filtered air through the pine system at 0.6 liters per minute to collect volatiles. After collecting volatiles, close the packed tubes at both ends with airtight storage caps.

Finally, rinse all PTFE tubing and glassware with 70%ethanol and heat in an oven at 230 degrees Celsius for two hours. Volatile profiles of maritime pine in vitro shoots obtained through hydrodistillation or distillation extraction were similar within the chemotype analyzed. Chemical profiles obtained through solid phase microextraction techniques showed variations depending on the amount of plant material and volatiles trapped.