This protocol allows for discovery of novel nematicides to combat Ditylenchus dipsaci by culturing technique and medium-throughput chemical screens. The simplicity of this technique allows for screening of thousands of compounds per week to identify compounds with nematicidal potential. This technique can identify new compounds to kill plant-parasitic nematodes of crops, and therefore, increase global food supply.
To begin, prepare 500 milliliters of nutrient agar, or NA media, with 23 grams per liter of NA and ultra-pure water. Using sterile technique, pour 25 milliliters of autoclaved NA media into 20 disposable 100-millimeter-diameter, 15-millimeter-deep Petri dishes. Prepare 500 milliliters of Gamborg B5, or GA media, containing 3.2 grams per liter of GA basal medium with minimal organics, 20 grams per liter of sucrose, 15 grams per liter of agar, and distilled water.
Using sterile technique, pour 50 milliliters of autoclaved GA media into 10 disposable Petri dishes. To perform seed sterilization, pour 150 pea seeds into a sterile two-liter beaker with a stir bar near a Bunsen burner flame on a lab bench. Add 200 milliliters of 95%ethanol to the seeds and stir vigorously on the stir plate for five minutes.
Then, pour off ethanol into a waste container. Pour bleach solution into the beaker to completely immerse the seeds. Stir vigorously on a stir plate for 20 minutes.
Then, pour off the bleach into a waste container. Pour distilled water into the beaker to immerse the seeds and stir vigorously on a stir plate for 20 minutes. After the final water wash, pour sterilized seeds into the glass Petri dish.
To check for contamination, transfer six seeds to each 10-centimeter NA plate in the laminar flow hood using sterilized forceps. Arrange the seeds around the plate's circumference. Wrap the plates individually in laboratory wrapping film and incubate them in the dark for three days at 26 degrees Celsius.
In a laminar flow hood, plate two non-contaminated seeds on each GA plate with sterilized forceps. Incubate at room temperature for 7 to 10 days to allow seeds to sprout. Prepare 50 milliliters of 20-gram-per-liter sucrose solution.
Filter sterilize the sucrose solution and set aside. In a laminar flow hood, cut a piece of agar containing root tissue from an existing culture plate. Pipette 500 microliters of sucrose solution on new GA plate with pea seedlings and place an agar cube on top of the sucrose.
Maintain the culture in a box lined with aluminum foil at room temperature. To maintain the culture, subculture the nematodes on fresh GA plates every eight to nine weeks. Nematodes are ready to be extracted after around eight weeks.
In a laminar flow hood, cut the agar and root tissue into one-centimeter cubes with a sterile scalpel. Transfer the agar cubes into the coffee filter-lined funnel and slowly pour distilled water on the agar to moisten the coffee filter. Remove the coffee filter-lined funnel from the beaker and fill the beaker with distilled water until the water level is just touching the bottom of the filter once the coffee filter-lined funnel is replaced.
Cover the coffee filter-lined funnel and beaker with aluminum foil. To collect the worms, remove the coffee filter-lined funnel and aspirate the top 40 milliliters of water from the collection beaker, not disrupting the settled worms. Collect the remaining liquid into a 15-milliliter conical centrifuge tube with a 10-milliliter plastic serological pipette.
To prepare the assay plates, pour autoclave-distilled water into a sterile trough and dispense 40 microliters of distilled water from the trough into each well of a flat-bottom 96-well plate with a multichannel pipette. Add chemicals from the 96-well chemical stock plates to the assay plates by pinning three times into the chemical plate. Then, transfer the pins 10 times into the assay plate.
Blot onto paper in front of the cleaning solution. To count the number of nematodes from the collection, first, re-suspend the collection and then pipette five microliters of the solution using low-retention tips onto a slide. Count the number of nematodes in five microliters using a dissection microscope.
Then, adjust the concentration to two worms per microliter using sterile, distilled water. Next, add 10 microliters of the sample to each well of the 96-well plates with a multichannel pipette and a trough. Wrap the plates with a damp paper towel and place them in a box.
Then, add an extra damp paper towel to stabilize and ensure minimal movement of the plates and affix on a sticky pad in a 20-degree-Celsius shaking incubator set to 200 RPM. Observe the plates on day five under a dissecting microscope. Count the number of mobile and total D.dipsaci in DMSO solvent controls and drug-treated wells.
If the worms are immobile, add two microliters of one-molar sodium hydroxide to a final concentration of 40-millimolar to the well to stimulate movement. Calculate the proportion of mobile worms. In the D.dipsaci screens, wells that reproducibly yielded 0%mobile worms are categorized as strong hits.
Three different drug plates were screened at a concentration of 60-micromolar, where each drug plate has three biological replicates. All three plates included 6%of DMSO-solvent-only controls. Many plates containing drugs from the small molecule library lacked any molecule with observable bioactivity against D.dipsaci.
Some plates had fully reproducible hits, while others containing characterized nematicides varied in their activity. The error bars represent the standard error of the mean. Here, fluopyram exhibited robust activity in the assay.
Fluopyram induced an apparent dose-dependent effect on D.dipsaci mobility with an EC50 of 9.3-micromolar. Oxamyl had no significant effect on mobility up to a 120-micromolar concentration. Oxamyl had no significant effect on mobility up to a concentration of 120-micromolar.
The addition of sodium hydroxide improved assay sensitivity in detecting immobile worms. 40-millimolar of sodium hydroxide was used as the assay endpoint to stimulate movement in capable individuals, and thus, distinguish resting worms from sick worms. This protocol has enabled identification of compounds with novel mechanisms of action against D.dipsaci and other species.
