These procedures are part of our routine soil processing for SCN egg counts.
Procedure for manual cyst extraction
After preparing a well-mixed soil sample, fill a bucket with 2 quarts of water (marked on inside of bucket).
Construct microsieves from 18-mm and 20-mm test tube caps and 25 micrometer pore nylon mesh.
UV-sterilize all microsieves and hatch trays.
Arrange the hatching trays in replicated boxes using a pre-generated random order. Fill each hatching tray with 12 ml of the appropriate test or control solution.
Pipette between 5,000 and 10,000 surface-disinfested SCN eggs (depending on experimental details) onto each microsieve.
To prepare the disinfestant solution of 0.5% chlorhexidine diacetate (Hibitane), dissolve 0.5 g into 100 ml sterile distilled water, or dissolve 2.5 g into 500 ml sterile distilled water
To disinfest the eggs:
Place the root samples in separate 100 or 150 ml glass beakers. Prepare 600 ml of a water-bleach solution by adding 100 ml household bleach (5% sodium hypochlorite or NaClO) to 500 ml distilled water. Add about 50 ml of water-bleach solution to each beaker containing a root sample.
Incubate roots in the water-bleach solution for 4 minutes, stirring with a metal spatula periodically. Rinse roots for 45 seconds or so in running tap water, then soak the roots in tap water for 15 minutes.
Soybean genotypes are evaluated for resistance to soybean cyst nematode (SCN), Heterodera glycines, by growing the plants in infested soil-sand mix for approximately 30 days, after which the numbers of adult SCN females and cysts on the roots is assessed. Alternatively, non-infested soil-sand mix may be infested with an SCN egg suspension.
After extracting the eggs from the cysts in the prepared soil sample, place 4 to 5 drops of 1 M HCl and approximately one eye-dropper full of acid fuchsin stain into each beaker of egg solution. Heat beakers containing eggs and stain in the microwave until the suspension is about to boil (approximately 3 minutes for 12 samples). Remove beakers from microwave and allow to cool. Place beakers in the refrigerator until they are counted.
Wash the cyst/sediment suspension (from the cyst extraction method) from the 100 ml beakers into the small PVC #60 sieve (250 micron diameter pores). Eggs of SCN average 47 microns by 100 microns in size.
Place a #200 sieve (75 micron diameter pores) over a #500 sieve (25 micron diameter pores) into the funnel below the rubber stopper, and start the drill press using the foot pedal. Bring the spinning rubber stopper into contact with the sieve surface.
One day before screening soil, place samples out to dry on drying racks, with one-quarter sheet of unused newspaper between the tray and sample. Sample bag and tag should be clipped to the tray used.
Investigators: Gustavo MacIntosh, Matthew E. O’Neal, Palle Pedersen, Felicitas Avendano, Gregory L. Tylka
Funding: Iowa Soybean Association