Protocols

Acid Fuchsin Stain Preparation

Procedure:

• To 750 ml distilled H2O add 3.5 g Acid Fuchsin and 250 ml glacial acetic acid.
• Stir well.

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Staining Roots for Plant-Parasitic Nematode Visualization

Procedure:

• 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 to 500 ml distilled water.
• Add approximately 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.
• Pour off the tap water, rinse with tap water one last time, then add approximately 40 ml of distilled water and approximately 7 or 8 drops of the acid fuchsin stain to each beaker.
• Place beakers containing roots and stain on a hot plate and heat until boiling fairly well.
• Take the beakers that have boiled from the hot plate and allow them to cool enough so that they are comfortable to touch.
• After cooling, carefully rinse roots in running tap water for approximately 1 minute, then carefully drain off most of the excess water.
• Add 10 to 20 ml of acidified glycerine to each beaker. Acidified glycerine is prepared by adding 5 to 10 drops of dilute 1.0 M HCl to regular glycerine.

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Purification of SCN Cysts by Sucrose Centrifugation

Procedure:

• Wash cyst/sediment suspensions from beakers into 50 ml plastic centrifuge tubes.
• Adjust the volume of liquid in each tube to approximately the same level (to avoid excessive centrifuge vibration).
• Place the tubes, 2 by 2, into opposite positions in the centrifuge.
• Centrifuge the tubes at 2000 RPM for 4 minutes. After 4 minutes, turn the centrifuge off and let the rotor coast to a stop.
• Save the supernatant and pour into the original sample beaker.
• Wipe the rim of the centrifuge tube to collect cysts stuck to the tube wall, and rinse into beaker.
• Add sucrose solution (1362 g sucrose / 1L tap water) to tubes until each is about 2/3 full, and stir or agitate the tubes to re-suspend the sediments and cysts. After stirring, add more sucrose solution to level off paired tubes.
• Centrifuge at 2000 RPM for 2 minutes. After 2 minutes, turn the centrifuge off and let the rotor coast to a stop.
• For each sample, pour the supernatant through a #60 or #80 mesh sieve. In addition, pour the supernatant from the 1st centrifuge step through the sieve. Rinse thoroughly with tap water.
• Collect cysts by rinsing them from the sieve back into the original beaker using a water bottle.
• Wash the plug out of the bottom of the tubes, and clean and rinse the tubes.

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Greenhouse Resistance Screening

Soybean genotypes will be 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 will be assessed. Subsequently, the reaction of individual plants of each soybean genotype to SCN will be classified based on the criteria presented by D.P. Schmitt and G. Shannon in "Differentiating Soybean Responses to Heterodera glycines races", 1992, Crop Science 32(1):275-277.

PROCEDURE:

Two or three seeds of each soybean genotype to be evaluated will be planted directly into individual replicate cone-tainers filled with soil-sand mix infested with Race 3 SCN/HG Type 0 (or selected other SCN populations if desired and available); a minimum of five replicates per genotype is recommended. Additionally, three seeds of Lee, PI548402, PI88788, PI90763, PI437654, PI209332, PI89772, PI548316, PI438489B, and Pickett (the HG Type and race differential genotypes) will be planted into separate replicate cone-tainers filled with the same SCN-infested soil-sand mix. All cone-tainers will be placed in an arbitrarily determined position in a greenhouse waterbath and incubated at constant 27oC under natural lighting conditions. Cone-tainers initially will be observed every day, and the second and third seedlings that emerge within each cone-tainer will be removed. Consequently, only the first seedling that emerges in each cone-tainer will be used in the evaluation. After approximately 30 days, the number of females and cysts on each soybean root will be determined by direct visual observation, and each replicate plant of each genotype will be classified into categories based on the number of females and cysts formed on the genotypes relative to the number formed on Lee or Lee74, which are considered by most soybean breeders to be universal SCN-susceptible soybean genotypes. An HG Type/Race test will be conducted to confirm the HG Type/Race of SCN population used in the screen. The unused seed of the soybean genotypes will be returned to the client with the report of the results or will be destroyed at the end of the experiment, whichever the client desires.

COSTS:

If time and greenhouse space permit, the staff of the Tylka Nematology Laboratory will perform greenhouse screening for SCN resistance for private companies on a contract basis. The cost for evaluation of soybean genotypes, as described above, utilizing two categories - resistant (< 10% of Lee 74) or not resistant (> 10% of Lee 74) - is $5.00 per individual cone-tainer. Categorizing genotypes as resistant (< 10% of Lee 74), moderately resistant (> 10% but < 30% of Lee 74), moderately susceptible (> 30% but < 60% of Lee 74), and susceptible (> 60% of Lee 74) is $10.00 per individual cone-tainer. The cost for obtaining actual counts of the number of SCN females formed on each plant root is $15.00 per cone-tainer. There may be a $150 charge for cone-tainers containing the HG Type and SCN race differential soybean genotypes; check with Greg Tylka or Chris Marett.. One half of the charge is due at the time the experiment is initiated and the balance of the fee is due after the results are received by the client. Checks should be made to Iowa State University and mailed to Dr. Gregory L. Tylka, Department of Plant Pathology, 351 Bessey Hall, Iowa State University, Ames, IA 50011.

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In-Vitro Hatching Studies

Procedure:

• 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 hatching trays in replicated boxes using 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 (depending on experimental details) surface disinfested SCN eggs onto each microsieve.
• Incubate the experiment at room temperature in darkness.
• At every-other day intervals, transfer the microsieves to duplicate hatching trays containing fresh solution, and count the hatched second-stage juveniles remaining in the original solution.

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Extracting Nematodes from Soil with a Baermann Funnel

Procedure:

• Assemble the Baermann funnel apparatus.
  • A clamped rubber tube is placed below the funnel
  • A piece of window screen (or similar material) is placed in the mouth of the funnel
  • The funnel is placed into a rack or holder

• Place a tissue-paper wrapped soil sample onto the screen material.

• Add water to the funnel setup until the screen and soil sample are immersed.
• Wait overnight (or longer if desired).
• Gather the first couple of drops of water from the bottom of the tube by slowly releasing the clamp on the tubing.
• Examine under the microscope. Note that this technique will work only with actively mobile, living nematodes.

 

 

 

 

 

 

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Surface Disinfestation of SCN Eggs

Disinfestant solution: 0.5% chlorhexidine diacetate (hibitane)

Solution Preparation:

• dissolve 0.5 g into 100 ml sterile distilled water
• dissolve 2.5 g into 500 ml sterile distilled water

Procedure:

1. Concentrate eggs in 50-ml centrifuge tube by centrifuging at 2,000 RPM for 3 minutes.
2. Under the laminar flow hood, pour off the water (taking care not to disturb the pellet of eggs), and add the hibitane solution.
3. Cover the tube opening with a square of parafilm, shake to resuspend the egg pellet, and incubate at room temperature for 15 minutes.
4. Centrifuge the egg suspension (as in Step 1) to concentrate the eggs and pour off the hibitane solution while working in the hood.
5. Add sterile distilled water to the tube, cover the tube, and shake to resuspend the eggs, then incubate for 10 minutes.
6. Centrifuge the egg suspension (as in Step 1), pour off the water, add fresh sterile distilled water and incubate one final time for 5 minutes.
7. Centrifuge to concentrate the eggs, pour off the water and decant the surface disinfested eggs into a suitable container.

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Routine soil processing for SCN egg counts

Screening Soil

Procedure:

• 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.

 

 

 

 

 

• Before beginning, check that the collection tray and rollers are clean.
• Turn the machine on using the side-mounted switch.
• Slowly pour a sample into the feed-chute.

 

 

 

 

• Clean the rollers using the wire brush. It is important to do this before collecting the sample to avoid possible contamination of other samples.
• Collect the sample from the tray and return to the sample bag. Used newspaper should be discarded and the collection tray should be cleaned before returning it to the machine.

 

 

 

 

 

• When complete, all samples should be returned to the cold room for storage.

Manual Cyst Extraction

Procedure:

• Obtain a well mixed soil sample.
• Fill a bucket with 2 quarts of water (marked on inside of bucket).

 

 

 

 

 

• Pour in the soil, break any clumps with your fingers, and mix the soil suspension well for 15 seconds.
• Pour the soil suspension through a #20 over a #60 sieve.

 

 

 

 

 

• Rinse the debris caught on the top sieve with water, and then discard its contents. Carefully wash the cysts and accompanying sediments from the #60 sieve into a clean beaker or directly into a 100 ml plastic grinding tube using as little water as possible.

Automated Cyst Extraction

Procedure:

• Add 400 ml tap water to graduated beaker.

 

 

 

 

 

• Raise volume to 500 ml with soil.
• Allow sample to sit for 15 minutes.
• Thoroughly stir sample.
• Allow sample to sit for 15 minutes.
• Add soil/water mixture to the machine beakers and raise volume to 1800 ml.

 

 

 

 

 

• Open valve to air drills and allow to stir for 15 seconds.

 

 

 

 

 

• Dump samples 15 seconds after stirring (through a #20 over a #60 sieve.)

 

 

 

 

 

• Collect what is caught on the #60 sieve.

 

 

 

 

 

Grinding Cysts to Release Eggs

Procedure:

• Wash the cyst/sediment suspension (from elutriator or manual extraction method) from the 100 ml beakers into the small PVC #60 sieve.

 

 

 

 

 

• Place a #200 over #500 sieve 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.

 

 

 

 

 

• Grind the material on the sieve lightly between the rubber stopper and sieve. Periodically rinse with the wash bottle. This will maintain lubrication between the sieve and pestle and also rinse eggs through the #200 sieve. Grind until nothing remains upon the small PVC sieve except sand.

 

 

 

 

 

• Release pressure on the foot pedal to stop the drill press, wait for the drill press to stop spinning, and rinse the rubber stopper.
• Wash the material on the #500 sieve back into the original 100 ml beaker.

 

 

 

 

 

• Carefully rinse all sieves under the faucet.
• Repeat for all remaining samples.
• 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.

Counting SCN Eggs

Procedure:

• Increase the volume of liquid in a sample to 100 ml with tap water.
• Examine the empty counting slide under the microscope to be certain that there are no eggs remaining in the slide from previous counts.
• Thoroughly mix the sample.
• Immediately after mixing the sample, draw sufficient volume into a glass pasteur pipet to fill the counting slide.
• Fill the counting slide from the pipet.

 

 

 

 

 

• Systematically move from cell to cell in the slide grid, counting all SCN eggs.

 

 

 

 

 

• Record total count and dilution.
• Pour the liquid from the slide back into the sample, and rinse the slide thoroughly with a water bottle.

Sources for Grinding Cysts to Release Eggs:

• Standard sieves:
  • Fisher Scientific
    1600 W. Glenlake Avenue
    Itasca, IL 60143
    (800) 223-9114 or
  • VWR Scientific
    P.O. Box 66929, O'Hare AMF
    Chicago, IL 60666
    (800) 932-5000
• The #60 (PVC) sieve:
  • David Soh
    (515) 294-5094
• Rubber stopper:
  • David Soh
    (515) 294-5094
• Motorized stirrer for rubber stopper:
  • Delta 10" drill press running at 2340 RPM (any brand would work).
• Chemicals:
  • Fisher Scientific
    1600 W. Glenlake Avenue
    Itasca, IL 60143
    (800) 932-5000

Sources for Counting SCN Eggs:

• Nematode counting slides: Chalex Corporation, 5004 228th Avenue S.E., Issaquah, Washington 98029, (425) 391-1169.