Part 2: Media for Pythium Culture

Clarified V8 juice agar or broth

Add some calcium carbonate (CaCO3) to V8 juice, stir thoroughly, and let sit at room temperature. Stir it up 2-3 times over the next hour or so.

On a colander or sieve lined with 2-3 layers of cheesecloth and held over another container, pour in the V8-CaCO3.

After most of the liquid has drained into the container, lift the cheesecloth and sediment into a strong plastic bag (like the bag that contained Petri plates). Squeeze the cheesecloth and sediment to force out more liquid. Pour the liquid through 2-3 layers of cheesecloth into the container.

Aliquot the liquid in 100 or 200ml volumes and freeze it until used.

20% V8 agar or broth. For use as broth or agar, add 100 ml of the clarified V8 to 400 ml of distilled water or 200 ml V8 to 800 ml of distilled water.

Autoclave that as broth, OR for agar, add about 10 g agar/500 ml of 20% clarified V8 broth and autoclave.

Handling the antibiotics for making selective media

In Moorman's experience, it works well to measure out the amounts of all dry antibiotics needed for 500ml of agar and store the mix in microcentrifuge tubes at -20°C. Then when you have 500 ml of molten sterile agar in a container that you can hold with your bare hand, dump in the dry antibiotics, swirl the container well, and pour plates.

This has worked very well for PARP, PARP + B, and TP. It also works well for NARF to weigh and store the NAR (nystatin, ampicillin, and rifampicin), add that to the molten agar and then add the liquid F (fluazinam) just before pouring the plates.

In Moorman's experience, it is not necessary to sterilize the antibiotics before adding them to the medium.

10% Soil Extract

Put a scoop of soil in a container and add 10 to 20 times that volume of tap water.
Stir very thoroughly. Skim off any floating material.
Stir thoroughly a few times over the next 24 hr.
Let the mixture sit quietly for a few hours to allow solids to settle out.
Slowly, slowly, decant the clear liquid into a clean container and throw away the mud.

Autoclave the decanted liquid for 1 hr.

Let the autoclaved liquid sit at room temp for 48 hr.

Autoclave again for 1 hr. Spore forming bacteria are not killed by one autoclaving.

Make 10% soil extract by diluting some of the above liquid with distilled water.
Put 50-100 ml aliquots into bottles and autoclave for 15 min.

Use the 10% soil extract to suspend boiled grass blades to be inoculated with Pythium.
The objective is to have a carbon-poor medium to encourage oospore, sporangium and zoospore development.
Pythium sporulates better on boiled grass blades in sterile 10% soil extract than on boiled grass blades in distilled water.

Corn Meal Agar

Corn meal agar from scratch. (Some batches of commercial corn meal agar do not support Pythium or Phytophthora growth well)

60 g corn meal
1 L distilled water

Steam in autoclave for 1 hr

Decant through 2-3 layers of cheesecloth.
Add the corn meal liquid in whatever amount you want to distilled water with some granulated agar.
If you want relatively clear agar, add 10-20 ml. If it is OK to have opaque agar, add more.

Autoclave this and pour plates

Fungicide resistance test

One of the most commonly used fungicides for Pythium management has been metalaxyl or mefenoxam (Subdue or Subdue MAXX). Some populations of Pythium are resistant to this chemical.

100 µg mefenoxam/ml (100 ppm) is often used as a discriminatory concentration

(Sanders, P. L. 1984. Failure of metalaxyl to control Pythium blight on turfgrass in Pennsylvania. Plant Disease 68:776-77).

If the isolate, on 100 ppm mefenoxam grows less than 50% the distance that the isolate grows on agar lacking mefenoxam, then it is considered sensitive to mefenoxam. The incubation time is 36-48 hours, depending upon the species and isolate. If 100 ppm mefenoxam does not slow growth of the isolate by at least 50% as compared to its growth on mefenoxam-free agar, the isolate is considered resistant to mefenoxam.

If many isolates are tested at one time, note the date and time of inoculation and the date and time of reading the plates. Mark the edge of the inoculum block and the edge of the colony at 2 random points, noting the date and time of marking. Then calculate the radial growth/hour. That way, you don't have to be too exact in the number of hours incubated.

If you do runs on different days with a variety of isolates, it is wise to always include one particular ‘standard’ isolate as an internal check in every run in order to determine that the test is running as 'normal'.

Method 1

Stock = 0.91 ml Subdue MAXX in 100 ml water = 10,000 µg mefenoxam/ml
1 ml stock in 100 ml molten agar = 100 µg/ml

Method 2

9.1 µl Subdue MAXX in 100 ml molten agar or 45.5 µl Subdue MAXX in 500 ml molten agar