Share

Part 1: Media for Pythium Culture

Water Agar

10-12 g granulated agar
500 ml distilled water

Rose Bengal Water Agar

Rose Bengal is a stain chemical readily available from scientific supply companies including VWR and Fisher.

10-12 g granulated agar
500 ml distilled water
0.08 g rose bengal

More than this amount of Rose Bengal will greatly inhibit or stop the growth of many species of Pythium. Therefore, put a little less in the agar rather than more.

OR

Rose Bengal Agar (Burr and Stanghellini. 1973. Phytopathology 63:1499-1501)

500 ml corn meal agar
0.05 g pimaricin
0.01 g streptomycin sulfate
0.07 g rose bengal
0.0025 g benomyl

Add the antibiotics/fungicides after the agar has been autoclaved and has cooled so that you can hold the bottle comfortably in your hand. Pour plates.

Most Pythium grow on this. 97% efficiency in detecting oospores. Mycelium, zoospores and sporangia also germinate.

PARP

(pimarcin, ampicillin, rifampicin, pentachloronitrobenzens)

(Jeffers, S. N, and Martin, S. B. 1986. Comparison of two media selective for Phytophthora and Pythium species. Plant Disease 70:1038-1043)

5 mg pimaricin OR 10 mg of Delvocid (50% pimaricin)
250 mg ampicillin                           
10 mg rifampicin                          
100 mg PCNB OR 133.3 mg Terraclor 75WP (75% PCNB) 

1 L corn meal agar (according to Jeffers and Martin) or clarified 20% V8 agar works well too.

PARP+B

(pimarcin, ampicillin, rifampicin, pentachloronitrobenzene, benomyl)

(Oudemans, P. V. 1999. Plant Disease 83:251-258.)

Benomyl will reduce Mortierella growth. Mortierella looks much like Pythium.

5 mg pimaricin OR 10 mg of Delvocid (50% pimaricin)
250 mg ampicillin                           
10 mg rifampicin                          
100 mg PCNB OR 133.3 mg Terraclor 75WP (75% PCNB)
10 mg benomyl                            

1 L corn meal agar (according to Oudemans) or clarified 20% V8 agar works well too.

NARF

(nystatin, ampicillin, rifampicin, and fluazinam)

Morita, Y., and Tojo, M. 2007. Modifications of PARP medium using fluazinam, miconazole, and nystatin for detection of Pythium spp. in soil. Plant Disease 91 (12):1591-1599

25 mg nystatin                             
150 mg ampicillin                           
5 mg rifampicin                           
5 µl Omega 500 (Syngenta; 40% fluazinam)

500 ml corn meal agar (according to Morita and Tojo) or clarified 20% V8 agar works well too.

Autoclave the agar.

Add antibiotics after the container has cooled enough to hold with a bare hand.

Pour plates.

Triple P

Pimaricin/Penicilin/Polymyxin - (Eckert and Tsao. 1962. Phytopathology 52:771-777)

(Some Pythiums don’t grow on this apparently because it inhibits spronagium germination.)

500 ml base media (CMA - corn meal agar)
0.1 g Delvocid (50% pimaricin)
0.025 g penicillin
0.025 g polymyxin

Autoclave the agar.

Add antibiotics after the container has cooled enough to hold with a bare hand.

Pour plates.

Clarified 20% V8 agar works well too.