March, 1995                                                                      PDF Version

Pythium Seed Rot and Damping-off Resistance
Pythium spp.

N.A. Altier, D.K. Barnes, J.A. Thies, and D.A. Samac


Growth chamber


9 cm-diameter petri plates
Medium............. 1.5% water agar (previously inoculated with Pythium)
Temp/Light........ 18 C; 14 hour daylength
No. of Seeds .... 25 per plate (replication)
No. of Reps....... 3 minimum



Baiting with alfalfa seedlings in infested soil. (See sources of inoculum section).
Storage/Temp.... Isolates stored on cornmeal agar (4 C) will remain viable for 4-6 months. Isolates stored in sterile water (room temperature) will remain viable for at least 12 months.
Production......... Cornmeal agar inoculated with an isolate from stored cultures and incubated at 24 C for 2-3 days.
Preparation........ A 3 mm-diameter disc of inoculum is removed from the periphery of the 2-3 day-old colony growing on cornmeal agar and placed in the center of a separate 9 cm-diameter petri plate containing 1.5% water agar. Plates are incubated 3 days at 24 C prior to plating seeds.


Plating Seed.... Surface-sterilized seeds are equidistantly spaced in a radiate pattern on Pythium-inoculated agar surface using a vacuum template. Test checks include surface-sterilized seeds placed on uninoculated plates of water agar for determining expected numbers of dead seed. Hard seed are not used in test calculations.
Location........... Environmentally controlled chamber maintained at 18C. Temperature during incubation is critical. Lower temperatures will increase disease severity and accuracy of disease rating may be compromized, higher temperatures favor alfalfa seedling growth causing less disease severity (1,2). Average soil temperature during alfalfa seeding dates for a target area may be considered when choosing a temperature to use during a plant resistance selection program.
Age at Rating..... 5 days after plating seeds.



Score each individual seedling.

1 Resistant....... healthy seedling: primary root free of necrosis; a slight discoloration of the primary root may occur
2 Resistant....... infected seedling: prirnary root tip necrotic but firm
3 Moderately Susceptible....... infected seedling: prirnary root tip soft and rotted
4 Susceptible.... dead seedling: germinated seed with emerged radicle rotted
5 Susceptible.... dead seed: ungerminated seed rotted

Ratings for each plate may be expressed as percentage Resistant Plants and as an Average Severity Index (ASI). The rating method is similar to one used to evaluate alfalfa germplasms and flax germplasms for seedling darnping-off caused by Rhizoctonia solani Kuehn (3,4).

Resist. Plants = 100 x total of seedlings in classes 1 and 2 
                       Number (N) of seeds expected to 
                       germinate in the ninoculated check 
                       (calculated by subtracting tbe number
                       of dead seed from the total number of
                       swollen seed)
ASI = [(N class 5 seeds-N dead seeds in ck.)5 + (N class 4)4 + (N class 3)3
                + (N class 2)2 + N class 1]/N    


  Approximate Average Severity Index
  W3 GR1 L3
Florida 77 2.6 4.2 4.8
Alfagraze 2.7 4.2 4.7
Wrangler 2.8 4.2 4.8
Saranac 4.5 5.0 5.0


  Approximate Expected Resistance (%)/Isolate
  W3 GR1 L3
Florida 77 55 0 1
Alfagraze 42 0 0
Wrangler 43 0 0
Saranac 0 0 0


  Acceptable Range of Resistance
Florida 77   45-65  
Alfagraze   32-52  
Wrangler   33-55  
Saranac   0-5  


Because the culture plate method maximizes inoculurn pressure, correlation to field reaction should be satisfactory if the laboratory and field isolates are similar.


The following Pythium species have been reported (1) to be highly pathogenic to alfalfa seedlings in North America: P. debaryanum Hesse, P. irregulare Buisman, P. paroecandrum Drechsler, P. splendens Braun, P. sylvaticum Carnpbell & Hendrix, and P. ultimumTrow. No races are known.

Name .........Deborah A. Samac
Address.......Department of Plant Pathology
                   University of Minnesota, USDA/ARS
                   495 Borlaug Hall, 1991 Buford Circle
                   St. Paul, MN 55108
Phone .........612-625-1243
FAX ............612-625-9728


1. Altier, N.A. 1993. Development of a culture plate method to evaluate and select alfalfa for resistance to Pythium seedling diseases. M.S. thesis. Univ. of Minnesota, St. Paul.

2. Altier, N.A., and J.A. Thies. 1992. p.63. A method to screen and evaluate alfalfa for seedling resistance to Pythium spp. In Proc. 33rd North Am. Alfalfa Improv. Conf. Atlanta, GA. (Abstr.).

3. Barnes, D.K., and N.A. Anderson. 1988. Rhizoctonia resistance. Laboratory and greenhouse methods. p. 100. In Proc. 31 st North Am. Alfalfa Improv. Conf. Beltsville, MA. (Abstr.).

4. Garza-Chapa, R. and N.A. Anderson. 1966. Behavior of single basidiospore isolates and heterokaryons of Rhizoctonia solani from flax. Phytopathology 56:1260-1268.


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