Oat Grain Inoculation Technique for S. sclerotiorum

Submitted by Anne Dorrance, Department of Plant Pathology, Ohio State University

Inoculum Preparation

A 3 mm diameter hole is cut with a hole-puncher in the center of the cotyledon. One oat kernal colonized by S. sclertiorum is placed in the cotyledon. Photo credit: Anne Dorrance

Reaction of test variety (middle) compared to susceptible standard Williams 82 (left) and partially resistant standard S19-90 (right).  Photo credit: Anne Dorrance. Click on the image to see a larger version.

Soak oats overnight in distilled water, then autoclave for 1h on two consecutive days in large flasks. Add three 5 mm agar plugs colonized by S. sclerotiorum to the oats and incubate at room temperature for approximately 10 days. Shake the flasks every three days to ensure even colonization of the oats. When the oats are well colonized but no sclerotia have formed (typically after 10 days), remove the oats from the flasks, dry on an air bench overnight, and store at 5°C.

Inoculation and Disease Rating

Plant fifteen to twenty soybean seeds in coarse vermiculite in 4-inch pots in the greenhouse (14 h light). Inoculate seedlings after approximately ten to twelve days when the unifoliates are beginning to expand. Cut a 3 mm diameter hole with a hole-puncher in the center of the cotyledon approximately 1 to 1.5 mm from the stem. Place one oat kernel colonized by S. sclertiorum in the cotyledon. Inoculate  ten to twelve seedlingsin each pot. 

Place the pots in a mist chamber (20°C, 12 hour light) for 48 hours at 100% relative humidity. Remove the pots from the moist chamber and place in the greenhouse. Take data on the number of plants that are dead and alive when the control pots (Williams 82) are approximately 80% dead, usually 24 hours after removal from the mist chamber. 

Every 10 soybean varieties or plant introductions are evaluated with control varieties (we use S-1990 and Williams 82). The entries are evaluated in a randomized complete block design with replicates in time due to limits in the mist chamber. 

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