Cut Stem Inoculation Technique for S. sclerotiorum

Submitted by Tri D. Vuong and Glen L. Hartman, University of Illinois

Plant Preparation

Mycelial plugs placed on cut stem of soybean plants. Photo credit: Tri Vuong and Glen. Hartman

Germinate seven soybean seeds of each entry in 15-cm clay pots containing a 1:1:1 mixture of soil: perlite: torpedo sand. Plant each entry in three replicate clay pots and grow in a greenhouse at 27 ± 1°C and 16 h daylength. Thin seven-day-old seedlings to 5 plants per pot, and continue to grow for 6 weeks, corresponding to V5 or V6 growth stage (Fehr et al., 1971).  

Inoculum Preparation

Maintain the isolate of Sclerotinia sclerotiorum in subculture at 4°C in the dark on potato dextrose agar (PDA) medium. Use the mycelium from this culture to prepare mycelial plugs. Cut a single mycelial plug from the culture using a 3-mm diameter cork-borer and place in the center of a fresh PDA plate. Incubate at 20°C for 48 h. Transfer three plugs from the margin of the colony to a new PDA plate and incubate at 20°C for 48 h. Cut plugs from the margin of the actively growing colony to inoculate plants.

Inoculation, Incubation, and Disease Rating

Sever the main stems of plants horizontally with a sterile razor blade 0.5cm above either the the fourth or fifth node. Carefully place a single mycelial plug, mycelial-side down, on the wound. The plug adheres to the cut surface by sap exudation.

Incubate the inoculated plants in a greenhouse mist chamber with approximately 80% humidity for 48 h. Maintain the chamber at 20 ± 1°C and cover with black mesh cloth (for 80% light reduction). After incubation, transfer the infected plants to an adjacent greenhouse room at 25 ± 1°C to allow disease symptoms to develop. Measure lesion lengths 7 and 14 days after inoculation. (DAI).  

Experimental Design

Arrange pots in a randomized complete block (RCB) design with three replications and repeat each experiment twice.  

References

Fehr, W. R., C.E. Caviness, D.T. Burmood, and J.S. Pennington. 1971. Stage of development descriptions for soybeans, Glycine max (L.) Merrill. Crop Sci. 11:929-931.

Submitted by: Tri D. Vuong, Department of Crop Sciences, University of Illinois; and Glen L. Hartman, Soybean/Maize Germplasm, Pathology and Genetics Research Unit, USDA-ARS and Department of Crop Sciences, University of Illinois, 1101 W. Peabody Drive, Urbana, IL 61801.

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