A Semi-selective Blue Medium to Trap
Sclerotinia sclerotiorum Ascospores

A semi-selective medium to trap ascospores of Sclerotinia sclerotiorum was developed based on the work done by Williams, J. and Stelfolk, D. (1979), Ben-Jephet, Y. and Bitton, S. (1985),  and Godoy, G. et al. (1990).  This medium is a modification of the one reported by Steadman et al  in 1995.

To make one liter of this semi-selective blue medium, add:

Potato dextrose agar	30 g
Streptomycin sulfate	100 mg
Penicillin-G sodium salt	100 mg
Calcium chloride	50 mg
Bromophenol blue	50 mg
PCNB (15 mg a.i.)	20 mg (PCNB 75 WP)
Etridiazole (35 mg a.i.)	100 mg (Trubam 35 WP)

Antibiotics and fungicides were added to the medium after sterilization (121 C at 15 psi for 30 minutes).

To trap ascospores, petri dishes containing the blue medium should be left open for one or two hours, between 9 to 11 a.m. After trapping, petri dishes should be incubated in the dark at room temperature (21 to 23 C).  After four days of incubation, petri dishes are evaluated to assess the number of S. sclerotiorum colonies growing in the semi-selective medium. Four-day old colonies of S. sclerotiorum originated from ascospores have a round shape with a mycelial clump in the center part of the colony that resembles a fried egg. A yellow halo is easily observed around S. sclerotiorum colonies as a result of the pH change of the medium. This change of coloration is the result of the oxalic acid that is produced by S. sclerotiorum colonies growing on the blue medium, making it more acidic (pH 3.5-4.0).  After ten to fifteen days of incubation, a small sclerotia may be observed at the edges of the colonies.  
When a high number of ascospores are trapped, the blue medium turm yellow faster, but it will go back to blue in 10 to 15 days. This is because the buffer calcium chloride precipitates the oxalic acid, forming calcium oxalate. This will raise the pH of the medium, turning it back to blue.

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Revised July 2001 by Plant Disease and Insect Clinic