Induction of Apothecia Formation and Ascospore Collection of
Sclerotinia sclerotiorum
W. A. Gutierrez,  H. D. Shew, and T. A. Melton 


 
 
The primary source of inoculum of Sclerotinia sclerotiorum can either be sclerotia (myceliogenic germination) or ascospores (released from apothecia by the carpogenic germination of sclerotia).

One of the important steps in the study of the pathogenicity of this fungus is the production of ascospores to simulate natural infection that occurs in the field.

Sclerotia Production

Sclerotia originated from cultures of S. sclerotiorum grown on carrot is used for production of ascospores.
- Infest 30g of sterile carrot disks/125ml flask with a mycelial disk of a Sclerotinia isolate.
- Incubate flask at room temperature.
- Harvest sclerotia after 15 to 20 days of incubation in sieves with 2 mm and 0.5 mm opening in running tap water, and let them dry for 48 hours.

Apothecia Induction

- Fill 120 ml. jar with 40 cc of dry sand and 10 cc of deionized water.
- Sterilize them for 30 min. (15 at 120 C).
- Disinfest sclerotia in 20% sodium hypochlorite solution for 1 min., then rinse with sterile water and allow to dry.
- Transfer 5 to 10 disinfested sclerotia into the sterile jar.
- Set jar at 4 C in the dark for 1 month, them place the jar in a 12 C growth chamber in the dark until the stipe is observed. When the stipe is 5 mm long (2 to 4 weeks), transfer the jars to a 16 C growth chamber with a 12 hour dark and light period, until apothecia are fully mature (2 to 6 weeks).

Ascospore Collection

- Mature apothecia are set in a growth chamber at 21 C and a 12 hour period of dark and light.
- Ascospores are collected using the Millipore membrane attached to a vacuum system.
- Store millipore membrane at 4 C in a dessicator.
 
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Revised July 2001 by Plant Disease and Insect Clinic