Updated 2015

 The first is a modification of the method of Nelson et al. (3) and is based on sclerotial production on cornmeal-vermiculite (outlined below):

1. Place the following in a 1 L beaker:
1. 108 g cornmeal;
2. 7 g vermiculite;
3. 0.75 g casamino acid;
4. d. 0.75 g yeast extract; and fill with 74 ml distilled water.
2. Cover the beaker loosely with plastic wrap, and microwave on high for 3 min.
3. Remove and immediately chop up the mixture with a spatula until cool.
4. Microwave uncovered for 20 sec and chop as above.
5. Place in glass containers (i.e. Pyrex dishes or beakers) covered with foil and autoclave for 15 min.

The alternative method used to produce sclerotia in our laboratory is on wheat grain:

1. Place 25 g of wheat grain in a vessel such as a conical flask or beaker.
2. Add 50 ml distilled water and autoclave.

Aseptically, place mycelial plugs that have been colonized by S. sclerotiorum into the cornmeal-vermiculite or grain and place at room temperature to allow for mycelial growth and production of sclerotia (~ 2 to 3 months).  In the latter protocol, wheat may be substituted for other types of grain but the shape of the wheat grains promotes the production of larger sclerotia.  Sclerotia can then be removed from either media by rinsing and sieving for use in the conditioning steps described below.

If naturally produced sclerotia can be obtained from sources such as diseased beans or sunflower omit the production steps described above.

Conditioning

Sclerotia must go through a conditioning process to induce carpogenic germination (1, 2).

To condition, place the sclerotia in cheesecloth bags and tie closed.  Place the bags in a bucket filled with tap water in a cold room (7°C). In our cold room, lights are continually on and air is bubbled through the water using an aquarium pump (Figure 1). Discard the water on a weekly basis and add fresh water to avoid algal build-up. Sclerotia should remain in the cold water bath for at least 4 weeks or longer as necessary. The time required for conditioning is likely to vary between S. sclerotiorum isolates.  If the conditioning time is too short, sclerotia will not rapidly produce apothecia. If the conditioning time is too long, the stipes will grow leggy in the dark cold room. After 8 weeks, check the bags weekly for sclerotia with initials that have protruded through the cheesecloth.  Some isolates will adequately produce apothecia without producing stipes in the cold water bath; however, to ensure rapid formation of apothecia, use only sclerotia with stipe initials forming for the next steps. Conditioned sclerotia may be stored for up to 2 years by air drying overnight and storing the sclerotia in a refrigerator (4-7°C) in glass Petri dishes sealed with parafilm.

Figure 1. Conditioning sclerotia in aerated water in a cold room. (Courtesy H.R. Dillard)

Carpogenic germination

To produce apothecia and ascospores from conditioned sclerotia, spread 45 cc of clean dry sand in each deep dish glass or plastic Petri plate (9 cm diameter by 2 cm depth) and shake gently to level the sand. Moisten the sand in each dish with distilled water and autoclave the dishes for 15 min. When cool, place 20-50 sclerotia (depending on size) on the sand surface of each dish, and press to embed the sclerotia. Add distilled water to each plate to saturation (standing water, but sclerotia are not submerged). Place the plates in a growth chamber at a constant temperature of 20°C and relative humidity of about 30%. If the humidity is too high condensation forms on the underside of the Petri dish lid which drips water onto the apothecia and results in rot. Maintain cool white fluorescent lights for 12 hr per day (0600 to 1800) providing light intensity of 100-132 microeinsteins m-2 sec-1 or 1000-1320 lux. Light intensity that is too low or too high will reduce or eliminate ascospore production.

After the initial watering, the sand in the Petri plates must be kept moderately moist to saturated. Check moisture content in the plates every 2 to 3 days.  If the sclerotia are conditioned properly, only 1 to 2 weeks are necessary for apothecia to begin to form.

Ascospore collection

Ascospores will be produced for at least three weeks. Mature apothecia are induced to forcibly discharge ascospores by lifting the Petri dish lid (Figure 2). Ascospores can be collected using the method described by Steadman (4). Ascospore density upon collection may be higher at least six hours into the 12-hour light cycle.  Do not discard plates prematurely because apothecia production time can vary and flushes of new apothecia are often produced.

Figure 2. In vitro production of apothecia on sterile sand and ascospore discharge of Sclerotinia sclerotiorum. (Courtesy H.R. Dillard)

References:

1. Cobb, A. C., and Dillard, H. R. 1996. A reliable method for producing ascospores of in vitro. Phytopathology 86: S94.  
2. Dillard, H. R., Ludwig, J. W., and Hunter, J. E. 1995. Conditioning Sclerotia of Sclerotinia sclerotiorum for carpogenic germination. Plant Dis. 79:411-415. 
3. Nelson, B., Duval, D., and Wu, H. 1988. An in vitro technique for large-scale production of sclerotia of Sclerotinia sclerotiorum. Phytopathology 78:1470-1472.
4. Steadman, J. R. 1974. A Simple Method for Collecting Ascospores of Whetzelinia sclerotiorum. Plant Dis. Rep. 58:190.