New York State Agricultural Experiment StationCornell Universityacc3@cornell.edu
Cobb, A.C., and H.R. Dillard, 2004. Production of Apothecia and Ascospores of Sclerotinia sclerotiorum. The Plant Health Instructor. DOI: 10.1094/PHI-T-2004-0604-01
Apothecia of Sclerotinia sclerotiorum are frequently used to study development and structure of asci and ascospore release. We produce sclerotia in the laboratory on cornmeal-vermiculite using the method of Nelson et al. (3), with the following modifications. The recipe is doubled and 24 additional ml of distilled water are added to a 1-liter beaker. Cover loosely with plastic wrap, and microwave on high for 3 min. Remove and immediately chop up the mixture with a spatula until cool. Microwave uncovered 20 sec and chop as above. Put in glass containers covered with foil and autoclave 15 min. If naturally produced sclerotia can be obtained from sources such as Sclerotinia-infected beans or sunflower omit the production step. Sclerotia must go through a conditioning process to induce carpogenic germination (1,2).
To condition, tie the sclerotia in cheesecloth bags and put the bags in a bucket filled with tap water in a cold room (7°C). Continuously bubble air through the water using an aquarium pump (Figure 1). Discard the water once each week and add fresh water. Condition at least 8 weeks or longer as necessary. If the conditioning time is too short, sclerotia will not rapidly germinate carpogenically. 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.
Figure 1. Conditioning sclerotia in aerated water in a cold room. (Courtesy H.R. Dillard)
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.
To produce apothecia and ascospores from conditioned sclerotia, spread 45 cc of clean dry sand in each deep dish glass 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. Ascospores will be produced for about three weeks or more. 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). 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)