I. Objectives: Many fungi rely on sunlight to synchronize spore production with the earth's day/night cycle and to orient the direction of spore discharge. One of the most dramatic examples is provided by the zygomycete fungus, Pilobolus umbonatus (pilobolus means "hat thrower" in Latin) This fungus, which is found in nature on horse dung, and that of other herbivores, uses water pressure to shoot its black sporangium away from the dung and onto nearby grass blades. Once on the grass blade, the sporangium may be eaten by another horse; the sporangium germinates in the dung, and the process is repeated. For Pilobolus, the timing and aiming of sporangium discharge are controlled by sunlight.

There are two ways that Pilobolus can be coaxed to show its reliance on sunlight, one uses natural isolates, the other is more controlled.

Natural method. The simplest demonstration of sporangial discharge toward light requires that you collect fresh horse dung from animals kept in pasture. Do not collect dung from stabled horses because they do not feed on grass covered with Pilobolus sporangia.

You can use this same demonstration to show that the timing of discharge needs light. If you cover the jar completely, no discharge will occur. If you uncover the jar, discharge will occur, but without all the sporangia aiming at the light.

Controlled method. If you do not live where dung can be collected from pastured animals, or if you prefer more control in your class demonstrations, you can work with cultured material. You will need facilities to sterilize glassware and media, and a temperature controlled incubator with fluorescent lights. Cultures of Pilobolus umbonatus are available from Carolina Biological Supply (Burlington, NC 27215) or the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland, 20852-1776) as ATCC 36717.

II. Materials:

• Natural method
• Glass cassarole dish and cover, 10 to 13 cm deep.
• North facing window sill
• Aluminum foil or black paper
• Horse dung
• Controlled method
• Sterile glass crystalizing dishes with covers (10 cm diameter, 8 cm deep)
• Sterile malt-peptone agar
• Pilobolus umbonatus culture
• Aluminum foil or black paper
• 21^oC incubator with timed fluorescent lights
• Malt-Peptone Agar
  Malt extract (Difco) 20 g/l
  Peptone (Difco) 5 g/l
  Agar 20 g/l
  Water mix powders first, then add water to desired final volume.

III. Procedures:

• A. Natural method
  1. Put the dung in clean covered glass jars lined with a layer of paper towels. Sprinkle the dung with water and add a little water to the bottom of the jar to provide moisture.
  2. Store the jar outside, away from any direct sunlight; we use the sill outsde of a north facing window. In five or six days, a robust lawn of Pilobolus sporangiophores should be obvious on the dung.
  3. Then cover the jar, including the bottom, with black construction paper and use a dissecting needle to poke a small hole (1-2 mm in diameter) in the middle of the paper covering the top of the glass dish (NOTE: do not use aluminum foil because it will reflect light inside the glass jar and confuse Pilobolus) . When the sporangia discharge, they will strike the glass lid just opposite the hole through which the light shines.
• B. Controlled method
  1. We grow Pilobolus in deep culture dishes (crystalizing dishes) filled to a depth of ca. 1 cm with malt-peptone agar medium.
  2. The fungus is transfered under sterile conditions to the sterile cool agar and incubated at 21^oC on a shelf beneath cool white fluorescent bulbs controlled to give 12h light and 12h dark.
  3. We leave 5 cm space between the bottom of the bulb and the top of the crystalizing dish.
  4. In 3 or 4 d, Pilobolus will grow out on the agar and begin producing sporangia; a new crop will apprear for about ten days.
  5. To make the "bullšs eye", exchange the old lid for a new sterile one and wrap the dish as for the natural method.
  6. Continue to incubate the culture under the lights for another ten days. The demonstration is most impressive in the morning. If your class will be held in the afternoon, cover the hole in the morning of the day your class meets to keep some of the sporangia from discharging.
• If you enjoy Pilobolus, you should give a thought to A.H.R. Buller, a revered Canadian mycologist who studied this fungus and its antics thoroughly.

IV. References:

Buller, A.H.R. 1934. Researches on Fungi, vol. 6. London, Longmans Green & Co. Pages

Webster, J. 1980. Introduction to Fungi, 2nd ed. Cambridge University Press. Pages 214-219.