I. Introduction: Although most biology students who have studied fungi have read about ballistospore discharge in basidiomycetes and the involvement of Buller's drop, few have actually observed the process of discharge. This is a pity because it is exciting and not diffcult to demonstrate although a few minutes of patient watching are necessary Here simple techniques for demonstrating discharge are described in cultures of Itersonilia perplexans, from basidiocarp slices of the Jews' ear fungus Auricularia auricula-judae and from other basidiomycetes.

II. Materials:

• Itersonilia perplexans culture (IMI 264396: International Mycological Institute, Bakeham Lane, Egham, Surrey TW20 9TY, UK).
• Corn Meal Agar (Difco).
• Petri dishes
• Basidiocarps of Auricularia auricula-judae. These are usually found on old branches of Sambucus niger (elder) and can be used fresh. Previously collected dried specimens, many months old, can he revived by wetting for a few hours before use. Other species of Auricularia such as A. mesenterica which grows on elm stumps may be substituted.
• Razor and pith (or carrot) for hand sectioning. Trap water agar (2% agar in tap water). Trap Water Agar supplemented with 3% mannitol. A long working distance x 20 microscope objective is an advantage but is not essential.

III. Procedure:

• A. Itersonilia perplexans methods
  1. Inoculate plates of Corn Meal Agar with Itersonilia perplexans 2-4 days before needed. Incubate at room temperature (about 20^oC).
  2. Mount the Petri dish containing the culture directly on the flat stage of the microscope, i.e. without a mechanical stage unless this permits the dish to be mounted level. Focus on the surface of the agar. The fungus will have developed a clamped mycelium with swollen lateral branches which terminate in 1-spored "basidia" formed at or just beneath the agar surface. From each basidium a single sterigma projects into the air and develops a ballistospore which is asymmetrical (Figure 1 A), shaped somewhat like a lemon. When the ballistospore is mature a clearly-defined hilar appendix can be seen projecting at the base of the more flattened (adaxial) face immediately above the hilum, the point of attachment of the spore to the sterigma (Figure 1 C). The appearance of Buller's drop is best seen in spores viewed from the aspect in which the hilar appendix is clearly visible in profile. Ballistospores formed from the basidia are termed primary ballistospores. Discharged primary ballistospores can germinate directly on fresh media to form a clamped mycelium but if they fall on to or close to an existing colony or on to nutrientpoor media such as Tap Water Agar they may germinate repetitiously to produce secondary ballistospores by forming a single sterigrna on which a secondary ballistospore develops by transfer of all the cytoplasm from the primary ballistospore (Figure 1 B). The secondary ballistospores are also discharged. This means that it is possible to follow the discharge process both of primary and of secondary ballistospores. A good way to subculture Itersonilia is to invert an actively sporulating culture in a Petri dish over a fresh plate of Corn Meal Agar for a few minutes to allow ballistospores to be projected onto it. This technique selects for the ability to produce ballistospores and helps preserve the culture in a suitabte state for demonstrating discharge. Subculturing by mycelial transfer may result in the cultures forming yeast-like colonies with few basidia. A population of secondary ballistospores of approximately the sarne age can also be obtained by inverting an actively sporulating plate culture of Itersonilia over a fresh plate of Tap Water Agar. The best way to follow discharge is to scan the surface of the culture with a low power objective (about 10x or 20x) to look for spores in profile which have already begun to develop Buller's drop at the hilar appendix. Because the interval between the appearance of Buller's drop and discharge is of the order of 40-50 sec. there is usually ample time to do this. Look for a "double" structure consisting of an ellipsoidal spore with a spherical body attached to it. If possible centre this and swing a higher power objective into place over it.
  3. Another technique is to cut out a slab about 1 cm square from an actively growing culture of Itersonilia and place it face down on a plate of Tap Water Agar. Incubate at room temperature (20�C) for a few hours or overnight. Basidia close to the cut edge of the slab will develop sterigmata projecting parallel to the surface of the Tap Water Agar. The development of a ballistospore from the first appearance of a swelling at the tip af the sterigma to discharge takes about 40-50 minutes. By mounting the whole Petri dish and scanning the edge of the block, it is possible to find mature ballistospores in profile view. The disadvantage of this technique is that, unless the developing spore is close to the upper surface of the cut-out culture slab, it may not be possible to focus it with a higher power objective because the working distance is too small. However one advantage of this method is that it allows the measurement of the horizontal distance of discharge by estimating the distance between the cut edge of the culture slab and the discharged spores which accumulate on the surface of the Tap Water Agar. This is done either by the use of a mechanical stage on the microscope or by a calibrated eyepiece graticule.
• B. Auricularia auricula-judae methods
  1. Using fresh basidiocarps cut out a small piece about l0 x 5 mm with a sharp scalpel or razor blade. If dried material is used allow it to take up water overnight by placing it in a closed dish on moist tissue paper. Place the basidiocarp piece between elder pith or carrot root and cut transverse sections by hand with a razor or razor blade. The sections do not need to be thin, say about 0.5 - 1mm thick. Place the basidiocarp slices on the surface of Tap Water Agar in a closed Petri dish and incubate at room temperature (20�C) for about 4 hours. Fresh ballistospores will develop during this time from the tips of epibasidia which project from the pink hymenial surface. Transfer the base of the Petri dish to the flat stage of the microscope and scan the hymenial edge of the slice to look for mature ballistospores which are sausage-shaped in lateral view showing the hilar appendix (Figure 3 A). Under higher power look for the largest, most mature spore and keep it under observation if necessary for several rninutes.
  2. When basidiocarp slices are placed on Tap Water Agar + 3% mannitol the water vapour concentration above the agar surface is lowered and this has an effect on the rate of expansion of Buller's drop and the time taken between the appearance of Buller's drop and spore discharge.

IV. Results expected:

• Itersonilia perplexans. With a little experience it should be possible to observe the later stages of development of Buller's drop and the disappearance of the spore as it is shot away. It should be noted that as Buller's drop expands to a diameter of about l0 �m, roughly equivalent to the breadth of the spore, there is no decrease in the dimensions of the spore (Figure 2). This has been interpreted as being due to the expansion of the drop by condensation of water vapour from a near-saturated atmosphere around hygroscopic rnaterial extruded at the hilar appendix. If suitable microscope equipment is available (e.g. a long working distance objective with sufflciently high magnification) it may be possible to see that shortly before and during the time that Buller's drop is expanding a second film of liquid, the adaxial drop, is also expanding along the adaxial face of the spore (see Figure 2). When the two drops coalesce Buller's drop moves very rapidly up the face of the spore, resulting in a momentum which carries the spore away. Sometimes the separation of the spore from the sterigma at the hilurn fails to occur at the time that the two drops coalesce. This causes the formation of a large lens-shaped drop on the adaxial face of the spore, followed, some seconds later, by the toppling of the spore from its perch as the attachment of the spore to the sterigma does eventually weaken (Webster and Kaeding, 1994). The horizontal distance of discharge has been estimated to be 1.5 mm.
• Auricularia auricula-judae. Using basidiocarp slices on Tap Water Agar, as with ltersonilia, it should be possible to see the appearance of Buller's drop, the adaxial drop and the disappearance of the ballistospore as it is shot away. The distance of discharge can be estimated by measuring the distance between the hymenial surface and the position of spores which accumulate on the surface of the Tap Water Agar. Using slices from the same basidiocarp placed on Tap Water Agar + 3% mannitol the spore output is considerably reduced. The rate of drop expansion is slowed down and the time interval between the appearance of Buller's drop and spore discharge is extended. Over Tap Water Agar the earliest time of discharge is 7 seconds after the appearance of Buller's drop but with 3% mannitol the earliest time observed is 22 seconds and sometimes spore discharge fails to occur. This is believed to be due to the failure of the two drops to expand sufflciently to bring them into contact with each other.
• Other basidiomycetes. Essentially the same method of demonstrating ballistospore discharge works equally well with other basidiomycetes especially if they have large spores. Some of these are illustrated in Figure 3 . I have used it, for example, with the agarics Laccaria striatula and Oudemansiella mucida by placing tangential slices of their caps on Tap Water Agar. Heterobasidiomycetes such as Exidia thuretiana and Ditiola peziziformis are also suitable but here sections are made by cutting basidiocarp slices with a scalpel. Rust fungi such as Puccinia graminis, P. malvacearum and Gymnosporangium spp. can be treated in a similar way. Straws with telia of P. graminis collected in spring are wetted and short lengths placed on Tap Water Agar. Sections through lesions of P. malvacearum on leaves or petioles of Malva or Althaea are placed on Tap Water Agar. Ballistospores develop in a few hours. Similarly when the telial horns of Gymnosporangium collected in spring on Juniperus are incubated on Tap Water Agar ballistospores are freely developed. The teliospores can be dissected out and also form ballistospores. This is another fungus which can be dried and reconstituted by moistening when required.

V. Cautionary Notes: Successful ballistospore depends on an absence of free water but requires an atmosphere saturated with water vapour around the ballistospores. For this reason spore discharge cannot be observed in material mounted in water under a cover slip but only in moist air. Bright microscope lighting will inhibit water vapour uptake as will a dry laboratory atmosphere. Always use freshly made up agar with a high water content consistent with the formation of a firm gel and avoid the use of media which have been stored for a long time. Replace Petri dish lids between observations to minimize evaporation.

VI. References:

Webster, J., Davey, R.A., Duller, G.A., and Ingold, C.T. (1984). Ballistospore discharge in Itersonilia perplexans. Transactions of the British Mycological Society 82: 13-29.

Webster, J., and Davey, R.A. (1985). A simple technique for demonstrating Buller's drop and ballistospore discharge from agaric gills. Transactions of the British Mycological Society 85: 513 -514.

Webster, J., Proctor, M.C.F., Davey, R.A., and Duller G.A. (1988). Measurement of the electrical charge on some basidiospores and an assessment of two possible mechanisms of ballistospore propulsion. Transactions of the British Mycological Society 91: 193-203.

Webster, J., Davey, R.A., and Turner J.C.R. (1989). Vapour as the source of water in Buller's drop. Mycological Research 93: 297-302.

Webster, J., and Chien, C-Y. (1990). Ballistospore discharge. Transactions of the Mycological Society of Japan. 31: 301-315.

Webster, J., and Kaeding, J. (1994). Ballistospore discharge. 16 mm film. Institut f�r den Wissenschaftlichen Film, Nonnenstieg 72, Gottingen, Germany.

Figure 1. Itersonilia perplexans.

• A.Clamped mycelium bearing a basidium with a single sterigma and mature primary ballistospore seen in lateral profile.
• B.Primary ballistospore which has developed a secondary ballistospore also seen in profile. Scale bar = 20 �m.
• C.Diagram of ballistospore labelled to show the terrns used.

Figure 2. Itersonilia perplexans. Two ballistospores showing the expansion of Buller's drop and the adaxial drop. The adaxial drop is visible in C3-C5 and can be seen by the change in the outline of the spore in Dl-D4. Note that there is no change in the linear dimensions of the spore as the two drops expand. From Webster et. al. ,(1984).

Figure 3. Ballistospores of a range of basidiomycetes. The spores are photographed in pairs. The number to the left of each photograph gives the time in seconds before discharge. The right hand photograph was taken 1 second before discharge. Note the adaxial drop in each of the right hand photographs.

• A: Auricularia auricula-judae.
• B: A. mesenterica.
• C: Exidia thuretiana.
• D: Oudemansiella mucida.
• E: Puccinia graminis.