MATERIALS AND METHODS
Teleomorphic materials were prepared for
examination by crushing the centrum contents beneath a coverslip. Material was mounted in
water in order to make critical measurements and to observe true colors. A drop of
Melzer's reagent [potassium iodine, 1.5 gm; iodine, 0.5 gm; distilled water, 20.0 ml;
chloral hydrate, saturated solution, 20.0 ml] was added to determine whether or not the
ascus apical ring blued (the amyloid iodine reaction); the reaction of the ring and its
morphology were noted. Centrum material was also mounted in 10% KOH to assess whether or
not the ascospore perispore--the outermost ascospore wall layer--is dehiscent. Finally,
fragments of stroma and perithecial wall were placed in 10% KOH on a microscope slide and
color of extracted pigment, if any, was compared with Rayner's (1970) A Mycological
Colour Chart. External stromatal colors were also recorded and coded after
Rayner (1970). Recording the internal stromatal colors was possible only under a
dissecting microscope. Since colors are subject to shifts under different light sources,
the internal colors were not coded.
For initiating cultures, a portion of the stromatal surface including the upper parts of perithecia were removed with a sterile razor blade. The contents of the exposed perithecia were scooped out and were spotted with a fine-tipped sterile forceps in Petri dishes containing Difco potato-dextrose agar to which 5 g/L Difco yeast extract was added [PDYA]. Hyphal tips emerging from the perithecial contents then were cut and transferred to fresh media. Therefore, the isolates obtained presumably originated from multiple ascospores. Isolates were cultured on Difco Oatmeal Agar [OA] and Scratch Malt Extract Agar [SMEA, Kenerley and Rogers (1976)] in 9-cm diameter plastic Petri dishes that were incubated on a laboratory bench at ca. 20 C under 12 hours fluorescent light. All culture descriptions are from colonies on OA, unless otherwise noted.
Single spore cultures were obtained by manually manipulating a sterile fine glass needle under a dissecting microscope. Ascospore masses were streaked onto 2% water agar. Single ascospores were manipulated by a glass needle into a clean area of the agar. A small knife was used to cut the agar around the single ascospore into a small block which was then transferred to OA plates.