Cyanide-Insensitive Respiration in Relation to Growth of a Low-Temperature Basidiomycete

Abstract

The cyanogenic low-temperature basidlomycete ( Shake cultures were grown on clarified V-8 Juice liquid medium. V-8 Juice (Campbell Soup Company) was centrifuged for 20 min at 10,000g, the resulting supernatant liquid was filtered successively through S and S filter paper (Schleicher and Schuell, Keene, NH) and Whatman Reeve Angel glass-fiber filters (Whatman, Inc.). The liquid medium contained 200 ml of the filtrate, 200 ml of 0.2 M glycine, and 36 ml 0.2 N NaOH/liter, at a final adjusted pH of 6.0. Cultures were started with aliquots of a homogenate of mycelial mats from two 1-to 3-week-old MYG still cultures (21). The mats were drained on nylon mesh, washed twice with 100 ml of cold sterile deionized H20, suspended in 100 ml of sterile deionized H2O, and homogenized (four times, 15 s each) in a chilled Waring Blendor. The amount of homogenate used to start cultures was determined from the average of the dry weights of four or five 5-ml aliquots (21). Dry weights were determined as described below, but with only a 2-h drying time. For cultures used in the FHL experiments, 4 to 7 ml of the homogenate, or homogenate diluted with sterile H20, containing approximately 1 mg dry weight of mycelium were added to 125-ml Erlenmeyer flasks containing sufficient V-8 Juice medium to give a culture volume of 25 ml. Cultures were incubated in the dark at 12 to 13°C on a reciprocating shaker at 100 rpm. With this culture method, exponential growth began 3 to 4 d after initiation of the cultures.To eliminate the excessive lag phase and lack of uniformity in size of mycelial fragments in the homogenate, 100-ml continuous shake cultures of the type described by Rawn and VanEtten (14) were established. Cultures started as above were homogenized after 48 to 72 h and 10-or 20-ml portions of the homogenate were used to start new cultures. This procedure was repeated until the homogenate produced was a fairly uniform suspension of hyphal fragments free of large clumps of mycelium. Continued blending and seeding maintained the fungus in this fragmented state. For growth and respiration experiments, several (usually two or three) l-d-old cultures were combined, and aliquots containing 0.9 to 1.4 mg dry weight of mycelium were used to start 25-ml cultures.These cultures were incubated at 14 to 16°C in the dark on a 1121 www.plantphysiol.org on April 27, 2019 -Published by Downloaded from