I N T R O D U C T I O NThe antibiotic polyoxin D specifically inhibits chitin synthase in fungi (Corbett, 1974). The development of sporophores of the basidiomycete Coprinus cinereus has been shown to require chitin synthase activity (Gooday, I972 ; Gooday, de Rousset-Hall & Hunsley, 1976). These authors found that sporophore development of C. cinereus was inhibited by concentrations of polyoxin D similar to those which inhibited the activity of chitin synthase preparations in vitro.The edible mushroom Agaricus bisporus (Lange) Imbach contains chitin in its cell walls (Kreger, 1954;Michalenko, Hohl & Rast, 1976). To determine if chitin synthase activity is involved in mycelial growth and fruit body expansion of A. bisporus, we have examined the effect of polyoxin D on these processes. We have also examined the effect of cycloheximide, a protein synthesis inhibitor.
METHODS
Organism. Agaricus bisporus strains ~6 2 1and ~6 4 9 were used. These are of direct commercial origin.Assessment of inhibition of colony growth. Cultures of A . bisportts strain ~6 2 1 were inoculated on to malt agar plates (Wood, 1976). Polyoxin D was incorporated into the plates before inoculation by spreading 0-2 ml of sterile filtered solutions of polyoxin D on to the surface of the medium. Concentrations are expressed as those present in the agar. After inoculation the plates were incubated at 25 "C and colony diameters were measured at intervals. Ten replicate plates were measured for each treatment.Treatment of growing and excised sporophores. Growing sporophores were treated by injecting sterile solutions of polyoxin D into the stipe base. Approximately 40 pl containing 200 pg polyoxin D was introduced into the sporophore. Alternatively, sporophores were harvested at about stage 2 of development (Hammond & Nichols, 1976) by cutting across the stipe approximately I cm below the velum. Measurements were made of the stipe length and cap diameter, using calipers. The sporophores were then placed with the cut stipe ends dipping into small beakers containing 2 ml of solutions of polyoxin D at 2,20 or roo pg ml-1, cycloheximide at roo pg ml-l or distilled water. The sporophores and beakers were kept in chambers at 90% relative humidity for 2 days. During this period all of the solution was imbibed. The sporophores were then removed and the cap and stipe were measured again.Chitin content. The treated sporophores were separated into cap, lower stipe and upper stipe tissues. Samples of these were homogenized in distilled water. Duplicate samples of the homogenate were removed and dried to constant weight. Other samples of the homogenate were treated with 10% (w/v) NaOH at roo "C for 30 min and centrifuged at 6000 g for 10 min. The pellets were treated with 2% (vlv) HC1 at 20 "C for I h, centrifuged and then
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