Shake cultures, in contrast to static cultures of Penicillium digitatum grown in lquid medium, were induced by methionine to produce ethylene. The induction was concentration-dependent, and 7 mm was optimum for the methionine effect. In the presence of methionine, glucose (7 mM) enhanced ethylene production but did not itself induce ethylene production. The induction process lasted several hours, required the presence of viable mycelium, exhibited a lag period for ethylene production, and was effectively inhibited by cydoheximide and actinomycin D.Thus, the methionine-induced ethylene production appeared to involve induction of an enzyme system(s). Methionine not only induced ethylene production but was also utilzed as a substrate since labeled ethylene was produced from [14Cjmethionine.Following induction by the fungus, filtrates of induced shake cultures also evolved ethylene in increasing amounts by both enzymic and nonenzymic reactions. Tracer experiments indicated that the ethylene released by the filtrate was derived from a fungal metabolite of methionine and not directly from methionine.It is now well established that ethylene, a plant hormone, is produced not only by higher plants (11,12) but also by a wide variety of microorganisms (7,8,20,22,25,26). Because of its association with citrus fruits (3), ethylene production by Penicillium digitatum Sacc. has been studied extensively; cultural conditions have been defined (6, 24) and attempts made to elucidate its biosynthetic pathway (9,10,23). When the fungus is cultured on liquid media under static conditions (without shaking), ethylene production is high and appears to be related to the surface development of a mycelial mat (24). However, in shake culture the mycelium grows as submerged ball-like structures and produces little or no ethylene although growth is equivalent or exceeds that in static culture (6,24). Ethylene produced by P. digitatum grown on liquid medium in static culture was shown (4, 9, 10) not to derive from methionine, the common precursor of ethylene in higher plants (11,12,25), but from 2-ketoglutarate and glutamate (4). Other, more recent studies of fungi (5, 18) and bacteria (20), however, have indicated that methionine may be associated with the production of ethylene in microorganisms. In this study we show that shake cultures of P. digitatum could be made to evolve ethylene. We show that methionine induced the ethylene-forming system and also was the precursor of ethylene. Our report describes characteristics of this system. MATERIALS AND METHODS P. digitatum (American Type Culture Collection No. 10030) was grown on modified Pratt's medium (24). For preparation of stock cultures, potato dextrose agar slants were inoculated with the fungus, held at 22 C for several days, and stored at 4 C. A spore suspension (105 spores/ml) used as inoculum for a given test was made from a stock culture with sterile deionized H2O. Test samples were prepared in triplicate or more, by aseptically pipetting 0.5 ml of the spore suspension i...