Mycologia, 82(3), 1990, pp. 295-305. ? 1990, by The New York Botanical Garden, Bronx,NY 10458-5126 EFFECTS OF CULTURE CONDITIONS ON MYCELIAL GROWTH AND LUMINESCENCE IN PANELLUS STYPTICUS David Bermudes Centerfor Great Lakes Studies, Universityof Wisconsin-Milwaukee, 600 East GreenfieldAvenue, Milwaukee, Wisconsin53204 Valerie L. Gerlach Marquette University,Department of Chemistry, Milwaukee, Wisconsin53233 AND Kenneth H. Nealson Centerfor Great Lakes Studies, Universityof Wisconsin-Milwaukee, 600 East GreenfieldAvenue, Milwaukee, Wisconsin53204 ABSTRACT A pure cultureofPanellus stypticuswas isolated froma maturebasidiocarp and studiedfor its growth and luminescenceabilities under various environmentaland nutritionalconditions. The culturewas non-luminousgrowing submerged in definedliquid media with or withoutagitation. After a two- to three-daylag period on solid substrata,luminescence increased exponentiallywith a doubling time of 4 hours while the increasein colony radial growthwas linear.On solid substrata,growth and total light emissionwere strongly correlated under most conditions studied. Optimal conditionsincluded darkness; 28 C; pH 3.8; glucose, maltose, trehalose,cellobiose or pectin as carbon source; and ammonia or asparagine as nitrogensource. Growthand luminescencewere inhibitedby ambient fluorescentlight. Dark-growncolonies were brightestin the centerwhile light-growncolonies were brightestat the pe? riphery.Cultures hydrolyzed starch and produced an extracellularphenoloxidase. Conditions forpro? duction of luminescentbasidiocarps by this cultureare described. Key Words: Panellus stypticus,luminescence, bioluminescence, defined media Understanding the physiology of luminescent and its biochemistry (Airth and McElroy, 1959; fungi and its relationship to light emission is im? Airth and Foerster, 1962,1964; Airth et al., 1966; portant for several reasons. First, luminescence Kuwabara and Wassink, 1966; Endo et al, 1970, is rapidly and sensitively measurable in vivo and Kamzolkena et al, 1983; Isobe et al, 1987,1988; thus constitutes a endogenous reporter system Nakamura et al, 1988; Shimomura, 1989) have that may help to reveal physiological interrela- been reported. However, the luminous fungus for tionships with environmental parameters. Sec? which physiology in relationship to its light emis- ond, expression of luminescence may be spatially sion is best understood has been reported as Col? resolved within a colony or basidiocarp and thus lybia velutipes (Curt. ex Fr.) Kummer (= Flam- used as a marker for gene expression and differ- mulina velutipes (Curt. ex Fr.) Sing.) (Airth and entiation. Finally, optimization of light emission McElroy 1959; Airth and Foerster, 1962, 1965; by fungal cultures in the laboratory may aid in Foerster et al, 1965; Airth et al, 1966), although studies of its biochemistry. it is of dubious identity (Wassink, 1978). When Several quantitative studies of the physiology strains of Flammulina velutipes obtained from of fungal luminescence (e.g., Hastings, 1952; Airth the Culture Collection at the Center for Forest and Foerster, 1960,1965; Berliner, 1961a, b, 1963; Mycology Research, University of Wisconsin- Berliner and Brand, 1962; Foerster et al, 1965; Madison (OKM 6261-sp) and by tissue culture Calleja and Reynolds, 1970a; Kamzolkena, 1982) were studied, neither basidiocarps nor mycelia 295 296 Mycologia produced measurable light (Bermudes, unpubl. ticus isolated by tissue culture were compared observ.). Other species of Collybia are also ap? for overall luminescence on MsY agar. The high? parently non-luminous [e.g., C. fusipes (Bull. ex est level of light emission was produced by cul? Fr.) Quel. and C. tuberosa (Bull. ex Fr.) Kummer] tures of P. stypticus isolated by tissue culture as assayed by the naked eye (Bothe, 1931, cited (unpubl. observ.), although others have reported in Harvey, 1952). Although we do not doubt the differentrelative intensities among various strains findings of the studies in which this fungus was of these species {e.g., Berliner, 1961b). Here we used, as suggested by Wassink (1978), we hence- report variations in growth and luminescence of forthrefer to this species in quotations. Without P. stypticus under various culture conditions. knowledge of the species studied, the work nec- essarily cannot be reproduced and many inter? materials and methods esting physiological data cannot be used. Further Culture conditions.?Panellus stypticus (strain studies ofthe physiology of luminous fungi may DB-Psl ATCC 66462) was obtained through tis? help to resolve this problem. sue culture techniques as described by Molina Airth and Foerster (1965) found that ammonia and Palmer (1982) and maintained on MsY me? or aspartic acid, glucose and pH 6.0 were optimal dia consisting of molasses (2.5% w/v) and yeast for light emission and that their isolate of "C. extract (0.5% w/v) with 1.5% agar. This and other velutipes" required the thiazole moiety of thia- cultures were selected for visible luminescence. mine. Reports vary on the level of luminescence Quantitative measurements of luminescence were in liquid as opposed to solid substrate cultures. made on fungi grown on various media as de? Airth and Wassink and Kuwabara (1966) (1961) scribed below. found no luminescence in liquid (submerged) shake cultures of Armillaria mellea [= Armillar- Light measurements. ?Luminescence was quan- iella mellea (Vahl in FI. Dan. ex Fr.) Karst.] while tified by placing material in a light-tighthousing Wassink and Kuwabara (1966) observed lumi? with a 25 mm aperture positioned 2 cm over an nescence in submerged cultures ofPanus stipticus EMI-type 9781A phototube coupled to a Pacific [= Panellus stypticus (Bull. ex Fr.) Karst.], My- Photometrics model 110 amplifier. Various cena polygramma [= Mycena zephirus (Fr. ex housings were equipped to accept Petri dishes, Fr.) Kummer], and Omphalia flavida [= Mycena culture tubes or scintillation vials. Fungal colo? citricolor (Berk. & Curt.) Sacc] grown on bread nies were usually smaller than the photometer crumb media. Calleja and Reynolds (1970b) re? aperture, thus readings generally represent total ported that on agar substrata luminescence by luminescence of the colony. In cases where the Armillaria mellea and Panus stypticus occurred colony expanded past the aperture opening, read? on all types of hyphae (primary, secondary, ter- ings continued to be taken from the center ofthe tiary, aerial, superficial and submerged) at some colony. All measurements were made between time in their development but not in actively 1200-1500 h. Light emission was calibrated us? growing apices. Diurnal periodicity of lumines? ing a radioactive light source as described by cence was observed in P. stypticus,A. mellea and Hastings and Weber (1963). The emission spec- M. polygramma (Berliner, 1961a; Calleja and trum maximum of the standard was 416 nm Reynolds, 1970a). Short-wave ultraviolet light (fungal emission maxima are approx. 530 nm; (280 nm) reversibly inhibited P. stypticus lumi? Spruit-van der Burg, 1950), and no correction nescence while longer wavelength (366 nm) ul? for variation in spectral sensitivity was made. traviolet was stimulatory (Airth and Foerster, The limit of detection was 2.2 x 105 quanta (q)/ 1960; Berliner and Brand, 1962). In Armillaria see. mellea, 280 nm ultraviolet light stimulated lu? Photography.?Photography of luminescence was minescence (Berliner, 1963). performed using Kodak Tri-X film in a Nikon Strains of Armillariella mellea (GB 795-s and camera with a 55 mm micro lens with aperture GB 895-s), Omphalotus olearius (DC ex Fr.) Sing. settings ranging from f2.8 to 8. Exposure time (900-22-s, HHB 7441-s, and HHB 2668-s) and ranged from 2-24 h. Panellus stypticus (OKM-3787-s, T-79-s, and RLG-6828-s) from the Culture Collection at the Light effects.?The effeetof ambient fluorescent Center for Forest Mycology Research, Univer? lighting on fungal light emission was determined sity of Wisconsin-Madison and strains of P. styp- by comparing cultures grown under darkened Bermudes et al.: Panellus Growth and Luminescence 297 conditions with those grown under ambient light. solution (see below) was added. pH was adjusted Subsequently, dark-incubated colonies were ex? to the desired value using 20% phosphoric acid. posed to ambient light and light-incubated col? Twenty-five ml of a 4 x phosphate-citrate buffer onies were darkened and the effeetof these treat? was added to create a stable buffered pH. This ments measured. solution was poured into glass Petri dishes and autoclaved, at which time gelation occurred; me? Temperature effects.? Temperature optimum for dia so prepared usually varied less than 0.03 pH both growth and luminescence was determined units. on dark-grown colonies. Colonies were grown under constant temperature conditions and col? Substrate utilization.?The effectof thiamine and ony size and total light output were determined. utilization of various carbon and nitrogen sources In addition, individual colonies grown at 22 C by P. stypticus was studied using a defined vari? were cooled and/or warmed at 2? increments for ation ofthe MMN medium as described by Moli- 20 minute intervals to temperatures ranging from na and Palmer (1982) lacking malt extract. The 4 to 38 C using a water jacket housing mounted basal MMN medium consisted of 0.25 g directly on the photometer. (NH4)2HP04 0.5 g KH2P04, 0.15 g MgS04? 7H20 0.05 g CaCl2, 0.025 g NaCl, 1.2 ml of 1% FeCl3 effeetof variables on lumi? pH optimum.?The solution per liter of distilled