Mycologia, 82(3), 1990, Pp

Home , Basidiocarp, Mycena polygramma

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 water. (NH4)2HP04 nescence were monitored on solid as P. media, was omitted for the nitrogen source utilization does not luminesce in cultures stypticus liquid study. Agar (Difco) at a concentration of 1.5% defined media. was deter? using Optimal pH w/v, unless otherwise specified, was used as the mined on both silica and substrata. Pre- gel agar solidifying agent for the medium. The basal me? showed that the liminary investigation pH op? dium was sterilized by autoclaving. Carbon and timum was below 4.2 and that did not agar nitrogen sources were added aseptically by ster- when autoclaved, due to acid solidify presumably ilization through a 0.22 /mifilter unless otherwise plates of low pH were hydrolysis. Agar prepared noted. Release of free glucose by autoclaved 100 mM buffer by autoclaving citrate-phosphate complex carbohydrates was determined colori- from 3.2 to 4.5 and ranging (McKenzie, 1974) metrically using the glucose oxidase method (Sig? defined nutrients from the The separately agar. ma). Absorbance was measured at 505 nm with final ofthe media was recorded to the nearest pH an LKB Ultrospec 4050. tenth pH unit. ? In order to be sure these results were not in- Enzyme activity. Ligninolytic and cellulolytic activities were on the above fluenced by hydrolysis ofthe agar, silica gels were assayed by growth also used as a solid substratum. Potassium sili- defined medium with cellulose as carbon source. Extracellular an indica- cate was prepared following Funk and Krulwich phenoloxidase activity, tor of was tested for the (1964) using 10 g powdered silica gel (100-200 ligninolytic activity, by o-tolidene described Leslie and Leon? mesh) dissolved in 7% KOH by heating on a hot reagent by ard and the addition of tannic acid plate and the volume adjusted to 100 ml with (1979) by at a concentration of 0.5 additional 7% KOH. Batch equilibration of al? (Fisher) g/L (Nicholson and Starch kaline potassium silicate (pH approx. 12) was Robinson, 1983). hydrolysis activity was detected 4-week-old colonies on performed by adding sufficientcation exchange by flooding with Gram-stain resin (Biorad G 50 W-X4) to bring pH to < 10, starch-containing agar plates iodine and then removing the resin by filtrationand adding (Smibert Krieg, 1981). back non-resin-treated potassium silicate to bring Basidiocarp production.? A modification ofthe pH to 10 (Sommers and Harris, 1968). The resin procedure for basidiocarp production by Pleu- was collected on a Buchner funnel using What? rotus ostreatus (Jacq. et Fr.) Kummer described man #2 filterpaper. The silica solution was mea? by Stamets and Chilton (1983) was utilized. The sured into 50 ml aliquots and autoclaved. During substrate consisted of a 15 x 30 cm piece of autoclaving the pH changed to approximately corrugated cardboard weighing approx. 40 g, 9.8 and a small amount of precipitate formed rolled up tightlyinto a 15 cm high cylinder bound which was removed by centrifugation. Subse- together with plastic coated electrical wire. The quent autoclaving did not alter the pH. To each cardboard cylinder was placed into a quart-sized 50 ml aliquot of autoclaved, ion-exchange treat? wide mouth canning jar and soaked with 100 ml ed silicate, 25 ml of 4 x -concentrated nutrient of MsY medium. Several holes were punctured 298 Mycologia Bermudes et al.: Panellus Growth and Luminescence 299 in the lid which was fittedwith a non-absorbent Table I cotton filter.This substrate was autoclaved and Temperature dependency of growth and allowed to cool. Panellus stypticus grown in liq? luminescence of panellus stypticus under dark uid media was aseptically homogenized by pas? CONDITIONS3 sage through a 16 gauge needle. Twenty ml of the mixture was uniformly introduced to the cardboard and incubated at 22 C and 90% rel? ative humidity for 4 weeks. The lids were then removed and several ofthe jars placed within a 10 gallon fish tank loosely fittedwith a plexiglass top. Humidity was maintained between 80-85% at 13-18 C by bubbling air through a water-containing beaker within the tank. A 15 watt flu? orescent light placed 30 cm from the side of the tank was set to a 12 h dark/light cycle.

results

Culture characteristics. ?Cultures were never luminescent when grown submerged on defined 1MsY medium 2% w/v. liquid media with or without agitation. However, plus agar on solid substrata, cultures were visibly luminescent. After transfer to a new substrate (MsY nescence at 22 C (Table I). However, lumines? there was a phase of 2-3 agar), lag days, during cence of 22 C cultures was increased 20% when which time luminescence often diminished be? the temperature of these cultures was raised to low the limit of detection. Subsequently, lumi? 28 C (Fig. 4). Additional transient temperature nescence increased exponentially with an aver? increase lowered luminescence, suggesting that age doubling time of approximately 4 hours while the lower luminescence at a sustained tempera? increase in radial growth proceeded linearly. ture of 28 C was not due simply to inhibition of On agar plates, young colonies were uniformly the enzyme system by high temperatures. luminescent while older colonies became differ- Growth under constant ambient fluorescent entiated. Older colonies grown under approxi? light at 22 C led to levels of luminescence reduced mately 12 h light/dark conditions formed con- by 38% (Table I). Dark-grown cultures exposed centric growth rings and showed maximum to fluorescent light for 5 minutes showed a rapid luminescence in one or two zones near the pe? increase in luminescence upon transfer to dark riphery of the colony, with the center consider- conditions (Fig. 5A). Emission was reduced 30% ably less bright (Fig. la, b). Colonies grown un? by exposure to light with recovery to 97% ofthe der dark conditions did not form conspicuous initial level in 40 min. Cultures exposed to flu? concentric growth rings and maintained the cen? orescent light for one hour showed an initial in? ter of the colony as the brightest region of lu? crease followed by a decrease and then a sec? minescence. However, they too had a zone of ondary increase (Fig. 5B). Cultures under increased brightness near the periphery (Fig. 2a, fluorescent illumination for three hours initially b). showed decreased levels of emission and then Light effects.? Dark-grown cultures of P. styp? increased emission by 30% within two h of being ticus exhibited optimum temperature for lumi- replaced in the dark (Fig. 5C).

Figs. 1-3. Growth formsand luminescence in Panellus stypticus.la. Culture grown under ambient light conditionson MsY agar exhibitingconcentric growth rings. lb. An autophotographofthe same cultureshowing the brightestluminescence at the periphery.2a. Culture grownunder dark conditions on MsY agar. Note the lack of concentricgrowth rings. 2b. Autophotographof the same cultureshowing brightest luminescence at the center.3a. Basidiocarps grownin the laboratory.3b. Autophotographof the same basidiocarps showingbio- luminescence. 300 Mycologia

Temperature(?C) Fig. 4. Effeetof temperatureshift on luminescence.Two separatecultures at 22 C adjusted by means ofthe photometershutter to an outputof 2.5 x 108q/sec were subjected to warmingand coolingat 2 degreeincrements for20 minutedurations. Arrows indicate the directionof temperatureshift (in degreesC). pHoptimum.?On silica gels, P. stypticusexhib? is probably due to the presence of organic con- ited maximal luminescence and growth at pH taminants in the agar. 3.8 (Table II; Fig. 6). Silica and agar gels gave Nitrogen source utilization is summarized in generally consistent results. Growth and lumi? Table IV. Ammonium and L-asparagine were nescence were greatlyreduced below 3.6 or above preferred substrata for both luminescence and pH 4.4, although specific luminescence (q/sec/ growth. Low levels of luminescence were cor? mm2) was not greatly affected; no growth or lu? related with very sparse yet broad colonies. minescence occurred at pH 6 or above. Enzyme activity.?The P. stypticus culture pos- Substrate utilization.?Thiamine (3 x 10~7 M) sessed extracellular phenoloxidase activity as de? decreased growth rate and maximum lumines? termined by the blue reaction with the o-tolidene cence on unbufferedglucose or cellulose substra- reagent (Leslie and Leonard, 1979) and intense ta but increased both on unbuffered lactose and darkening in the Bavendamm test as described sucrose media. by Nicholson and Robinson (1983). Studies with various carbon sources revealed Basidiocarp production. ? Bioluminescent basid? that pectin, lactose, maltose, glucose, cellobiose iocarps were initiated 4 weeks after cardboard and trehalose gave high levels of luminescence substrate inoculation. Many of the structures while organic and amino acids were poor carbon produced possessed incomplete caps. Fully substrata (Table III). Complex carbon sources formed mushrooms (Fig. 3a) were brightly lu- were found to release low levels of free glucose minescent (Fig. 3b) and produced viable spores when autoclaved making these experiments more which germinated into luminous mycelia. Lu- difficultto control and evaluate. Although cul? minous basidiocarps continued to be produced tures on cellulose were lumines- grown weakly for 9 months. cent when compared to those grown on free sug? and often resulted in the ars, growth persisted DISCUSSION production of basidiocarp primordia. Media lacking added carbon sources supported ex- As occurs in other fungi (Tan, 1978), light tremelysparse (yet widely extended) radial growth stronglyinfluenced P. stypticus. Both growth and which gave very low levels of luminescence. This luminescence data show that ambient fluorescent Bermudes et al.: Panellus Growth and Luminescence 301

3.23.4 3.6 3.84.0 4.2 4.4 4.6 4.7 5.2 5.6 5.8 6.0 6.26.4 6.6 6.8 7.0

Fig. 6. Effectsof pH on growthand luminescence ofPanellus stypticus.Comparison of% maximumtotal luminescenceand % maximum growthat varyingpH. Based upon both agar and silica gel experiments.

have been due to decay of endogenous fluores- cence {e.g., Cormier and Totter, 1966, or Lavelle 0 6 12 18 24 30 36 42 48 54 60 66 72 78 et al, 1972, cited in Wassink, 1978) while the TIME(min.) positive effeetof darkening these colonies quickly ensues, causing the level to increase over a Fig. 5. Lightand dark effectson lightemission by of hours of 28-day-oldcultures ofPanelllus stypticus.5A. A dark- period (Fig. 5C). Comparisons light grownculture on MsY agar exposed to fluorescentlight emission among fungi must be comparable in for 5 minutes,then placed under dark conditions (ar? their previous light exposure conditions. Berliner and monitored for luminescence. 5B. A dark- row) and Brand (1962) found that UV light of 280 nm cultureon MsY to fluorescent grown agar exposed light was detrimental to luminescence of for 1 hour,then placed under dark conditions(arrow) particularly and monitored for luminescence. 5C. A dark-grown P. stypticus and suggested that it may involve a culture on MsY agar exposed to fluorescentlight 3 photolabile component ofthe luciferase system. hours,then placed under dark conditions(arrow) and The putative luciferin from Mycena citricolor monitoredfor luminescence. isolated by Kuwabara and Wassink (1966) had an absorption maxima at 270 and 320 nm. Al? though photolytic destruction of luciferin might light inhibits this fungus (Table 1) and caused variations in its pattern of luminescence (Figs. either or dark- 1,2). Young colonies, light-grown Table II were undifferentiated while grown, relatively Effects of pH on light production and mycelial older colonies exposed to the light had their growth in Panellus stypticus* brightest zone of light emission at the periphery and dark-grown colonies had their brightest zone at the center (Figs. 1, 2). Unless utilization is made of small undifferentiated colonies unoc- cluded by the photometer aperture, quantitative measurement of light emission is complicated by the geometry of the photometer in use. Dark- grown colonies must be assayed without expo? sure to ambient light or they sufferconsiderable loss in their level of light emission although they quickly regain most ofthe initial level (Fig. 5A). Light-grown colonies must be assayed rapidly, as the photometer chamber represents a change to darkened conditions and an increase in lu? a Medium: MsY; silica gel; citrate-phosphatebuffer. minescence occurs (Fig. 5B). The initial drop of pH afterautoclaving was within 0.03 units of initial luminescence after3-hour exposure (Fig. 5C) may pH. 302 Mycologia

Table III Growth and luminescence of P. stypticus grown on various carbon sources3

a Medium is basal MMN plus carbon source, 1% w/v;nitrogen source, 1.9 mM (NH4)2HP04; citrate-phosphate buffer:Final concentrationcitric acid 33 mM, Na2HP04 40 mM; agar, 1.5%; pH 4.2 ? 0.05 unless otherwise noted. Carbon sources sterilizedby filtration(0.22 nm) except forthose marked by asterisk,which were auto? claved. nd = not determined.

be expected to give brief reductions in the level the same time each day. A single report on sea- of luminescence followed by recovery due to de sonal variation of luminescence in Armillariella novo synthesis, the cause and effectrelationships mellea (Kamzolkena, 1982) furthersuggests the of these facts are unknown. absolute need for simultaneously grown control Some luminescent fungi (Armillariella mellea, cultures. Mycena polygramma and Panellus stypticus) re- Absence of luminescence in liquid cultures on portedly exhibit pronounced diurnal periodicity defined media creates several difhculties. Large (Berliner, 1961a; Calleja and Reynolds, 1970a). quantities of luminescent mycelia are more dif? In the investigation by Berliner (1961a), diurnal ficult to obtain. Determination of pH optima periodicity was not found in Armillaria fusipes requires solid substrata which entail special prep? (= Armillariella mellea), Clitocybe illudens (= aration. Further, the use of radial extension as Omphalotus olearius), Mycena galopus (Fr.) an indicator of overall growth on such solid sub? Quelet, or Mycena citricolor. Maximum lumi? strata is also problematical. While it gives results nescence occurs between 6 and 9 PM regardless stronglycorrelated with maximal light intensities as to whether the cultures are incubated in con? for the best substrata, the poorest substrata often tinuous light, continuous darkness, or a normal have broad radial extensions with extremely dif- day-night cycle. This fact makes it imperative fuse hyphae, representing little biomass. This that time of day be noted in day-to-day com- problem is somewhat rectified by determination parisons. Preferably, measurements are made at of luminescence per unit area. Bermudes et al. : Panellus Growth and Luminescence 303

Table IV Growth and luminescence of P. stypticus grown on various nitrogen sources3

a Medium: basal MMN (minus nitrogen)plus carbon source,0.056 M D-glucose; finalconcentration of nitrogen 3.8 mM; agar 2% w/v; pH = 4.25 ? 0.05. Citrate-phosphatebuffer: final concentrationcitric acid 29 mM, Na2HP04 42 mM. Carbon and nitrogensources sterilizedby filtration(0.22 ^m pore size). b nd = not determined.

Species of luminescent fungi reviewed by Was? deficiencies for thiamine are common among sink (1978) are generally noted as occurring in fungi (Moore-Landecker, 1972) they do not con- wood-decay environments. Singer (1986) noted stitute a reliable taxonomic marker. Both fungi that P. stypticus is opportunistic in tree wounds. preferred glucose and ammonia, and each had a Thus it is not surprising that these fungi prefer preference for a particular amino acid; L-aspartic glucose and similar sugars or polymers such as acid for "C. velutipes" (Airth and Foerster, 1965) pectin as carbon sources. Based on cellulose uti? and L-asparagine for P. stypticus. lization, cellulolytic activity is low in the strain Panellus stypticusis among the most common studied (Table III) but is presumed to occur based and brightlyluminescent North American fungi. on continued growth and luminescence of basid? It is easily grown on defined or undefined media iocarps on cardboard substrata long after free and produces basidiocarps in culture on inex- num? sugars would be exhausted. Based on the pres? pensive substrata. It has been included in a ence of phenoloxidase activity, like its relative ber of studies on luminescence including some Panus tigrinus (Bull. ex Fr.) Sing. (Leslie and concerning its genetics (Macrae, 1937,1942) and Leonard, 1979), Panellus stypticus is probably biochemistry (Airth and Foerster, 1964; Naka? ligninolytic. However, radiolabeled lignin and mura et al, 1988; Shimomura, 1989) and should cellulose degradation assays would be required be useful for further studies of bioluminescent to confirm these assumptions. fungi. It is doubtful that P. stypticus studied here is the "Collybia velutipes" described by Airth and acknowledgments Foerster (1965; Foerster et al, 1965). Panellus This work was the National Institute of low for most supportedby stypticus has a pH optimum 3.8, of EnvironmentalHealth Sciences National Service fungi and did not grow at pH 6 or above while Research Award Grant #ES 07043 and NASA Grant "Collybia velutipes" did not grow at pH below #144Z212 for (DB) and the National Science Foun- Research for 5 (Airth and Foerster, 1965). "Collybia velutipes" dation Experience Undergraduates(for VG). We thankAlbert M. Boman forliving specimens required the thiazol moiety of thiamine (Airth ofPanellus and MartynJ. Dibben, Milwaukee P. stypticus and Foerster, 1965) which is not required by Public Museum fortheir identification. We are grateful stypticus although it enhanced growth and lu? to ElizabethDorworth at theUniversity of Wisconsin- minescence under some conditions. However, as Madison Centerfor Forest Mycology Research forpro- 304 Mycologia viding culturesof luminescentfungi. We also thank ofclear silica gelsthat can be streaked./. Bacteriol. Rebecca Asbek and Jo Linda Denoyer fromthe Uni? 88: 1200-1201. versityof Wisconsin-MilwaukeeYoung Scholars Pro? Harvey, E. N. 1952. Bioluminescence. Academic gram forassistance in photometricmeasurements. Press, New York. 649 p. Hastings,J. W. 1952. Oxygenconcentration and bio? literature cited luminescenceintensity. 1. Bacteria and fungi./. Cell. Comp. Physiol.39: 1-30. Airth,R. L. 1961. Characteristicsof cell-freefungal -, and G. Weber. 1963. Total quantum fluxof bioluminescence.Pp. 262-273. In: Lightand Life. isotropic sources. /. Opt. Soc. Amer. 53: 1410? Eds., W. D. McElroy and B. Glass. The Johns 1415. Hopkins Press, Baltimore,Maryland. Isobe, M., D. Uyakul,and T. Goto. 1987. Lamptero- -, and G. E. Foerster. 1960. Some aspects of myces bioluminescence?1. Identificationof ri? fungalbioluminescence. /. Cell Comp. Physiol. boflavinas the lightemitter in the mushroom,L. 56: 173-182. japonicus. J. Biolum. Chemilum. 1: 181-188. -, and-. 1962. The isolation of catalytic -,-, and-. 1988. Lampteromyces components required for cell-freefungal biolu? bioluminescence ?2. Lampteroflavin, a light minescence.Arch. Biochem. Biophys. 97: 567-573. emitterin the luminous mushroom,L. japonicus. -, and-. 1964. Enzymes associated with TetrahedronLett. 29: 1169-1172. bioluminescencein Panus stypticusluminescens Xamzolkena,O. V. 1982. Oscillation of biolumines? and Panus stypticusnon-luminescens. J. Bacteriol cence in submergedculture of Armillaria mellea 88: 1372-1379. (Fr.) Kumm. Mikologiia Fitopatologiia 16: 323- -, and-. 1965. Light emission fromthe 325. luminousfungus Collybia velutipesunder different -, V. S. Danilov, and E. S. Egorov. 1983. The nutritionalconditions. Amer. J. Bot. 52: 495-505. natureof luciferasefrom the bioluminescentfun? -,-, and P. Q. Behrens. 1966. The lu? gus Armillariella.Doklady Akademii Nauk SSSR minous fungi.Pp. 203-223. In: Bioluminescence 271: 750-752. in Progress.Eds., F. H. Johnsonand Y. Haneda. 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