Luminescent Intensity of Cultured Mycelia of Eight Basidiomycetous Fungi from Japan
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Mushroom Science and Biotechnology, Vol. 24 (4) 176-181, 2017 Copyright © 2017, Japanese Society of Mushroom Science and Biotechnology Regular Paper Luminescent intensity of cultured mycelia of eight basidiomycetous fungi from Japan Yoshie TERASHIMA1)*, Hitoshi NEDA2) and Masaru HIROI3) 1) Tropical Biosphere Research Center, University of the Ryukyus, 1-banchi Senbaru, Nishihara, Nakagami-gun 903-0213, Japan 2) Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan 3) Koriyama Women’s University, Kaisei 3-25-2, Koriyama, Fukushima 963-8503, Japan （Received 22 July 2016 / Accepted 29 November 2016） Abstract Using eight species of fungi collected in Japan, we determined the intensity of light emitted by cultured mycelia based on multiple isolates from a single fungal species after two different culture periods and at two different temperatures. The highest luminescent intensity was produced by isolates of Favolaschia peziziformis followed by Dictyopanus gloeocystidiatus, Armillaria mellea, Omphalotus japonicus, Pleurotus nitidus, Mycena chlorophos, Mycena manipularis, and Armillaria tabescens. There were significant differences within the three species that had more than five isolates, D. gloeocystidiatus, O. japonicus, and P. nitidus. All P. nitidus isolates showed greater intensitiy after incubation for 7 d than for 14 d. In contrast, incubation for 14 d yielded greater intensity in M. chlorophos, M. manipularis, and O. japonicus. For one isolate of D. gloeocystidiatus, one isolate of M. manipularis, and four isolates of O. japonicus, the luminescence were significantly more intense at 25℃ than at 15℃. Only one isolate each of D. gloeocystidiatus, M. chlorophos, and P. nitidus had lower intensity at 25℃ than 15℃. Eight isolates from four species showed the highest intensity at 520 nm. The intensity produced by one isolate of A. mellea gradually decreased during measurement for 16 h. Key words: Dictyopanus gloeocystidiatus, Favolaschia peziziformis, Luminescent mushroom, Omphalotus japonicus, Pleurotus nitidus the reaction between luciferin and luciferase, which is Introduction the same source for luminescence by the firefly Luciola The emission of light by living organisms has attracted cruciata7, 8). Stevani et al.4) demonstrated the involvement human attention since the time of Aristotle (384-322 of an NAD(P)H-dependent reductase and membrane- BC). This light emission phenomenon is called biolumi- bound luciferase in a two-step reaction triggered by the nescence, a term first used by Harvey1). It can be defined addition of NAD(P)H and molecular oxygen to generate as the emission by living organisms of cold light, which green light. However, no luminescent substances have is visible to the human naked eye. Bioluminescence is yet been extracted from any luminescent fungi, except distributed in approximately 700 genera from 16 major possibly from the basidiome of Mycena chlorophos9). distantly related phyla, including bacteria, fungi, dinoflag- The light emitted from the basidiomes of fungi appears ellates, and marine and terrestrial animals. green or blue to the human eye. This wavelength is At present, 80 species of fungi are known to be bio- approximately 530 nm according to Bondar et al.10) They luminescent2). In 2008, Desjardin et al.3) recognized 64 focused on fungal bioluminescence defined as a chemical species of bioluminescent fungi belonging to three reaction that occurs to yield constant light emission with distinct evolutionary lineages: Omphalotus, Armillaria, a maximum intensity in the range of 520-530 nm, where and mycenoids. By 2013, 71 fungal species were reported the chemiexcitation step is catalyzed by luciferase10). as bioluminescent4), belonging to four distinct evolution- The emission of light by mycelia is known in many ary lineages in the order Agaricales: 52 mycenoids, five species of fungi. Indeed, only the mycelium is luminescent from Armillaria, 12 from Omphalotus, and two from the in many species, whereas both the mycelium and Lucentipes, Gerronema viridilucens and Mycena lucentipes. basidiomes emit light in others. Very rarely, the basidiome Luminescent fungi can be found in tropical and temperate has been reported to be luminescent whereas the mycelium areas, where high humidity and a warm climate favor is nonluminescent. The distribution of luminous basidi- mycelial growth and reproductive growth4). Nine species ome tissues among fungal species is not uniform3); the have been described in Japan5, 6). whole basidiome is luminescent in some species, whereas The mechanism of bioluminescence is attributed to only certain parts emit light in other species. In Mycena lamprospora and Mycena pruinoso-viscida var. raboulensis, only the basidiospores are known to emit light, only the *Corresponding author. E-mail: [email protected] lamellae glow in G. viridilucens, the pilei and lamellae are 日本きのこ学会誌 MUSHROOM SCIENCE AND BIOTECHNOLOGY 177 Table 1. Fungal isolates used. Isolate No.1)/ Abbre- Scientific name Japanese name Location Original No. viation provided Armillaria mellea Naratake AM1 Fukushima Koriyaka city Osemachikozu TBRC74 AT1 Fukushima Koriyaka city Otsuki Park TBRC72 Armillaria tabescens Naratakemodoki AT2 Fukushima Nihonmatsu city Take hot spring TBRC73 DG1 Okinawa Kunigami village Mt. Yonaha TBRC 1 DG2 Okinawa Kunigami village Mt. Nishime TBRC28 DG3 Okinawa Ishigaki city Yonehara TBRC44 Dictyopanus Suzumetake DG4 Okinawa Taketomi town Iriomote island TBRC61 gloeocystidiatus DG5 Okinawa Kunigami village Mt. Yonaha TBRC69 DG6 Okinawa Kunigami village Mt. Yonaha TBRC70 DG7 Gunma Kiryu city Umeda cho TBRC71 FP1 Okinawa Shimajiri-gun Minami-Daitoh TBRC14 Favolaschia FP2 Okinawa Yonaguni-mahi Mt. Urabe TBRC47 Enasirassitake pezizaeformis FP3 Okinawa Yonaguni-mahi Mt. Urabe TBRC50 FP4 Tokyo Ogasawara village Nagatani TBRC65 MAFF MC1 Tokyo Ogasawara village Chichijima Mycena chlorophos Yakotake 30579 MC2 Tokyo Ogasawara village Hahajima TBRC33 Mycena manipularis Amihikaritake MM1 Okinawa Kunigami village Mt. Yonaha TBRC16 OG1 Yamanashi Narusawa village Mt. Fuji TBRC29 OG2 Ibaraki Tsukuba city Mt. Tsukuba TBRC30 OG3 Iwate Hachimantai city Appi plateau TBRC31 Omphalotus OG4 Tochigi Nasusiobarakogen city Shiobara TBRC32 Tsukiyotake guepiniformis OG5 Ehime Saijo city Mt. Ishiduchi TBRC62 OG6 Ibaraki kitaibaraki city Ogawa research forest TBRC63 OG7 Ehime Saijo city Mt. Ishiduchi TBRC64 OG8 Fukushima Koriyaka city Bandaiatami TBRC75 PN1 Okinawa Taketomi town Iriomote island TBRC 2 PN2 Okinawa Taketomi town Iriomote island TBRC 3 PN3 Okinawa Taketomi town Iriomote island TBRC 4 PN4 Okinawa Taketomi town Iriomote island TBRC 5 Pleurotus nitidus Shirohikaritake PN5 Okinawa Taketomi town Iriomote island TBRC26 PN6 Okinawa Ishigaki city Banna park TBRC41 PN7 Okinawa Ishigaki city Yonehara TBRC42 PN8 Okinawa Ishigaki city Banna park TBRC43 PN9 Okinawa Ishigaki city Mt. Omoto TBRC45 Lentinula edodes Shiitake Cont Hokken 600 1) Provisional isolates number provided by University of the Ryukyus. luminescent in M. chlorophos and Mycena asterina, and Materials and Methods only the stipes glow in M. lucentipes. Previous studies have investigated the luminescence Fungal isolates mechanism and assessed how culture conditions (culture We used 34 isolates from eight species in Japan that temperature, medium pH, medium composition, etc.) affect are classified as luminescent, Armillaria mellea, Armillaria the intensity of the light emitted. However, only a single tabescens, Dictyopanus gloeocystidiatus, Favolaschia representative isolate was used for each species in these peziziformis, Mycena chlorophos, Mycena manipularis, studies3, 4). Thus, we used eight species of fungi collected Omphalotus japonicus, and Pleurotus nitidus (Table 1). The in Japan and observed the intensity of light emitted by Hokken 600 (Hokken Co. Ltd.) variety of Lentinula edodes, cultured mycelia from multiple isolates of a single fungal which is a well-known edible but nonluminescent mush- species after different culture periods and temperatures. room in Japan, was used as a control in all experiments. We used photographs to record the luminescent Mycelial preparation and measurement intensity based on color changes. We also determined To determine the intensity of the luminescence the wavelength of the light produced by representative produced by the different species and isolates, all of the isolates and the changes in light intensity during isolates were inoculated onto PDA medium in Petri dishes measurements obtained over 16 h in one Armillaria mellea (Φ = 40 mm) with five replicates each and were incubated isolate. for 14 d. The luminescent intensity of a mycelial colony 178 Vol.24 No. 4 6 g) 5 0 g) 1 10 14 d, 15 /m /m ec ec 5 /s 5 /s 14 d, 25 2 10 3 10 cm cm 4 n/ n/ 10 4 to to 10 ho ho 3 10 (p (p 3 y y 10 it it 2 ns ns 10 2 te te 0 in in 0 1 1 10 0 0 1 1 2 3 4 1 2 1 2 1 2 3 4 5 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 1 1 2 1 2 3 4 5 6 7 8 9 1 2 1 1 2 3 4 Luminescent Luminescent PN PN PN PN PN DG DG OG OG OG OG MC MC AT AT DG DG DG DG DG DG DG FP FP FP FP MM OG OG OG OG OG OG OG OG PN PN PN PN PN PN PN PN PN AM MC MC MM AM AT DG FP MC MM OG PN C o nt DG MC MM OG PN Cont Strain Strain Fig. 1. Luminescent intensity of mycelia of 35 isolates from nine Fig. 3. Luminescent intensity of mycelia of 15 isolates from six species species cultured for 14 days at 25℃. cultured for 14 days at 15℃ or 25℃. AM, AT, DG, EP, MC, MM, OG, and PN refer to the species The abbreviations are the same as those used in Fig. 2. abbreviations given in Table 1. Bars represent standard deviation (n = 3). Bars represent the standard deviation (n = 3). Asterisks indicate significant differences in the luminescent intensity of mycelia cultured at 15℃ and 25℃ (p < 0.05). 6 10 g) /m 5 ec 7 d, 15 10 /s produced by mycelia of A. mellea were obtained for 16 h.