Initiation of Basidioma Formation of Rare and Medicinal Macromycetes in Pure Culture

Науковий вісник Східноєвропейського національного університету імені Лесі Українки. Серія: Біологічні науки, 2019, 3 (387) УДК 582.28 Initiation of Basidioma Formation of Rare and Medicinal Macromycetes in Pure Culture Maryna Sukhomlyn1, Vitaliia Didenko1, Nadiia Tsvyd1*, Yurii Petrychuk2 Educational and Scientific Centre «Institute of Biology and Medicine», Taras Shevchenko National University of Kyiv, Kyiv, Ukraine 2 Hutsulshchyna National Nature Park, Kosiv, Ukraine Correspondence: suhmary@ukr.net 1 Abstract. More than 5000 species of Ascomycetes and Agaricales fungi are known nowadays. However, 30 species of them are intensively cultivated and have been used into the agriculture. One of the reasons of a small number of species that can be grown in industrial culture are unclear conditions for the basidiomes formation. Basidiomes and mycelium include various nutritional substances that are used in food, medicine, cosmetics, etc. Recently, the biosynthesis of fungal biologically active substances with immunomodulation, radioprotective, antitumor, antiviral and other properties is under investigation by many scientists. Only 15 % of all products of medical mushrooms are made from extracts of mycelium. Verification of conditions of mushroom cultivation will significantly expand the range of species that could serve as a potential object for biotechnology. 29 species of macromycetes from the pure Cultures Collection of Fungi (FCKU) of Educational and Scientific Centre «Institute of Biology and Medicine» Taras Shevchenko National University of Kyiv were used in our experiment. The initiation basidiomes formation of 29 macromycetes on different substrates in pure culture was studied. The optimal substrate for the studied species was husk of sunflower seeds. Most fungi formed basidiomes on it. If mushrooms did not formed basidiomes on this substrate, they fastest it overgrown or developed primordia or sclerotium-shaped structures. The shaping of the basidiomes, primordia or sclerotiashaped structures was observed in 28 species. Three of these species were listed in the Red Book of Ukraine (Grifola frondosa, Leucoagaricus barsii, Sparassis crispa) and others rare species for Ukraine (Ceriporia viridans, Hericium cirrhatum, Sarcodontia crocea, Sparassis laminosa). Key words: basidiomes formation, medicinal macromycetes, pure cultures, rare species, sclerotium-shaped structures. Ініціація формування плодових тіл рідкісних та лікарських грибів в умовах чистої культури Марина Сухомлин1, Віталія Діденко1, Надія Цвид 1, Юрій Петричук2 Навчально-науковий центр «Інститут біології та медицини» Київського національного університету імені Тараса Шевченка, Київ, Україна 2 Національний природний парк «Гуцульщина», Косів, Україна Адреса для листування: suhmary@ukr.net 1 Отримано: 18.02.19; прийнято до друку:20.03.19; опубліковано: 28.06.19 Резюме. Вивчено ініціацію утворення плодових тіл 29 видів грибів на різних субстратах в умовах культури. У результаті скринінгу цих макроміцетів у 28 видів спостерігали формування плодових тіл, примордіїв або склероцієподібних структур. Також плодові тіла формували (Grifola frondosa, Leucoagaricus barsii, Sparassis crispa) три види, що занесені до Червоної книги України, та декілька рідкісних видів для України (Ceriporia viridans, Hericium cirrhatum, Sarcodontia crocea, Sparassis laminose). Ключові слова: формування плодових тіл, лікарські гриби, чиста культура, рідкісні види, склероцієподібні структури. © Сухомлин М., Діденко В., Цвид Н., Петричук Ю., 2019 17 Lesia Ukrainka Eastern European National University Scientific Bulletin Series: Biological Sciences, 2019,3 (387) Species names were agreed according to the Introduction current names in the International Database of More than 5000 species of Ascomycetes and Mushrooms. The cultures are stored in test tubes Agaricales fungi are known nowadays. Only with a potato-glucose agar medium (PGA) in an approximately 2,000 species are considered edible equipped room, 4-8 °C temperature. We used (Lakhanpal, Rana, 2005). However, 30 species of different nutrient media, substrates and substrates them are intensively grown and have been entered combinations for the basidiomes formation. into the culture (Akata et al., 2012; Buchalo, 1988; The nutrient media are potato-glucose agar Krupodorova et al., 2012). medium (PGA) and liquid potato-glucose nutrient Basidiomes and mycelium include various medium (PG). nutritional substances that are used in food, The substrates are husks of pumpkin, husk of medicine, cosmetics, etc. (Ikekawa et al., 2004; sunflower seeds, boiled wheat grains, pine Gao et al., 2005; Carvaja et al.l, 2012). Medical sawdust, ground coffee beans. properties were described for many macromycetes The substrate combinations are mixtures of (Dovgiy et al., 2013; Krupodorova et al., 2012; ground coffee beans and husk of sunflower seeds, Solomko, Lomberg, 2005; Wasser, 2010). a mix of pine sawdust with husk of sunflower Recently, the biosynthesis of fungal biologically seeds, a mix of a husk of sunflower seeds with active substances with immunomodulation, sawdust of hardwoods. radioprotective, antitumor, antiviral and other 50g substrates have put into 250 ml Erlenmeyer properties is an investigation by many scientists flask or flat-bottomed round flask. Each flask with (Engler, 1998; Takaku et al., 2001; Zjawiony 2004; substrates was filled with 50 ml of water. The Mariga et al., Vaz et al., 2010; Jeena et al., 2014). moisture content of the solid media was adjusted to The medicinal mushrooms products derived from 65 % (w/w) on a wet basis. 50 g of the liquid basidiomes have been commercially grown or pick potato-glucose nutrient medium have poured into up from the wild. Only 15 % of all products of 250 ml flat-bottomed round flask. All nutrient medical mushrooms are made from extracts of media, substrates and substrate combinations were mycelium (Lindequist et al., 2005). However, in prepared according to generally accepted methods our opinion, almost all mushrooms may be and were autoclaved at 120°C for 30 min. medical, but we don’t know it precisely yet. (Buchalo, 1988; Ohta and Fujiwara, 2003). Now scientists are studying intensely fungi but Mycelia agar-plugs (3–5 mm diam.) cut from their special interest is an investigation of the periphery of the growing colony and were put mycorrhizal fungi (Sanmee et al., 2010; Akata et to flasks with nutrient media, substrates and al., 2012; Endo et al., 2014). One of the reasons a substrate combinations. After then, inoculated small number of species that can be grown in flasks were incubated in darkness at an optimal industrial culture are unclear conditions for the temperature for mycelium growing of these basidioma formation. Verification of conditions species. When the surface of the medium was mushrooms cultivation will significantly expand covered by mycelium, incubation conditions were the range of species that could be a potential object changed to daylight with temperature to 16–18°C for biotechnology. So, the study of the mushrooms for fruit body formation. Maximum light intensity basidiomes formation in culture is important in was 1600–2000 lux daily. many aspects. The purpose of our work was the The Thiamine (Vitamin B1) (0,25 mg added to initiation of basidioma formation of rare and each flask) or selenium (Se) (0,125 g in each flask) medical mushrooms in culture. have put in some substrates for initiation basidiomes formation. The beginning of Materials and Мethods basidiomes formation was determined of primordium appearance. The 29 species of macromycetes from the pure Same basidiomes of studying mushrooms Cultures Collection of Fungi (FCKU) of Educahave formed spores that were checking by tional and Scientific Centre Institute of «Biology microscope. We used statistical analysis to test our and Medicine» Taras Shevchenko National data. The entire process was repeated with five University of Kyiv have used in our experiment replicates. (tab.1). Collection FCKU was registered in the World Federation of Cultures collections and Results includes 51 species: 19 species of them are edible, 5 – inedible, 1 - poisonous, 4 - included in the Red The investigated species of fungi, their location Data Book of Ukraine (2009) and 4 are rare source and the success of basidiomes formation in species that need additional protection. vitro are shown in table 1. 18 http://journalbio.eenu.edu.ua Науковий вісник Східноєвропейського національного університету імені Лесі Українки. Серія: Біологічні науки, 2019, 3 (387) Table 1 Successful of Basidiomes Formations of Studied Macromycetes in Vitro Species Origin Date of Collection Edibility Rarity Basidiomes in Pure Culture 1 2 3 4 5 6 Ascocoryne sarcoides Kiev region September, 2010 NK F October, 2006 E SS, R June, 2012 NE F September, 2008 NE SS June, 2005 E F June, 20011 NE F October, 2003 E RB F June, 2008 E R F Kiev region October, 2011 P F Kiev region October, 2011 NE F June, 2008 NE SS June, 2007 E F November, 2008 E September, 2009 E F September, 2005 NE F May, 2008 E May, 2009 E SS April, 2012 E SS May, 2004 E RB SS RB SS Armillaria mellea Ceriporia viridans Chondrostereum purpureum Coprinus micaceus Coprinellus domesticus Grifola frondosa Hericium cirrhatum Hypholoma fasciculare Hypholoma sublateritium Lentinus cyathiformis Lentinus tigrinus Leucoagaricus barsii Marasmius scorodonius Meripilus giganteus Morchella crassipes Morchella elata Morchella esculenta Morchella steppicola Mutinus caninus Oudemansiella longipes Oudemansiella radicata Phallus impudicus Pleurotus calyptratus Polyporus squamosus Розділ І. Ботаніка Kherson region, Chornomorsky State Biosphere Reserve Kaniv, Cherkasy region, Kaniv Nature Reserve Kiev region Volnovakha district, Donetsk region Kaniv, Cherkasy region, Kaniv Nature Reserve Sloviansky district, Donetsk region Kaniv, Cherkasy region, Kaniv Nature Reserve Kaniv, Cherkasy region, Kaniv Nature Reserve Cherkasy region Trakhtemyriv village, Kaniv distric, Cherkasy region Kiev region Volnovakha distric, Donetsk region Kaniv, Cherkasy region, Kaniv Nature Reserve Lutsk Kiev region, RB RB F SS Volnovakha district, Donetsk region Kaniv, Cherkasy region, Kaniv Nature Reserve Kyiv, Holosiyvsky National Nature Park Kaniv, Cherkasy region, Kaniv Nature Reserve Kaniv, Cherkasy region, Kaniv Nature Reserve June, 2007 NE September, 2013 NE F June, 2007 E SS June, 20011 E N Kiev region October, 2012 E SS Sosnytsia district, Chernihiv region October, 2010 E F 19 Ініціація формування плодових тіл рідкісних та лікарських грибів в умовах чистої культури Continuation of Table 1 1 Pycnoporus cinnabarinus Sarcodontia crocea Sparassis crispa Sparassis laminosa 2 3 4 Kiev region September, 2011 NE October, 2010 NE R F September, 2013 E RB F September, 1996 E R F Sosnytsia district, Chernihiv region Kiev region Artemivsk district, Donetsk region 5 6 F Note. Edibility: E – edible species, N – inedible species, O – poisonous species; Rarity: R – rare species, RB – species listed in the Red Data Book of Ukraine; Successful fruit formation: SS – species that formed primordia or sklerotium-shaped structures, F– species, which formed basidiomes in a pure culture, N – species that did not form basidiomes. Primarily, all of investigated species have basidiomes formation on the three type of substrates such as husk of sunflower seeds, PGA and PG. The time (days) for overgrowth of the substrate by mycelium, appearance of primordia and basidiomes of all species are shown in table 2. Table 2 Fouling Substrate and Basidiomes Formation of Studied Species (Days) Type of Substrates PG 20 Basidioma Appearance Full Fouling Substrate The Appearance of Primordia Basidioma Appearance Full Fouling Substrate The Appearance of Primordia Basidioma Appearance 1 Ascocoryne sarcoides Armillaria mellea Morchella crassipes Morchella elata Morchella esculenta Morchella steppicola Chondrostereum purpureum Coprinus micaceus Coprinellus domesticus Grifola frondosa Ceriporia viridans Hericium cirrhatum Hypholoma fasciculare Hypholoma sublateritium The Appearance of Primordia Species Husk of Sunflower Seeds Full Fouling Substrate PGA 2 3 4 5 6 7 8 9 10 10±0,6 14±0,3 16±0,3 13±0,9 - - 14±0,4 20±0,9 34±1,2 28±2,5 36±1,5 - 32±3,5 - - 43±0,2 56±1,3 - 5±0,7 14±1,3 - 6±0,8 - - 13±0,3 2 ±0,4 - 6±0,9 16±1,3 - 7±0,9 20±0,4 - 13±0,3 21±0,4 - 7±0,4 18±1,3 - 9±0,4 - - 13±0,3 21±0,4 - 8±0,3 21±2,3 - 9±0,3 - - 13±0,3 21±0,4 - 8±0,8 - - 8±1,5 - - 10±0,3 32±0,2 - 14±0,4 20±0,3 21±0,8 16±0,7 - - 19±0,9 25±0,6 26±0,2 13±0,5 19±0,3 20±0,4 14±0,5 - - 19±0,6 24±0,2 25±0,2 15±0,8 15±1,0 25±0,7 32±1,2 80±0,3 108±0,2 22±1,1 - 118±0,9 25±0,7 24±1,6 76±0,2 33±2,2 79±0,9 44±2,8 14±0,5 18±0,8 21±0,7 35±0,6 41±0,7 45±0,3 20±0,5 25±0,9 30±0,5 18±0,9 - - 30±0,2 - - 33±0,1 39±0,2 45±0,8 33±1,7 22±1,9 47±2,9 32±1,3 - - 42±2,2 48±2,4 54±1,8 Сухомлин М., Діденко В., Цвид Н., Петричук Ю. Науковий вісник Східноєвропейського національного університету імені Лесі Українки. Серія: Біологічні науки, 2019, 3 (387) Continuation of Table 2 1 Lentinus tigrinus Lentinus cyathiformis Leucoagaricus barsii * Marasmius scorodonius * Meripilus giganteus Mutinus caninus Oudemansiella longipes Oudemansiella radicata Phallus impudicus Pleurotes calyptratus Polyporus squamosus Pycnoporus cinnabarinus Sarcodontia crocea Sparassis crispa Sparassis laminosa 2 11±0,3 3 20±0,6 4 29±1,8 5 43±0,3 6 - 7 - 8 14±0,3 9 35±0,9 10 39±0,3 14±0,5 - - 18±1,8 - - 22±0,2 137±0,4 19±1,3 - - 16±2,6 - - 32±2,3 91±4,9 96±5,3 15±0,7 - - 49±0,3 - - 31±0,3 37±0,9 41±0,3 10±0,7 14±1,2 20±0,9 13±0,3 - - 10±0,3 16±0,8 18±0,5 10±0,4 21±0,6 - 12±0,3 24±0,9 - 16±1,2 28±1,7 - 14±0,4 - - 21±2,8 - - 18±1,4 31±2,5 37±3,1 12±0,6 32±1,7 - 22±3,4 - - 16±0,4 23±0,2 - 32±3,4 - - 23±2,3 - - 25±2,4 - - 10±0,7 - - 18±1,3 - - 11±1,3 132±0,3 - 10±0,4 20±1,3 - 16±0,8 - - 17±0,1 35±0,3 43±0,1 9±0,8 20±1,5 25±2,1 20±0,7 32±0,3 53±1,5 14±0,5 50±0,7 63±0,1 17±0,5 25±0,7 30±0,7 20±0,3 - - 25±0,8 36±0,7 41±0,9 16±0,2 20 ±0,7 23 ±0,7 22 ±0,4 41 ±0,7 46 ±0,7 17 ±0,1 35 ±0,3 43 ±0,1 16±0,2 - - 13±0,5 - - 23±0,2 25±1,8 33±0,4 - Note. Column «The appearance of primordial» contains the results of appearance primordia or sclerotium-shaped structures; * – macromycetes generated sporocarps after coverage by soil and additional moisture into medium. According to the table 2, M. gianteus had the fastest mycelium growing. Mycelium of this mushroom wholly covered the sunflower husk substrate and PG medium during 10 and 13 days of cultivations respectively. F. velutipes had the slowest mycelium growing. The sunflower husk substrate have had completely covered F. velutipes mycelium after 66 days cultivations and 45 days after PG medium cultivation accordingly. Mycelium of almost investigated species, except Phallus impudicus L., was forming primordias and sclerotium-shaped structures on the tested substrates (fig.1). Some species formed primordia even before full fouling of the substrate by the mycelium. M. giganteus have formed primordia on the sunflower husk substrate the fasted amount investigation mushrooms after 16 days of cultivations. Ascocoryne sarcoides (Jacq.) J. W.Groves & D. E. Wilson, Coprinus micaceus (Bull.: Fr.) Vilgalys, Hopple & Jacq. Johnson, Coprinellus domesticus (Bolton) Vilgalys, Hopple & Jacq.Johnson, Grifola frondosa (Dicks.) Gray, Розділ І. Ботаніка Hericium cirrhatum (Pers.) Nikol., Hypholoma fasciculare (Huds.: Fr.) P. Kumm., Lentinus tigrinus (Bull.) Fr., Leucoagaricus barsii (Zeller) Vellinga, Marasmius scorodonius (Fr.) Fr., Meripilus giganteus Karst., Oudemansiella longipes (Quél.) M. M. Moser, Pycnoporus cinnabarinus (Jacq.) P. Karst., Sarcodontia crocea (Schwein.) Kotl., Sparassis crispa (Wulfen) Fr. and Sparassis laminosa Fries formed a well-developed basidiomes on the husk of sunflower seeds (fig. 2). The early basidiomes of M. giganteus looked like light-coloured corals, but later they acquired in a dark red colour. The most massive and intensive basidiomes formation had in P. cinnabarinus (fig. 2 D) on the husk of sunflower seeds substrate. This fungus had basidiomes bright orange colour on the walls of a flask. As we see from Figure 2 basidiomes of L. tigrinus has a stem of 3–4 cm length and cap of 2–6 cm diameter. The basidiomes of S. crocea looked like sharp spines of creamy colour with 1 to 2 cm in size and located in groups throughout the substrate surface (fig. 2 F). 21 Ініціація формування плодових тіл рідкісних та лікарських грибів в умовах чистої культури Fig. 1. Primordium and Sclerotium-Shaped Structure of Macromycetes in Culture. Primordia of Lentinus Tigrinus (A), Polyporus Squamosus (E), Hypholoma Sublateritium (B). Sclerotium-Shaped Structure of Mutinus Caninus (C), Chondrostereum Purpureum (D), Oudemansiella Radicata (F), Morchella Steppicola (G) and Hyphae Sclerotium-Shaped Structure on a Scanning Electron Microscope (H) Fig. 2. Basidiomes Formation of Lentinus Tigrinus (A), Sparassis Laminosa (B), Oudemansiella Longipes (C), Pycnoporus Cinnabarinus (D), Grifola Frondosa (E), and Sarcodontia Crocea (F) on Husk of Sunflower Seeds 22 Сухомлин М., Діденко В., Цвид Н., Петричук Ю. Науковий вісник Східноєвропейського національного університету імені Лесі Українки. Серія: Біологічні науки, 2019, 3 (387) of sunflower seeds substrate. Also, this plate was M. scorodonіus formed brown basidiomes with twisted at the ends and formed small spines in the a stem of 4–5 cm length and cap of 1 cm diameter center of the plate (fig. 3 D). basidiomes on PGA (fig. 3 A). Also, this fungus had a garlic smell had the form elongated strands with white color which inherent in these species. Some macromyspines (fig. 3 F). cetes generated sporocarps after additional coveThe ten of twenty-nine studied species don’t rage by soil and added moistures (fig. 3 A and C). form basidiomes. Several species developed basidiomes on liquid The basidiomes formation of rare medium PG or PGA (fig. 3 B, D, E, F). macromycetes S. laminosa and G. frondosa was H. cirrhatum formed basidiomes on all type of investigated in different substrates more details tested media. The basidiomes look likes a white (table 3). colour plate on a liquid nutrient medium and husk Fig. 3. Generation of Sporocarps After Additional Coverage By Soil and Additional Moisture by Marasmius Scorodonіus (A) and Leucoagaricus Barsii (C). Basidiomes Formation by Grifola Frondosa (B), Hericium Cirrhatum (D) on Liquid Medium (PG); Ascocoryne Sarcoides (E), Hericium Cirrhatum (F) on PGA Table 3 Fouling Substrate and Basidiomes Formation by S. Laminosa and G. Frondosa (Days) S. Laminosa Type of Substrates HPS PS+HSS H + HSS CB CB+HSS WG G. Frondosa Full Fouling Substrate The Appearance of Primordia Basidioma Appearance Full Fouling Substrate The Appearance of Primordia Basidioma Appearance 7 ± 0,6 11 ± 0,4 9 ± 0,3 4 ± 0,7 27 ± 0,4 24 ± 0,8 24 ± 1,9 80 ± 2,1 – – – – – 85 ± 2,9 – – – – 14 ± 0,5 14 ± 0,3 14 ± 1,3 12 ± 0,8 33 ± 0,5 32 ± 0,5 40 ± 0,8 70 ± 0,2 49 ± 0,4 – 55 ± 0,5 28 ± 0,3 43 ± 1,4 74 ± 1,2 53 ± 0,6 – 59 ± 0,4 – Note. HPS – husk of pumpkin seeds; WG – wheat grain; CB – milled coffee beans; CB+HSS – mix of milled coffee beans with husk of sunflower seeds; PS+HSS – mix of pine sawdust with husk of sunflower seeds; H + HSS – mix sawdust of hardwood with husk of sunflower seeds. Розділ І. Ботаніка 23 Ініціація формування плодових тіл рідкісних та лікарських грибів в умовах чистої культури The coffee beans substrate was the fastest primordia or sclerotium-shaped structures covered by mycelium of S. laminosa and G. (Armillaria mellea (Vahl) P. Kumm., frondosa through 4 and 12 days respectively. Chondrostereum purpureum (Pers.) Pouzar, However, on this substrate, S. laminosa and G. Lentinus cyathiformis (Schaeff.) Bres., Morchella frondosa did not form primordia and basidiomes. crassipes (Vent.) Pers., Morchella elata Fr., Although S. laminosa and G. frondosa slowly Morchella esculenta Fr., Morchella steppicola overgrown substrates, but basidiomes formed at Zerova, Mutinus caninus, Pleurotus calyptratus the faster on husk of sunflower seeds. (Lindblad ex Fr.) Sacc., Polyporus squamosus The only suitable substrate for fruiting S. (Huds.) Fr. and Oudemansiella radicata (Relhan) laminosa was a mix of pine sawdust and husk of Singer) are promising for further research the sunflower seeds. However, the basisiomes initiation basidiomes formation in a pure culture. formation takes a long time (80 days) on a mix of pine sawdust with husk of sunflower seeds. Reference The favorable substrates for basidiomes 1. Akata, I.; Kalyoncu, F.; Solak, M. H.; Kalmis, formation by G. frondosa were pumpkin seed E. Growth of mycelium of three ectomycorrhizal husks and a mix of hardwood sawdust and husk of macrofungi, Infundibulicybe geotropa, sunflower seeds. Its basidiomes were as small Tricholoma anatolicum and Lactarius deliciosus in bushes that were initially white, later becoming culture media containing various carbon sources. brown at all types of substrate. The largest number African Journal of Microbiology Research; 2012, of basidiomes of G. frondosa was formed on a mix 6(12), 3042–3046. of pine sawdust with husk of sunflower seeds, but 2. Buchalo, A. S. Higher Edible Basidiomycetes they were very small. Large basidiomes are formed in Pure Culture; Naukova Dumka: Kiev; 1988. on a mix of milled coffee beans with husk of 3. Carvajal, A. E. S. S.; Koehnlein, E. A.; Soares, sunflower seeds. A. A.; Eler, G. J.; Nakashima, A. T. A.; Bracht, A.; Peralta, R. M. Bioactives of fruiting bodies and The effect of Thiamine (Vitamin B1) on the submerged culture mycelia of Agaricus brasiliensis (A. basidiomes formation was tested on S. laminosa. blazei) and their antioxidant properties. LWT - Food Vitamin B1 accelerated fouling substrate Science and Technology; 2012, 46, 493–499. mycelium (10 ± 1,5 days) and the mass of 4. Diduh, Ya. P. Red Book of Ukraine. Flora; basidiomes. The term of basidiomes formation was Hlobalkonsaltynh: Kyiv, 2009. 9 ± 1,3 days. 5. Dovgiy, R. S.; Pozur, V. V.; Svyatetska, V. N.; The added Selenium in each of the flasks with Suhomlyn, M. N.; Livinska, O. P.; Makarenko, A. N. husk of sunflower seeds did not affect on the Immunobiological action of extracts and medicines, basidiomes formation of Mutinus caninus (Huds.) obtained from mushrooms Ganoderma lucidum, Fr., but speeded up fouling substrate by mycelium Cordyceps sinensis and Leucoagaricus macrorhizus. Bulletin of the problems of biology and medicine (In (average 9 ± 2,0 days). Discussion As a result screening of 29 macromycetes species on the ability to produce basidiomes in pure culture, the capacity of 28 of them was installed, including possibility to form primordia or sclerotium-shaped structures. Basidiomes were obtained in three species, which are included in the Red Data Book of Ukraine (G. frondosa, L. barsii, S. crispa) and four rare species (Ceriporia viridans (Berk. & Broome) Donk, H. cirrhatum, S. crocea, S. laminosa) in pure culture. The good substrate for the studied species appeared husk of sunflower seeds. Most fungi formed basidiomes on it. If mushrooms are not formed basidiomes on this substrate, they fastest it overgrown or developed primordia or sclerotiumshaped structures. There is the positive effect of Vitamin B1 on the indicators of the basidiomes formation for example S. laminosa. The species that formed 24 Ukraine); 2013, 2(2), 44–49. 6. 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