Micromorphological characteristics of the species of Pholiota (Strophariaceae, Basidiomycota) in pure culture

The article presents results of the research on micromorphology of vegetative mycelia of eight species of the genus Pholiota from the IBK Mushroom Culture Collection of the M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine. Using scanning electron microscopy and light microscopy, the micromorphological structures specific to these species were studied in order to enable their identification in pure culture. New data on micromorphology of Pholiota species are given. A set of micromorphological structures was observed for this genus, namely clamp connections, chlamydospores, arthrospores, anastomoses, crystals on hyphae, hyphal rings, rhizomorphs, pellicle spots, hyphae ornamentation, and secretory hyphae. For the first time a detailed study of microstructures of P. alnicola, P. limonella, P. nameko, P. populnea, and P. subochracea was conducted. Secretory hyphae and vacuolized mycelia in pure culture were noticed only for P. populnea. For P. subochracea, various hyphae ornamentation on vegetative mycelium was demonstrated. New information about the presence of hyphal rings for three Pholiota species, P. alnicola, P. limonella, and P. subochracea, is presented.


Introduction
About 470 species of the genus Pholiota (Fr.) P.Kumm. are known worldwide (http://www.mycobank.org), 25 of them have been reported for Ukraine (Zerova, 1979;Dudka et al., 2009). Mushrooms received attention due to combination of nutritious properties with health-stimulating and medicinal effects (Kim et al., 2006). Anticarcinogenic, antioxidant, antimicrobial and immunomodulating properties of the components isolated from mycelia and fruit bodies of this genus have been established (Cho et al., 2003;Kim et al., 2006;Zhang et al., 2009). Using mushrooms as a source of preventive and therapeutic agents and their cultivation in the industrial scale have been realized after many years of fundamental research of biology of macrofungi, peculiarities of their growth and development, and nature and mechanisms of metabolic and enzymatic activity (Wasser, 2010). Fruit bodies of P. adiposa and P. nameko are cultivated in the countries of Southeast Asia in the industrial scale (Pegler, 2003;Gizaw, 2015).
Taxonomic features of macrofungi can be used for their identification in pure culture, therefore research on their micromorphological structures is important. Micromorphological characteristics of Basidiomycetes include a set of microscopic features such as presence of clamp connections or pseudo-clamp connections, width and types of hyphae according to the traditional classification by Stalpers (1978), presence of anastomoses and various structures formed during differentiation of the hyphae in culture (hyphal rings, rhizomorphs, inlaid hyphae, crystals on hyphae, etc.), presence of asexual reproduction structures (Mykhaylova, 2014).
This study was focused on micromorphological structures of eight species of the genus Pholiota preserved in the IBK Mushroom Culture Collection.

Materials and methods
Eight strains of Pholiota species from the IBK Mushroom Culture Collection of the M.G. Kholodny Institute of Botany, National Academy of Sciences of the Ukraine were investigated (Bisko et al., 2016). Some of these strains were obtained in 2017-2018 (Table 1).
Mushroom cultures were grown on glucose peptone yeast agar media; g/l: glucose -25; peptone -3; yeast extract -3; MgSO 4 -0.25; KH 2 PO 4 -1; K 2 HPO 4 -1; agar-agar -22; pH -6.0) in Petri dishes at temperature 26 ± 1 ºC. Mycelia were taken from the cultures in the phase of active growth (3-10 days) and from the long-term cultivation of Pholiota species (2 months). Vegetative mycelium microstructures were studied using Zeiss light microscope (LM) and scanning electron microscopy (SEM). Samples were prepared for light microscopy using distilled water, 10% KOH or preparative mixture (glycerin: ethanol: water = 1: 1: 1) (Bilay, 1982). Samples were prepared for SEM using the modified method of Quattelbaum and Carner (Quattelbaum, Carner, 1980). Four sterilized square 4×4 mm pieces of the cover glass were placed 1-3 cm away from inoculum into Petri dishes. The cover glasses were removed from the agar media, when mycelia overgrew the surface, and then transferred to a microscopic slide. The slide was then placed into a sealed glass vessel fixed with osmium tetroxide vapor (1% solution) for 8 hours. On fixation, the slides were transferred to an empty Petri dish to dry out for 72 hours. After drying out, samples were covered with gold in the vacuum spray gun JII-4X with rotation (Buchalo, Didukh, 2005). The specimens were examined using the Jeon JSM-6060 LA Scanning Electron Microscope (Japan) and studied at a magnification ×1000 -10000.

Results and discussion
An important taxonomic feature for identification of macrofungi in pure culture is the presence of a unique structure that occur on the mycelium of many species of Basidiomycota clamp connections (Stalpers, 1978;Buchalo, Didukh, 2005). Though the clamp connections are exclusive to this phylum, not all species possess these structures. Regular clamp connections were observed for vegetative mycelia in all studied Ukraine, Kyiv Region, Kyyliv, on soil, 2017 species ( Fig. 1), which agrees with the literature data (Buchalo, Didukh, 2005;Buchalo et al., 2009Buchalo et al., , 2011Dyakov et al., 2011). Vegetative mycelium of Pholiota species in pure culture consist of the thin-walled, moderately branched, regularly septated, uncolored hyphae of 1-3 μm in diameter, with anastomoses formed between hyphae (Table 2, Fig. 2, A) and pellicle spots on vegetative mycelium (Table 2, Fig. 2, B). Hyphae fusion is carried out through anastomoses and pellicle spots on vegetative mycelium, which is a well-known phenomenon in the phylum Basidiomycota (Buchalo et al., 2009).
The ability to form crystals is widely known for many species of Basidiomycota which sometimes also depends on the nutrient media and age of the mushroom cultures (Buchalo et al., 2009;Dyakov et al., 2011). Crystal formation was observed in almost all investigated species of Pholiota (Table 2, Fig. 8). The morphology of the crystals is very different. We observed prismatic, cubic, hexahedral, sometimes of the undefined shape crystals. Maximum length of the crystals was 12.2 μm, minimum -0.5 μm, width -0.3-6.1 μm.
Thus, presence of clamp connections, ability to form anastomoses and pellicle spots on vegetative mycelia, formation of conidial sporulation, arthrospores and chlamydospores, ability to secrete crystals different in shape and size were observed for various investigated species. Our results confirm some earlier literature data (Buchalo, Didukh, 2005;Buchalo et al., 2009Buchalo et al., , 2011Dyakov et al., 2011). Micromorphological characteristics of the studied Pholiota species are shown in Table 2.

Conclusions
The as P. alnicola, P. limonella, P. nameko, P. populnea, and P. subochracea for morphological characteristics and identification of these taxa in pure culture. The presence of all typical for the genus features has been noted in various studied species. New data about micromorphological features of Pholiota species in pure culture were obtained. Only for P. populnea the existence of secretory hyphae and vacuolized mycelium and for P. subochracea -the hyphae ornamentation on vegetative mycelium were noticed. New information about the presence of hyphal rings for three Pholiota species, P. alnicola, P limonella and P. subochracea, is provided.  Pholiota subochracea 2535 + * -+ * + * + * + * -+ * + * "+" structures found; "-" structures not found; * new data for this species