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A verification of Sparassis crispa (Wulfen) Fr. strains of the IBK Mushroom Culture Collection using molecular-genetic and cultural-morphological methods were done. For all S. crispa strains a complete determination of the nucleotide sequences of the internal transcribed spacer: ITS1, 5.8S and ITS2 regions of rRNA, as well as the partial determination of 18S and 28S sequences surrounding the ITS, was performed. As a result of the search in the gene bank, the S. crispa samples deposited there showed 99-98% identity with the sequences we received, thus confirming the species of the examined strains. On the basis of the sequences we received, investigated strains were registered in NCBI GenBank. To confirm the taxonomic affiliation of the strains we studied their cultural and morphological characteristics. Microstructures of vegetative mycelium by optical and scanning electron microscopy were investigated. In all S. crispa cultures we observed the hyphae with regular one-sided gapless clamp connections, numerous secretory cells on the surface of the hyphae, anastomoses, filamentous strands and films. We studied the growth rate and morphology of the strains on seven agar nutrient media. According to the radial growth rate of S. crispa cultures can be placed to the very slowly growing mushrooms, growth rate of 0,5−2,8 mm/day. We found the selective media such as malt agar with the addition of pine sawdust (SS) and larch sawdust (SM) as most favorable for the vegetative growth and generative stage for all strains, temperature of incubation was 26 ± 0.1 °C. For mycelial growth the critical temperature was 39 ± 0.1 °C for S. crispa strains 312, 314 and 40 ± 0.1 °C for the strains 304, 2004. We confirmed belonging of studied strains to S. сrispa species according to established morphological and cultural characteristics. This result coincided with the results of DNA typing. Thus, the obtained mycelium growth parameters on nutrient media, micro- and macromorphological characteristics can be used as additional taxonomic characteristics of S. сrispa culture in the vegetative stage of growth. The strain S. сrispa 314 may become a potential producer for new fungal biotechnologies in Ukraine in the near future and reintroduction of this species in the nature. |
