The Conservation and Study of Macromycetes in the Komarov Botanical Institute Basidiomycetes Culture Collection-Their Taxonomical Diversity and Biotechnological Prospects.

Culture collections (CCs) play an important role in the ex situ conservation of biological material and maintaining species and strains, which can be used for scientific and practical purposes. The Komarov Botanical Institute Basidiomycetes Culture Collection (LE-BIN) preserves a large number of original dikaryon strains of various taxonomical and ecological groups of fungi from different geographical regions. Started in the late 1950s for the investigation of Basidiomycetes' biological activity, today, in Russia, it has become a unique specialized macromycetes collection, preserving 3680 strains from 776 species of fungi. The Collection's development is aimed at ex situ conservation of fungal diversity, with an emphasis on preserving rare and endangered species, ectomycorrhizal fungi, and strains useful for biotechnology and medicine. The main methods applied in the collection for maintaining and working with cultures are described, and the results are presented. Some problems for the isolation and cultivation of species are discussed. The taxonomical structure and variety of the strains in the collection fund are analyzed, and they show that the taxonomical diversity of fungi in the LE-BIN is commensurable with the largest CCs in the world. The achievements from the ex situ conservation of the diversity of macromycetes and the main results from the screening and investigation of the collection's strains demonstrate that a number of strains can be prospective producers of enzymes (oxidoreductases and proteases), lipids, and biologically active compounds (terpenoids, phthalides, etc.) for biotechnology and medicine.

Lomasomes and Other Fungal Plasma Membrane Macroinvaginations Have a Tubular and Lamellar Genesis.

The plasma membrane of filamentous fungi forms large-sized invaginations, which are either tubes or parietal vesicles. Vesicular macroinvaginations at the ultrastructural level correspond to classical lomasomes. There is an assumption that vesicular macroinvaginations/lomasomes may be involved in macrovesicular endocytosis. The original aim of this study was to test for the presence of macroendocytosis in xylotrophic basidiomycetes using time-lapse and Z-stacks fluorescent microscopic technologies. However, the results were unexpected since most of the membrane structures labeled by the endocytic tracer (FM4-64 analog) are various types of plasma membrane macroinvaginations and not any endomembranes. All of these macroinvaginations have a tubular or lamellar genesis. Moreover, under specific conditions of a microscopic preparation, the diameter of the tubes forming the macroinvaginations increases with the time of the sample observation. In addition, the morphology and successive formation of the macroinvaginations mimic the endocytic pathway; these invaginations can easily be mistaken for endocytic vesicles, endosomes, and vacuole-lysosomes. The paper analyzes the various macroinvagination types, suggests their biological functions, and discusses some features of fungal endocytosis. This study is a next step toward understanding complex fungal physiology and is a presentation of a new intracellular tubular system in wood-decaying fungi.

Contribution to Studies on Rare Medicinal Mushroom Sparassis crispa (Agaricomycetes) in Culture and Its Production of Phenol Compounds under Various Cultivation Conditions.

Three Sparassis crispa strains from the Komarov Botanical Institute Basidiomycetes Culture Collection (LE-BIN) were studied on various agar and liquid media for growth and phenol compound production. On agar media, the strains produced crystals of various shapes and sizes that glowed in ultraviolet light and were visible to the naked eye. Using gas chromatography-mass spectrometry, the crystals were identified as sparassol (methyl 2-hydroxy-4-methoxy-6-methylbenzoate). Other phenol compounds (i.e., methyl ester of sparassol and methyl ester of orsellinic acid) were also found in S. crispa mycelium. Phenol compounds were detected both in the mycelium and culture filtrate of all studied strains cultivated on various agar and liquid media, at different acidity and duration, but their composition and ratio varied. The initial pH of cultivated medium did not have a large influence on phenol compound production, unlike culture growth duration, which correlated directly with sparassol concentration in mycelium. Production of the phenol compounds presumably connected with constitutive processes of Sparassis metabolism, but the intensity of their production and accumulation in mycelium depends on the strain, conditions, and cultivation time. Strain LE-BIN 2902 is considered a promising sparassol producer.

Diversity of ESI-MS Based Phosphatidylcholine Profiles in Basidiomycetes.

Phosphatidylcholines (PC) are the main membrane lipid constituents comprising more than 50% of total glycerophospholipids. They coordinate a number of cell functions, particularly cell growth, homeostasis, secretion, recognition and communication. In basidial fungi PC are synthesized via the Kennedy pathway as well as through methylation of phosphatidylethanolamines (PE) and then undergo remodeling in Lands cycle that replaces fatty acids in PC molecules. The molecular profile of PC is determined by the genetic features that are characteristic for every species and depend on the environment. Here we present the results of ESI-MS based analyses of PC profiles of 38 species of basidiomycetes belonging to Agaricales (12), Polyporales (17), Russulales (5), Gleophyllales (2), Cantharellales (1), Auriculariales (1), Phallales (1). Although the variety of PC molecular species of basidiomycetes is rather diverse (20-38 molecular species in every profile), only 1-3 main molecular species represent 70-90% of total PC content. The most abundant of them are C36:4 and C36:3, followed by C34:1, C34:2, C36:5, C36:2. In the majority of basidiomycetes, C36:4 reaches up to 50-70% of total PC molecular species. Based on the results of hierarchical cluster analysis four main types of PC profiles which characterized the studied fungi independently from their taxonomic position, ecology, trophic status, and hyphal differentiation have been revealed. Comparative analyses of studied fungi using PCA method have shown that species of Polyporales differ from those of Agaricales by higher variability of PC profiles.

Evaluation of the carbonylation of filamentous fungi proteins by dry immune dot blotting.

The development of mycological gerontology requires effective methods for assessing the biological age of fungal cells. This assessment is based on the analysis of a complex of aging and oxidative stress markers. One of the most powerful such markers is the protein carbonylation. In this study, the already known method of dry immune dot blotting is adapted for mycological studies of the content of protein carbonyl groups. After testing the method on a number of filamentous fungi species, some features of the accumulation of carbonylated proteins in mycelium were established. Among these features: (i) a weak effect of exogenous oxidative stress on the accumulation of carbonyls in a number of fungi, (ii) reversibility of the carbonyl accumulation, (iii) possibility of arbitrary regulation of carbonyl content by fungus itself and (iv) the influence of hormesis. In addition, two polar strategies for the accumulation of carbonyl modification were revealed, named Id-strategy (Indifferent) and Cn-strategy (Concern). Thus, even the analysis of one marker allows making some preliminary general assumptions and conclusions. For example, the idea that fungi can freely regulate their biological age is confirmed. This feature makes fungi very flexible in terms of responding to environmental influences and promising objects for gerontology.

Lipid and Metabolite Profiling of Serpula lacrymans Under Freezing Stress.

Basidiomycete fungus Serpula lacrymans is one of the most dangerous indoor fungus causing dry rot of timber. The physiology of this fungus deserves more attention as a basis for development of methods of dry rot treatment. We observed an increase in the freezing resistance of S. lacrymans after pre-cultivation of mycelia at elevated temperatures. To examine the biochemical mechanisms underlying this phenomenon the lipid composition and metabolite profiling of mycelia subjected to freezing and thawing were investigated. An analysis is made of the growth rate and metabolism of "daughter" cultures derived from a frozen mycelia. According to the results, sphingolipids and water-soluble metabolites such as mannitol, glycerol, sugar alcohols, some amino- and organic acids are able to function as protective compounds providing a cross-resistance between heat shock and freeze-thaw stress in S. lacrymans.

Fungal Adaptation to the Advanced Stages of Wood Decomposition: Insights from the Steccherinum ochraceum.

Steccherinum ochraceum is a white rot basidiomycete with wide ecological amplitude. It occurs in different regions of Russia and throughout the world, occupying different climatic zones. S. ochraceum colonizes stumps, trunks, and branches of various deciduous (seldom coniferous) trees. As a secondary colonizing fungus, S. ochraceum is mainly observed at the late decay stages. Here, we present the de novo assembly and annotation of the genome of S. ochraceum, LE-BIN 3174. This is the 8th published genome of fungus from the residual polyporoid clade and the first from the Steccherinaceae family. The obtained genome provides a first glimpse into the genetic and enzymatic mechanisms governing adaptation of S. ochraceum to an ecological niche of pre-degraded wood. It is proposed that increased number of carbohydrate-active enzymes (CAZymes) belonging to the AA superfamily and decreased number of CAZymes belonging to the GH superfamily reflects substrate preferences of S. ochraceum. This proposition is further substantiated by the results of the biochemical plate tests and exoproteomic study, which demonstrates that S. ochraceum assumes the intermediate position between typical primary colonizing fungi and litter decomposers or humus saprotrophs. Phylogenetic analysis of S. ochraceum laccase and class II peroxidase genes revealed the distinct evolutional origin of these genes in the Steccherinaceae family.

Role of lipids in the thermal plasticity of basidial fungus Favolaschia manipularis.

In this study, we examined the lipid composition of two strains of the tropical basidiomycete Favolaschia manipularis (Berk.) Teng, which differ in their adaptive potential to high (35 °C) and low (5 °C) temperatures. The results suggest that adaptation to extreme temperatures involves a change in the molecular composition of sterols, in addition to other well-known mechanisms of regulating membrane thickness and fluidity, such as changes in the lipid unsaturation and in the proportion of bilayer- and non-bilayer-forming lipids. It was demonstrated for the first time that adaptation to high temperature stress in fungi is accompanied by the accumulation of 9(11)-dehydroergosterol and ergosterol peroxide. Furthermore, increased thermal plasticity correlates with high storage lipid (triglycerides) content, accumulation of phosphatidic acid in the membrane, and an equal proportion of bilayer and non-bilayer lipids in the membrane.

The Brown Roll-Rim Mushroom, Paxillus involutus (Agaricomycetes), as a Promising Biomedical Research Resource.

The brown roll-rim mushroom (Paxillus involutus) quickly produces biomass in nature, although, being a mycorrhizal fungus, it is rather poorly maintained in culture. Information about its toxic properties is controversial. Until the mid-20th century, the species was considered as an edible fungus; however, data later accumulated regarding its poisonous properties, leading to the term "Paxillus syndromeP. involutus. Since mushrooms can have quite a few unidentified antigens complementary to B-lymphocyte receptors, this is a hidden danger of using unfractionated mushroom raw materials for preventive and oncotherapy purposes, and we hope that this article stimulates immunological groups worldwide to identify the "X" antigen related to the Paxillus syndrome. Oncotherapy effects of the known bioactive complexes of P. involutus are associated with a specific inhibition of some growth receptors of the cancer cell, whereas experimentation with purified substances of P. involutus and various families of growth receptors of cancer cell has good prospects. A clear speciation is fixing within the P. involutus complex. The key for identification of species of P. involutus complex is given and cultural characteristics of P. involutus strains kept at Komarov Botanical Institute Basidiomycetes Culture Collection are presented.

White-rot basidiomycetes Junghuhnia nitida and Steccherinum bourdotii: Oxidative potential and laccase properties in comparison with Trametes hirsuta and Coriolopsis caperata.

White-rot basidiomycetes from the poorly studied residual polyporoid clade of Polyporales order Junghuhnia nitida (Pers.) Ryvarden and Steccherinum bourdotii Saliba & A. David grow as secondary xylotrohps on well decomposed woody materials. The main objective of the current study was to compare oxidative potential, growth, production of oxidative enzymes and laccase properties of J. nitida and S. bourdotii with that of typical primary xylotrohps Trametes hirsuta (Wulfen) Lloyd and Coriolopsis caperata (Berk.) Murrill, belonging to the core polyporoid clade. For the first time we report species J. nitida and S. bourdotii as active laccase producers. New laccases from J. nitida and S. bourdotii were purified and characterized. They had an identical molecular weight of 63 kDa and isoelectric points of 3.4 and 3.1, respectively. However, the redox potential of the T1 copper site for both J. nitida (610 mV) and S. bourdotii (640 mV) laccases was lower than those for T. hirsuta and C. caperata laccases. The new laccases showed higher temperature optima and better thermal stability than T. hirsuta and C. caperata laccases. Their half-lives were more than 40 min at 70 °C. The laccases from J. nitida and S. bourdotii showed higher affinity to syringyl-type phenolic compounds than T. hirsuta and C. caperata laccases. The oxidative potential of studied fungi as well as the properties of their laccases are discussed in terms of the fungal life-style.

Cultural and Metabolomic Studies of a New Phtalides Producer, Lignomyces vetlinianus (Agaricomycetes).

Culture characteristics and metabolomic profiling (on the basis of gas chromatography-mass spectrometry) of 3 strains of Lignomyces vetlinianus were studied. Growth rate, macromorphology, and micromorphology of mycelia grown on various media are described. More than 60 compounds were detected in the mycelial extracts, including amino acids, organic acids that are active during the tricarboxylic acid cycle, sugars, fatty acids, sugar alcohols, and sugar acids. Principal component analysis of low-molecular-weight compounds in mycelial methanol extracts of L. vetlinianus strains at different stages of growth demonstrated that the pattern of mycelial metabolomes grouped by age of the culture indicates a significant relation between the development of the culture and the specificity of its metabolite spectrum. Slow-growing cultures develop gradually and are characterized by several changes in metabolite states. The pattern of points is grouped more tightly for fast-growing strains. The production of crystal-like aggregates was observed for aging mycelia at the stationary phase of growth. These aggregates were isolated from mycelia and identified as clusters of 4,6-dimethoxy-phthalide. The molecular structure of this substance was confirmed by nuclear magnetic resonance analysis. The results show that the concentration of 4,6-dimethoxy-phthalide increased during cultivation. Fruiting bodies contained very small amounts of 4,6-dimethoxy-phthalide compared with amounts in mycelia. It can be assumed that L. vetlinianus is a powerful natural producer of phthalides of biotech-nological interest and can be used as a model to study phenolic metabolism in fungi.

Typification and Characterization of Trametes multicolor (Agaricomycetes), a Perspective Species of Medicinal Mushrooms.

Nomenclature revision and enlarged taxonomical descriptions are still needed for some well-known species whose interpretation is complicated by many nomenclature or taxonomical problems. The polyporoid fungus widely known as Trametes ochracea (= Coriolus zonatus) belongs to such a problematic group. At the same time, recent data show that this species, like its sister species T. versicolor, seems to be a perspective subject for fungal biotechnology and pharmacology. This article is devoted to stabilizing the nomenclature of the species in question via lectotypification and epitypification of Boletus multicolor. It will clarify the name T. multicolor as applied to this species is nomenclaturally correct and useful, free of further problems. An expanded species description and cultural characterization of epitype materials are presented.

Draft Genome Sequence of the Fungus Trametes hirsuta 072.

A standard draft genome sequence of the white rot saprotrophic fungus Trametes hirsuta 072 (Basidiomycota, Polyporales) is presented. The genome sequence contains about 33.6 Mb assembled in 141 scaffolds with a G+C content of ~57.6%. The draft genome annotation predicts 14,598 putative protein-coding open reading frames (ORFs).

Diacylglyceryltrimethylhomoserine content and gene expression changes triggered by phosphate deprivation in the mycelium of the basidiomycete Flammulina velutipes.

Diacylglyceryltrimethylhomoserines (DGTS) are betaine-type lipids that are phosphate-free analogs of phosphatidylcholines (PC). DGTS are abundant in some bacteria, algae, primitive vascular plants and fungi. In this study, we report inorganic phosphate (Pi) deficiency-induced DGTS synthesis in the basidial fungus Flammulina velutipes (Curt.: Fr.) Sing. We present results of an expression analysis of the BTA1 gene that codes for betaine lipid synthase and two genes of PC biosynthesis (CHO2 and CPT1) during phosphate starvation of F. velutipes culture. We demonstrate that FvBTA1 gene has increased transcript abundance under phosphate starvation. Despite depletion in PC, both CHO2 and CPT1 were determined to have increased expression. We also describe the deduced amino acid sequence and genomic structure of the BTA1 gene in F. velutipes. Phylogenetic relationships between putative orthologs of BTA1 proteins of basidiomycete fungi are discussed.

Antiviral activity of polyporoid mushrooms (higher Basidiomycetes) from Altai Mountains (Russia).

Antiviral activity against type A influenza virus of birds A/chicken/Kurgan/05/2005 (H5N1) and humans A/Aichi/2/68 (H3N2) was investigated for aqueous extracts from mycelium of 11 basidial fungi species collected in the Altai Mountains (Altai Republic, Russia). The most perspective strains for producing antiviral medicines are studied strains Daedaleopsis confragosa, Datronia mollis, Ischnoderma benzoinum, Trametes gibbosa, T. versicolor, Laricifomes officinalis, and Lenzites betulina.