Mycelial Cultures Research Articles

Micromycetes Trichoderma for plants protection against alternariosis

Currently there is an increased presence of Alternaria sp. mycotoxins in raw food materials and foods. In order to reduce the development of Alternaria fungi and to reduce their harmfulness, it is important to use biological preparations on the base of the microorganisms for plant protection. The aim of the research is to select strains of Trichoderma sp. fungi promising to control alternariosis in plants crops. The antagonistic activity of the mycelial culture of Trichoderma towards Alternaria was studied by dual culture method. The antifungal activity of the culture liquid was evaluated by agar diffusion method. The culture liquids containing metabolites of three strains, inhibited the growth of the pathogen by forming inhibition zones with a radius of 13-16 mm. Germination of wheat seeds on infectious background of soil contamination with the pathogen A.alternata identified micromycetes to create a biological product against alternariosis. Biological effectiveness of isolates 2N and 13N used for seeds protection against Alternaria is 87.3% and 83.6%, respectively.

Responses of Bacterial Community Structure, Diversity, and Chemical Properties in the Rhizosphere Soil on Fruiting-Body Formation of Suillus luteus.

Mycorrhiza helper bacteria (MHB) play an important role in driving mycorrhizal formation. There are few reports on the relationship between bacteria and fruiting growths. Taking mycorrhizal rhizosphere soil from sporocarps of the S. luteus and non-mycorrhizal rhizosphere soil of the host plant (Larix gmelinii), we measured the bacterial community structure and diversity and chemical properties to clarify the effect of bacteria on fruiting-body formation. The bacterial diversity was significantly higher in mycorrhizal rhizosphere soil (p < 0.05) than that in non-mycorrhizal rhizosphere soil. The relative abundance of Burkholderia, Bradyrhizobium, Pseudomonas, and Rhizobium was significantly higher (p < 0.05) in mycorrhizal rhizosphere soil than in non-mycorrhizal rhizosphere soil. The soil organic matter (SOM), total nitrogen (TN), total phosphorus (TP), total potassium (TK), ammonium nitrogen (AN), available phosphorus (AP), available potassium (AK), and the activity of catalase, urease, and phosphatase in mycorrhizal rhizosphere soil were significantly higher (p < 0.05) than those in non-mycorrhizal rhizosphere soil. A redundancy analysis (RDA) showed that dominant bacteria are closely related to soil enzyme activity and physicochemical properties (p < 0.05). The boletus recruits a large number of bacteria around the plant roots that speed up nutrient transformation and increase the soil nutrient content, providing an important guarantee for mycelium culture and fruiting-body formation. These findings provide ideas for the nutritional supply of boletus sporocarps and lay the theoretical foundation for the efficient artificial cultivation of boletus.

Lanostane triterpenoids from mycelial cultures of the basidiomycete Ganoderma weberianum

Eight undescribed lanostane triterpenoids ( 1 – 8 ) were isolated from cultures of the basidiomycete Ganoderma weberianum TBRC-BCC 60642. The structural types of lanostane constituents were different between mycelial cultures and fruiting bodies. Lanostanes 2 , 3 and 6 showed moderate antimalarial activity against Plasmodium falciparum K1 (IC 50 10–15 μM). • Eight new lanostanes were isolated from cultures of Ganoderma weberianum. • The structures were elucidated by HRESIMS and NMR spectroscopic methods. • Three new lanostanes possessed antimalarial activity.

CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITY OF CULTIVATED MYCELIUM OF WOOD-DESTROYING BASIDIOMYCETE FOMES FOMENTARIUS (L.) FR.

The biomass of the mycelium of the xylotrophic basidiomycete Fomes fomentarius strain Kr-112 was obtained by submerged cultivation. The highest yield of dry biomass (6.9±0.3 g/l) was observed during submerged cultivation in a nutrient medium with starch and corn extract. Dry extracts were obtained from the biomass of the cultivated mycelium of the fungus and characterized by their chemical composition. The highest content of total protein (27±1 mg/g), phenolic compounds (13.8±0.5 mg/g) and flavonoids (13±1 mg/g) was shown in the ethanol extract of the cultured mycelium Fomes fomentarius grown in glucose-peptone medium. The maximum content of polysaccharides (379±16 mg/g) was shown by an ethanol extract from the mycelium of Fomes fomentarius grown in a medium with starch and corn extract. Extracts from the mycelium of Fomes fomentarius grown in a glucose-peptone medium showed the highest antioxidant activity. An ethanol extract obtained from a cultured mycelium grown in a glucose-peptone medium-exhibited antiviral activity in vitro against herpes simplex virus type 2 and avian influenza virus A/chicken/Kurgan/05/2005 (H5N1). The animal protection factor was 60% in experiments in vivo when orally administered ethanol extract of mycelium grown in a glucose-peptone medium in mice infected with the human influenza A/Aichi/2/68 (H3N2) virus in the prophylactic scheme.

Contrasting regulation of live Bacillus cereus No.1 and its volatiles on Shiraia perylenequinone production

BackgroundFungal perylenequinones (PQs) are a class of photoactivated polyketide mycotoxins produced by plant-associated fungi. Hypocrellins, the effective anticancer photodynamic therapy (PDT) agents are main bioactive PQs isolated from a bambusicolous Shiraia fruiting bodies. We found previously that bacterial communities inhabiting fungal fruiting bodies are diverse, but with unknown functions. Bacillus is the most dominant genus inside Shiraia fruiting body. To understand the regulation role of the dominant Bacillus isolates on host fungus, we continued our work on co-culture of the dominant bacterium B. cereus No.1 with host fungus Shiraia sp. S9 to elucidate bacterial regulation on fungal hypocrellin production.ResultsResults from "donut" plate tests indicated that the bacterial culture could promote significantly fungal PQ production including hypocrellin A (HA), HC and elsinochrome A-C through bacterial volatiles. After analysis by gas chromatograph/mass spectrometer and confirmation with commercial pure compounds, the volatiles produced by the bacterium were characterized. The eliciting roles of bacterial volatile organic compounds (VOCs) on HA production via transcriptional regulation of host Shiraia fungus were confirmed. In the established submerged bacterial volatile co-culture, bacterial volatiles could not only promote HA production in the mycelium culture, but also facilitate the release of HA into the medium. The total production of HA was reached to 225.9 mg/L, about 1.87 times that of the fungal mono-culture. In contrast, the live bacterium suppressed markedly fungal PQ production in both confrontation plates and mycelium cultures by direct contact. The live bacterium not only down-regulated the transcript levels of HA biosynthetic genes, but also degraded extracellular HA quickly to its reductive product.ConclusionOur results indicated that bacterial volatile release could be a long-distance signal to elicit fungal PQ production. Biodegradation and inhibition by direct contact on fungal PQs were induced by the dominate Bacillus to protect themselves in the fruiting bodies. This is the first report on the regulation of Bacillus volatiles on fungal PQ production. These findings could be helpful for both understanding the intimate fungal–bacterial interactions in a fruiting body and establishing novel cultures for the enhanced production of bioactive PQs.Graphical

Antimicrobial and Cytotoxic Cyathane-Xylosides from Cultures of the Basidiomycete Dentipellis fragilis.

In our continued search for biologically active metabolites from cultures of rare Basidiomycota species, we found eight previously undescribed cyathane-xylosides from submerged cultures of Dentipellis fragilis, which were named dentifragilins A–H. In addition, the known cyathane derivatives striatal D and laxitextine A were isolated. All compounds were characterized by high-resolution electrospray ionization mass spectrometry (HR-ESIMS) as well as by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy. Several of the compounds exhibited significant activities in standardized cell-based assays for the determination of antimicrobial and cytotoxic effects. The discovery of cyathanes in the genus Dentipellis has chemotaxonomic implications, as this class of diterpenoids has already been shown to be characteristic for mycelial cultures of the related genera Hericium and Laxitextum, which are classified as Dentipellis in the family Hericiaceae.

Studies on the secondary metabolism of Rosellinia and Dematophora strains (Xylariaceae) from Iran

The xylariaceous genus Dematophora has recently been resurrected and segregated from Rosellinia based on a molecular phylogeny and morphological characters. This was an important taxonomic change because Dematophora in the current sense contains several important pathogens, while Rosellinia is limited to mainly saprotrophic species that have an endophytic stage in their life cycle and may even have beneficial effects on the host plants. During our ongoing work on the functional biodiversity of the Xylariales, we have encountered new strains of rosellinoid Xylariaceae from Iran and have studied their mycelial cultures for secondary metabolites in an attempt to establish further chemotaxonomic affinities. In the process, we isolated and identified 13 compounds, of which rosellisteroid (1), the cichorine derivative 2, and the alkaloid 3 are new. Out of these, nine were tested for their antimicrobial affinities with cytochalasin E (6) exhibiting weak activity against Schizosaccharomyces pombe. The cytotoxicity of three cytochalasin derivatives was examined and their effects on the F-actin cytoskeletal organization studied by fluorescence microscopy using fluorescent phalloidin. Cytochalasin E (6) and Δ6,12-cytochalasin E (7) showed strong and irreversible action on actin, while cytochalasin K (8) exhibited weaker, reversible effects.

Estimation of the most suitable nitrogen concentration for sporocarp formation in Laccaria japonica colonizing Pinus densiflora seedlings through in vitro mycelial culture.

Many ectomycorrhizal (ECM) fungi produce commercially valuable edible sporocarps. However, the effects of nitrogen (N) application on ECM fungal sporocarp formation remain poorly understood. In this study, we investigated the effect of application of various N concentrations (0, 5, 25, 50, 100, and 200mg/L) on the growth of Laccaria japonica mycelia in vitro for 1month. The results showed that L. japonica mycelial biomass was highest in the 50mg/L treatment and was significantly inhibited at N concentrations higher than 200mg/L. Next, we investigated the effects of N application on mycorrhizal colonization and sporocarp formation in L. japonica colonizing Pinus densiflora seedlings in pots. The seedlings were watered with nutrient solutions containing 0, 5, 25, 50, or 100mgN/L. The biomass, photosynthetic rate, and mycorrhizal colonization rates of the seedlings were measured at 45days (first appearance of primordia), 65days (sporocarp appearance on the substrate surface), and 4months after seedlings were transplanted. The numbers of primordia and sporocarps were recorded during the experimental period. Total carbon (C) and N content were determined in seedlings at 4months after transplantation, and in L. japonica sporocarps. Both mycelial growth and sporocarp production reached their maximum at an N application concentration of 50mg/L, suggesting that the most suitable N concentration for ECM fungal sporocarp formation can easily be estimated in vitro during mycelial growth. This finding may help determine the most suitable N conditions for increasing edible ECM fungus sporocarp production in natural forests.

Meroterpenoids Possibly Produced by a Bacterial Endosymbiont of the Tropical Basidiomycete Echinochaete brachypora.

A mycelial culture of the African basidiomycete Echinochaete cf. brachypora was studied for biologically active secondary metabolites, and four compounds were isolated from its crude extract derived from shake flask fermentations, using preparative high-performance liquid chromatography (HPLC). The pure metabolites were identified using extensive nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). Aside from the new metabolites 1-methoxyneomarinone (1) and (E)-3-methyl-5-(-12,13,14-trimethylcyclohex-10-en-6-yl)pent-2-enoic acid (4), the known metabolites neomarinone (2) and fumaquinone (4) were obtained. Such compounds had previously only been reported from Actinobacteria but were never isolated from the cultures of a fungus. This observation prompted us to evaluate whether the above metabolites may actually have been produced by an endosymbiontic bacterium that is associated with the basidiomycete. We have indeed been able to characterize bacterial 16S rDNA in the fungal mycelia, and the production of the metabolites stopped when the fungus was sub-cultured on a medium containing antibacterial antibiotics. Therefore, we have found strong evidence that compounds 1–4 are not of fungal origin. However, the endofungal bacterium was shown to belong to the genus Ralstonia, which has never been reported to produce similar metabolites to 1–4. Moreover, we failed to obtain the bacterial strain in pure culture to provide final proof for its identity. In any case, the current report is the first to document that polyporoid Basidiomycota are associated with endosymbionts and constitutes the first report on secondary metabolites from the genus Echinochaete.

Fungal Degradation of Extractives Plays an Important Role in the Brown Rot Decay of Scots Pine Heartwood.

Scots pine heartwood is known to have resistance to wood decay due to the presence of extractives, namely stilbenes and resin acids. However, previous studies have indicated that these extractives are degradable by wood decaying fungi. This study aimed to investigate the relationship between extractive degradation and heartwood decay in detail and to gain insight into the mechanisms of extractive degradation. Mass losses recorded after a stacked-sample decay test with brown rot fungi showed that the heartwood had substantial decay resistance against Coniophora puteana but little resistance against Rhodonia placenta. Extracts obtained from the decayed heartwood samples revealed extensive degradation of stilbenes by R. placenta in the early stages of decay and a noticeable but statistically insignificant loss of resin acids. The extracts from R. placenta-degraded samples contained new compounds derived from the degraded extractives: hydroxylated stilbene derivatives appeared in the early decay stages and then disappeared, while compounds tentatively identified as hydroxylated derivatives of dehydroabietic acid accumulated in the later stages. The degradation of extractives was further analysed using simple degradation assays where an extract obtained from intact heartwood was incubated with fungal mycelium or extracellular culture fluid from liquid fungal cultures or with neat Fenton reagent. The assays showed that extractives can be eliminated by several fungal degradative systems and revealed differences between the degradative abilities of the two fungi. The results of the study indicate that extractive degradation plays an important role in heartwood decay and highlight the complexity of the fungal degradative systems.

The development of extracellular vesicle markers for the fungal phytopathogen Colletotrichum higginsianum.

Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14‐3‐3 protein (Bmh1) as potential EV markers and generated transgenic strains expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV interkingdom communication between plants and fungi.

Optimization of White-Rot Fungi Mycelial Culture Components for Bioremediation of Pharmaceutical-Derived Pollutants

White-rot fungi can degrade a wide spectrum of environmental pollutants, including pharmaceuticals, which are not efficiently removed from wastewater by conventional methods, e.g., the activated sludge method. However, the treatment of wastewater with the use of fungal cultures (mycoremediation) also has significant limitations: among others, the need to use appropriate, often-expensive culture media. We aimed to screen 18 media ingredients, including seven agrifood byproducts for Armillaria mellea, Phanerochaete chrysosporium and Pleurotus ostreatus in submerged cultures to select the low-cost medium optimal for biomass production and laccase activity. We screened nine mathematic models to describe the relation of fungal growth and the amount of the selected byproduct in media. Finally, we tested the ability of the strain with the highest mycelial growth and enzyme-producing ability in the selected medium to degrade eight drug contaminants. Three media variants composed of byproducts provided both efficient growth and laccase production: corn steep liquor + poplar, dried distillers grains with solubles + poplar and corn steep liquor 50%. Among the investigated growth models, the Han–Levenspiel equation described well the specific growth rate in function of the nominal substrate concentration in one-component media. Pleurotus ostreatus, the fungus with the highest ligninolytic enzyme activity, cultured in medium composed of corn steep liquor, removed six of eight drug contaminants with a removal degree of 20–90% in 48 h. The obtained data on the optimal culture media consisting of insoluble components provide initial data for upscaling the process and designing an appropriate type of bioreactor for the process of removing drug contaminants from water.

First Report of Epicoccum layuense Causing Leaf Brown Spot on Camellia oleifera in Hefei, China.

Camellia oleifera, a major tree species for producing edible oil, is originated in China. Its oil is also called ''eastern olive oil'' with high economic value due to richness in a variety of healthy fatty acids (Lin et al. 218). However, leaves are susceptible to leaf spot disease (Zhu et al. 2014). In May 2021, we found circular to irregular reddish-brown lesions, 4-11 mm in diameter, near the leaf veins or leaf edges on 30%-50% leaves of 1/3 oil tea trees in a garden of Hefei City, Anhui Province, China (East longitude 117.27, North latitude 31.86) (Figure S1 A). To isolate the causal agents, symptomatic leaves were cut from the junction of diseased and healthy tissues (5X5 mm) and treated with 70 % alcohol for 30 secs and 1 % NaClO for 5 min, and subsequently inoculated onto PDA medium for culture. After 3 days, hyphal tips were transferred to PDA. Eventually, five isolates were obtained. Then the isolates were cultured on PDA at 25°C for 7 days and the mycelia appeared yellow with a white edge and secreted a large amount of orange-red material to the PDA (Figure S1 B and C). Twenty days later, the mycelium appeared reddish-brown, and sub-circular (3-10 mm) raised white or yellow mycelium was commonly seen on the Petri dish, and black particles were occasionally seen. Meanwhile, the colonies on the PDA produced abundant conidia. Microscopy revealed that conidia were globular to pyriform, dark, verrucose, and multicellular with 14.2 to 25.3 μm (=19.34 μm, n = 30) diameter (Figure S1 D). The morphological characteristics of mycelial and conidia from these isolates are similar to that of Epicoccum layuense (Chen et al.2020). To further determine the species classification of the isolates, DNA was extracted from 7-day-old mycelia cultures and the PCR-amplified fragments were sequenced for internal transcribed spacer (ITS), beta-tubulin and 28S large subunit ribosomal RNA (LSU) gene regions ITS1/ITS4, Bt2a/Bt2b and LR0R/LR5, followed by sequencing and molecular phylogenetic analysis of the sequences analysis (White et al. 1990; Glass and Donaldson 1995; Vilgalys and Hester 1990). Sequence analysis revealed that ITS, beta-tubulin, and LSU divided these isolates into two groups. The isolates AAU-NCY1 and AAU-NCY2, representing the first group (AAU-NCY1 and AAU-NCY5) and the second group (AAU-NCY2, AAU-NCY3 and AAU-NCY4), respectively, were used for further studies. Based on BLASTn analysis, the ITS sequences of AAU-NCY1 (MZ477250) and AAU-NCY2 (MZ477251) showed 100 and 99.6% identity with E. layuense accessions MN396393 and KY742108, respectively. And, the beta-tubulin sequences (MZ552310; MZ552311) showed 99.03 and 99.35% identity with E. layuense accessions MN397247 and MN397248, respectively. Consistently, their LSU (MZ477254; MZ477255) showed 99.88 and 99.77% identity with E. layuense accessions MN328724 and MN396395, respectively. Phylogenetic trees were built by maximum likelihood method (1,000 replicates) using MEGA v.6.0 based on the concatenated sequences of ITS, beta-tubulin and LSU (Figure S2). Phylogenetic tree analysis confirmed that AAU-NCY1 and AAU-NCY2 are closely clustered with E. layuense stains (Figure S2). To test the pathogenicity, conidial suspension of AAU-NCY2 (106 spores/mL) was prepared and sterile water was used as the control. Twelve healthy leaves (six for each treatment) on C. oleifera tree were punched with sterile needle (0.8-1mm), the sterile water or spore suspension was added dropwise at the pinhole respectively (Figure S1 E and F). The experiment was repeated three times. By ten-day post inoculation, the leaves infected by the conidia gradually developed reddish-brown necrotic spots that were similar to those observed in the garden, while the control leaves remained asymptomatic (Figure S1 G and H). DNA sequences derived from the strain re-isolated from the infected leaves was identical to that of the original strain. E. layuense has been reported to cause leaf spot on C. sinensis (Chen et al. 2020), and similar pathogenic phenotypes were reported on Weigela florida (Tian et al. 2021) and Prunus x yedoensis Matsumura in Korea ( Han et al. 2021). To our knowledge, this is the first report of E. layuense causing leaf spot on C. oleifera in Hefei, China.

New data in Porotheleaceae and Cyphellaceae: epitypification of Prunulus scabripes Murrill, the status of Mycopan Redhead, Moncalvo & Vilgalys and a new combination in Pleurella Horak emend.

Mycopan is a genus established for Hydropus scabripes by Redhead, Moncalvo & Vilgalys (in Redhead 2013). They considered the genus to be distinct based on morphology and the phylogenetic analysis by Moncalvo et al. (2002) which included a sequence of Hydropus scabripes (AF042635, DAOM 192847) unrelated to the type species of Hydropus (H. fuliginarius). Subsequent sequences of material identified as Hydropus scabripes are not conspecific with the sequence of DAOM 192847. We consider this sequence (obtained from a mycelium culture) to be misidentified. We investigated the true phylogenetic position of authentic Mycopan including genera previously included in Cyphellaceae and Porotheleaceae. Sixteen collections of M. scabripes from Europe and North America were studied on morphological and molecular basis (nrITS and nrLSU sequences). No sequences were obtained from the holotype of Mycopan scabripes, and we designate an epitype to fix the interpretation of this species and the genus Mycopan. Mycopan is maintained as a good genus nested within Cyphellaceae as sister to the mycenoid genus Atheniella. The misidentified Hydropus scabripes AF042635 (DAOM 192847) represents a different species that is closely related to the holotype (and a new Italian collection) of Hebelomina microspora and the monospecific genus Pleurella described from New Zealand. Consequently, Hebelomina microspora is transferred to the emended genus Pleurella, which is sister to Baeospora within the Cyphellaceae. Additionally, based on these phylogenetic results, an updated taxonomic arrangement of Cyphellaceae and Porotheleaceae is proposed, emphasizing once again the polyphyletic nature of Hydropus and Gerronema.

First report of the fungus Gnomoniopsis smithogilvyi causing cankers on sweet chestnut (Castanea sativa) in Ireland

In autumn 2020 cankers and branch dieback (Figure 1) were found in a five-year-old gene bank plantation of Castanea sativa in Wicklow, Ireland. The planting material had been imported as grafted saplings from the United Kingdom. Isolations were made from the cankers by surface sterilising the outer bark near the cankers with 70% ethanol. Pieces (5 × 5 × 5mm) of woody material were removed and soaked in 2% NaOCl for one minute, followed by two one-minute distilled water washes. After dabbing dry using paper towels the pieces were placed aseptically onto the surface of malt extract agar (MEA; Difco™) and incubated at 21°C in the dark for three weeks. After several days, hyphal tips were taken from the fungal colonies and transferred onto 2% potato dextrose agar (PDA; DifcoTM). DNA was extracted from the mycelium of seven-day old cultures and the ITS region was sequenced using primers ITS5 and ITS4. Gnomoniopsis smithogilvyi (syn. G. castaneae) (Figure 2) was detected. Microscopic observations agreed with the published descriptions of Shuttleworth et al. (2012) and Visentin et al. (2012). The ITS sequences were deposited in GenBank under the accession numbers OM683278 and OM683279. Sixty three-year-old C. sativa saplings were inoculated with either of two G. smithogilvyi isolates, or a control sterile PDA plug (Table 1). After surface sterilising with 70% ethanol, a stem wound was made using a sterile 6 mm cork borer, 10 cm above soil level. A 6 mm mycelial plug of a three-week-old isolate of G. smithogilvyi was placed onto the wound. This was then covered in moist sterilised cotton wool, wrapped in plastic film, and covered by aluminium foil. Control plants were inoculated with sterile PDA plugs. Treatments were randomised and grown outside, between mid-August to mid-November (mean temperature 14.9°C; mean relative humidity 85.5%). After six weeks dark necrotic lesions (Figure 3; Table 1) were observed on all saplings which had been inoculated with G. smithogilvyi isolates, and G. smithogilvyi was re-isolated from all lesions. Gnomoniopsis smithogilvyi caused girdling on numerous saplings (Figure 4). The identity of the reisolated colonies was confirmed as G. smithogilvyi by morphological assessment and ITS sequencing, fulfilling Koch's postulates. All control saplings were found to be asymptomatic, and no G. smithogilvyi was isolated from these (0/8 attempted re-isolations). Gnomoniopsis smithogilvyi causes cankers on C. sativa hybrids in Spain, and on C. sativa in five European countries (Shuttleworth et al., 2016; Lewis et al., 2017; Lione et al., 2019). It also causes chestnut rot in C. sativa in Australia, France, Italy, New Zealand, Portugal and Switzerland (Visentin et al., 2012; Shuttleworth et al., 2016; Coelho & Gouveia, 2021). This is the first report of G. smithogilvyi causing cankers on C. sativa in Ireland. Follow up surveys and tests are planned to fully understand the distribution of G. smithogilvyi in Ireland.

Extraction possibilities of lipid fraction and authentication assessment of chaga (Inonotus obliquus)

Chaga (Inonotus obliquus) (Fr.) Pilát is a black perennial fungus that grows on adult birch trunks and has been traditionally used as a health promoting remedy in different countries. The lipophilic compounds of chaga were isolated applying conventional (Soxhlet), intensive (ultrasound assisted, accelerated solvent extraction) and environmentally friendly (supercritical fluid) extraction methods utilizing both polar and non-polar solvents. Soxhlet extraction showed the highest inotodiol yield (231 mg/100 g chaga) using cyclohexane. For authentication of chaga samples, isotope ratio mass spectrometry (IRMS) and multi-elemental analysis was performed. The extraction yield and profile of lipids, sterols and triterpenoids depend on the chaga origin. The amount of lipids in chaga samples varied in the range from 0.5 to 1.0%. The antimicrobial activity of chaga extracts against pathogenic and opportunistic pathogenic microorganisms was demonstrated. The results showed that extraction using supercritical CO2 delivered yields comparable to those obtained by conventional and intensive extraction techniques. Amount of target compounds (sterols and triterpenoids) obtained using supercritical CO2 extraction could represent the future for sustainable industrial-scale production of biologically active ingredients with antimicrobial activity. Authentication using IRMS allowed to differentiate between natural (fruiting bodies) and cultivated (mycelium) chaga.

Bioactivity and Mycochemical Profile of Extracts from Mycelial Cultures of Ganoderma spp.

Fungal mycelium cultures are an alternative to natural sources in order to obtain valuable research materials. They also enable constant control and adaptation of the process, thereby leading to increased biomass growth and accumulation of bioactive metabolites. The present study aims to assess the biosynthetic potential of mycelial cultures of six Ganoderma species: G. adspersum, G. applanatum, G. carnosum, G. lucidum, G. pfeifferi, and G. resinaceum. The presence of phenolic acids, amino acids, indole compounds, sterols, and kojic acid in biomass extracts was determined by HPLC. The antioxidant and cytotoxic activities of the extracts and their effects on the inhibition of selected enzymes (tyrosinase and acetylcholinesterase) were also evaluated. The total content of phenolic acids in the extracts ranged from 5.8 (G. carnosum) to 114.07 mg/100 g dry weight (d.w.) (G. pfeifferi). The total content of indole compounds in the extracts ranged from 3.03 (G. carnosum) to 11.56 mg/100 g d.w. (G. lucidum) and that of ergosterol ranged from 28.15 (G. applanatum) to 74.78 mg/100 g d.w. (G. adspersum). Kojic acid was found in the extracts of G. applanatum and G. lucidum. The tested extracts showed significant antioxidant activity. The results suggest that the analyzed mycelial cultures are promising candidates for the development of new dietary supplements or pharmaceutical preparations.

Cultivation Technologies for Production of Medicinal Mushroom Biomass: Review.

Since most medicinal mushrooms are rare in nature, artificial cultivation and farming of their fruiting bodies as well as biotechnological cultivation of their fungal biomass in bioreactors on solid substrates and in liquid media has been established. This review compares the benefits and weaknesses of all three capabilities. Cultivation technologies are presented, including traditional cultivation via farming of fruiting bodies on wood logs and on sawdust-based substrates and modern biotechnological cultivation of mycelia in bioreactors by submerged and solid-state cultivation technologies. Our findings indicate that farming is cost-effective but large-scale production is time-consuming. In addition, solid-state cultivation is a comprehensive well-controlled technology that is close to the natural growth process and is suitable for veterinary use and use in food supplements. Finally, submerged liquid-state cultivation of mushroom mycelia is fast and well controlled.

Efficient Transport System of Cultivated Mushroom Mycelium Enables its Derived Carbon with High Performance Electrochemical Desalination Capability

Efficient Transport System of Cultivated Mushroom Mycelium Enables its Derived Carbon with High Performance Electrochemical Desalination Capability

Antimicrobial Activity of Fomitopsis Officinalis (Vill.) Bondartsev &amp; Singer in Pure Culture

Background. According to the World Health Organization antibiotic resistance is among the top ten threats to human health, food safety and development. Today antibiotic resistance has reached alarmingly high levels all over the world. Meanwhile, the increase in the synthetic drugs' production has led to the pathogenic mycobiota's rapid adaptation to the created chemicals, which have a narrow focus of application. That is why in modern biotechnology and pharmacology much attention is paid to natural producers of biologically active compounds, in particular – to xylotrophic fungi. It has been experimentally proven that the xylotrophic macromycete Fomitopsis officinalis or tinder fungus can be considered to be a promising producer of pharmacological substances with a broad spectrum of action. Studies of active metabolites, contained in the mycelial mass, culture fluid of the medicinal xylotrophic macromycete F. officinalis, and determination of their biological action remain relevant. Objective. The objective was to determine the antimicrobial activity of culture fluid and mycelial mass of F. officinalis different strains from the mushrooms collection (IBK Mushroom Culture Collection of the M.G. Kholodny Institute of Botany, NAS of Ukraine) against gram-negative and gram-positive bacteria species. Methods. An in vitro study of the antimicrobial activity of ethyl acetate extracts of culture fluid and aqueous-ethyl extracts of mycelial mass for F. officinalis strains IBK-5004, IBK-2497, IBK-2498 against gram-positive Staphylococcus aureus (B-918), Bacillus subtilis (В-901) and gram-negative Escherichia coli (B-906), Bacillus subtilis (B-900), Klebsiella pneumoniae (M-123) bacteria by disc-diffusion method was conducted. Results. High antimicrobial activity of tinder fungus culture fluid and mycelial mass extracts against Staphylococcus aureus was established after the 21st day of cultivation, while on the 28th day the zone of growth retardation was maximal (15–25 mm). The highest indices were recorded in F. officinalis IBK-5004 (20–25 mm) and IBK-2498 (20–24 mm) strains. Antimicrobial activity against Klebsiella pneumoniae in culture fluid extracts was manifested on the 21st and 28th days of cultivation. The highest antimicrobial activity against Klebsiella pneumoniae was observed in the culture fluid of the strain F. officinalis IBK-5004, the diameter of the growth retardation zone was 18 mm on the 28th day of cultivation. Mycelial mass's extracts showed moderate activity on the 14th day of cultivation (7-8 mm); maximal activity was recorded on the 28th day (12–22 mm). The most active strain was Fomitopsis officinalis IBK-2498. No antimicrobial activity against test organisms was detected in the following studied strains: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis. Conclusions. It has been established that the mycelial mass and culture fluid extracts of F. officinalis IBK-5004, IBK-2497, IBK-2498 strains have high antimicrobial activity against Staphylococcus aureus and moderate antimicrobial activity against Klebsiella pneumoniae on the 21st and 28th day of cultivation.