Discovery of novel secondary metabolites from the basidiomycete Lentinus cf. sajor-caju and their inhibitory effects on Staphylococcus aureus biofilms.

Three novel derivatives of microporenic acid, microporenic acids H-J, were identified from submerged cultures of a Lentinus species obtained from a basidiome collected during a field trip in the tropical rainforest in Western Kenya. Their structures were elucidated via HR-ESIMS spectra and 1D/2D NMR spectroscopic analyses, as well as by comparison with known derivatives. Applying biofilm assays based on crystal violet staining and confocal microscopy, two of these compounds, microporenic acids H and I, demonstrated the ability to inhibit biofilm formation of the opportunistic pathogen Staphylococcus aureus. Thereby, they were effective in a concentration range that did not affect planktonic growth. Additionally, microporenic acid I enhanced the anti-biofilm activity of the antibiotics vancomycin and gentamicin when used in combination. This opens up possibilities for the use of these compounds in combination therapy to prevent the formation of S. aureus biofilms.

A Novel O- and S-Methyltransferase from Pleurotus sapidus Is Involved in Flavor Formation.

Increasing consumer aversion to non-natural flavoring substances is prompting a heightened interest in enzymatic processes for flavor production. This includes methylation reactions, which are often performed by using hazardous chemicals. By correlation of aroma profile data and transcriptomic analysis, a novel O-methyltransferase (OMT) catalyzing a respective reaction within the formation of p-anisaldehyde was identified in the mushroom Pleurotus sapidus. Heterologous expression in E. coli followed by purification allowed for further characterization of the enzyme. Besides p-hydroxybenzaldehyde, the proposed precursor of p-anisaldehyde, the enzyme catalyzed the methylation of further hydroxylated aromatic compounds at the meta- and para-position. The Km values determined for p-hydroxybenzaldehyde and S-adenosyl-l-methionine were 80 and 107 µM, respectively. Surprisingly, the studied enzyme enabled the transmethylation of thiol-nucleophiles, as indicated by the formation of 2-methyl-3-(methylthio)furan from 2-methyl-3-furanthiol. Moreover, the enzyme was crystallized at a resolution of 2.0 Å, representing the first published crystal structure of a basidiomycetous OMT.

The Nitrogen Content in the Fruiting Body and Mycelium of Pleurotus Ostreatus and Its Utilization as a Medium Component in Thraustochytrid Fermentation.

Following the idea of a circular bioeconomy, the use of side streams as substitutes for cultivation media (components) in bioprocesses would mean an enormous economic and ecological advantage. Costly compounds in conventional media for the production of the triterpene squalene in thraustochytrids are the main carbon source and complex nitrogen sources. Among other side streams examined, extracts from the spent mycelium of the basidiomycete Pleurotus ostreatus were best-suited to acting as alternative nitrogen sources in cultivation media for thraustochytrids. The total nitrogen (3.76 ± 0.01 and 4.24 ± 0.04%, respectively) and protein (16.47 ± 0.06 and 18.57 ± 0.18%, respectively) contents of the fruiting body and mycelium were determined. The fungal cells were hydrolyzed and extracted to generate accessible nitrogen sources. Under preferred conditions, the extracts from the fruiting body and mycelium contained 73.63 ± 1.19 and 89.93 ± 7.54 mM of free amino groups, respectively. Cultivations of Schizochytrium sp. S31 on a medium using a mycelium extract as a complex nitrogen source showed decelerated growth but a similar squalene yield (123.79 ± 14.11 mg/L after 216 h) compared to a conventional medium (111.29 ± 19.96 mg/L, although improvable by additional complex nitrogen source).

Enzymatic machinery of wood-inhabiting fungi that degrade temperate tree species.

Deadwood provides habitat for fungi and serves diverse ecological functions in forests. We already have profound knowledge of fungal assembly processes, physiological and enzymatic activities, and resulting physico-chemical changes during deadwood decay. However, in situ detection and identification methods, fungal origins, and a mechanistic understanding of the main lignocellulolytic enzymes are lacking. This study used metaproteomics to detect the main extracellular lignocellulolytic enzymes in 12 tree species in a temperate forest that have decomposed for 8 ½ years. Mainly white-rot (and few brown-rot) Basidiomycota were identified as the main wood decomposers, with Armillaria as the dominant genus; additionally, several soft-rot xylariaceous Ascomycota were identified. The key enzymes involved in lignocellulolysis included manganese peroxidase, peroxide-producing alcohol oxidases, laccase, diverse glycoside hydrolases (cellulase, glucosidase, xylanase), esterases, and lytic polysaccharide monooxygenases. The fungal community and enzyme composition differed among the 12 tree species. Ascomycota species were more prevalent in angiosperm logs than in gymnosperm logs. Regarding lignocellulolysis as a function, the extracellular enzyme toolbox acted simultaneously and was interrelated (e.g. peroxidases and peroxide-producing enzymes were strongly correlated), highly functionally redundant, and present in all logs. In summary, our in situ study provides comprehensive and detailed insight into the enzymatic machinery of wood-inhabiting fungi in temperate tree species. These findings will allow us to relate changes in environmental factors to lignocellulolysis as an ecosystem function in the future.

Improvement in the Stability and Enzymatic Activity of Pleurotus sapidus Lipoxygenase Dissolved in Natural Deep Eutectic Solvents (NADESs).

Natural deep eutectic solvents (NADESs) can serve as solvents for enzymes, are biodegradable, and have low toxicities. Eight NADESs with different hydrogen bond acceptors and donors were tested to improve the stability and activity of a lipoxygenase from Basidiomycete Pleurotus sapidus (LOXPSA). Betaine:sorbitol:water (1:1:3, BSorbW) and betaine:ethylene glycol (1:3, BEtGly) had the best impact on the peroxidation of linoleic acid and the side reaction of piperine to the vanilla-like scented compound piperonal. The yield of piperonal in NADESs increased by 43% in BSorbW and 40% in BEtGly compared to the control. The addition of BSorbW also enhanced the enzyme's stability at various temperatures and increased its activity during incubation at 60 °C. The demonstrated improvement in lipoxygenase activity and stability indicates versatile applications in industry, expanding the potential uses of the enzyme.

A New Species of Ganoderma (Ganodermataceae, Polyporales) from Southern China and Optimum Condition for Mycelia Production.

The present study sought to propose Ganoderma guixiense sp. nov. as a new species based on phenotypic and genotypic evidence. Phylogenetic analyses were carried out based on the internal transcribed spacer (ITS), the large subunit of nuclear ribosomal RNA gene (nLSU), and the second subunit of RNA polymerase II (RPB2) sequence data. G. guixiense has been characterized by pileate basidiomata, long stipe, in addition to reddish-black zonate pileal surface. Basidiospores are broadly ellipsoid with one end tapering at maturity, and measuring 9-12.8 × 6.5-9.3 µm. Basidia are oval to subglobose. This study marks the first exploration of the biological characteristics of G. guixiense. The result indicated that the optimal medium of mycelial growth was observed on malt extract agar (MEA) and yeast extract peptone dextrose agar (YPD) while the optimal temperature was found to be 25-30 °C with pH range of 6-7.

Three new Dioszegia species (Bulleribasidiaceae, Tremellales) discovered in the phylloplane in China.

The genus Dioszegia is comprised of anamorphic basidiomycetous yeasts and is classified in the family Bulleribasidiaceae of the order Tremellales. Currently, 24 species have been described and accepted as members of the genus, although its diversity and global distribution have not been thoroughly investigated. In this study, yeasts were isolated from plant leaves collected in the Guizhou and Henan Provinces of China and identified through a combination of morphological and molecular methods. Phylogenetic analyses of the combined ITS and LSU sequences coupled with morphological studies revealed three novel species, D.guizhouensissp. nov., D.foliicolasp. nov., and D.aurantiasp. nov., proposed here. Additionally, our phylogenetic analyses suggest that the recently discovered species D.terrae is a synonym of D.maotaiensis. This study presents detailed descriptions and illustrations of three new Dioszegia species and highlights distinctions between them and their close relatives. The findings of this study contribute to our knowledge of the biodiversity of Dioszegia, offering a foundation for future research.

Two new Erythrobasidium species inhabiting the phyllosphere discovered in the Baotianman Nature Reserve in China.

The genus Erythrobasidium is kind of species-scarce and undersampling basidiomycetes. Currently, only six species have been accepted into the genus and the diversity still remains incompletely understood. In this study, five Erythrobasidium strains were isolated in the surface of plant leaves collected from the Baotianman Nature Reserve, Henan Province, central China. Phylogenetic analyses of the small ribosomal subunit (SSU) rRNA gene, the internal transcribed spacer (ITS) region, the D1/D2 domain of the large subunit (LSU) rRNA gene, and the translation elongation factor 1-α (TEF1-α) gene coupled with morphological studies were employed to characterize and identify these isolates. As a result of these, two new species, namely E. turpiniae sp. nov. and E. nanyangense sp. nov., were delimited and proposed based on morphological and molecular evidence. A detailed description and illustration of both new species, as well as their differences with the close relatives in the genus are provided. An identification key for Erythrobasidium species is also provided. This study provides further insights into our understanding of Erythrobasidium species.

Influence of Salinity on the Extracellular Enzymatic Activities of Marine Pelagic Fungi.

Even though fungi are ubiquitous in the biosphere, the ecological knowledge of marine fungi remains rather rudimentary. Also, little is known about their tolerance to salinity and how it influences their activities. Extracellular enzymatic activities (EEAs) are widely used to determine heterotrophic microbes' enzymatic capabilities and substrate preferences. Five marine fungal species belonging to the most abundant pelagic phyla (Ascomycota and Basidiomycota) were grown under non-saline and saline conditions (0 g/L and 35 g/L, respectively). Due to their sensitivity and specificity, fluorogenic substrate analogues were used to determine hydrolytic activity on carbohydrates (ß-glucosidase, ß-xylosidase, and N-acetyl-ß-D-glucosaminidase); peptides (leucine aminopeptidase and trypsin); lipids (lipase); organic phosphorus (alkaline phosphatase), and sulfur compounds (sulfatase). Afterwards, kinetic parameters such as maximum velocity (Vmax) and half-saturation constant (Km) were calculated. All fungal species investigated cleaved these substrates, but some species were more efficient than others. Moreover, most enzymatic activities were reduced in the saline medium, with some exceptions like sulfatase. In non-saline conditions, the average Vmax ranged between 208.5 to 0.02 µmol/g biomass/h, and in saline conditions, 88.4 to 0.02 µmol/g biomass/h. The average Km ranged between 1553.2 and 0.02 µM with no clear influence of salinity. Taken together, our results highlight a potential tolerance of marine fungi to freshwater conditions and indicate that changes in salinity (due to freshwater input or evaporation) might impact their enzymatic activities spectrum and, therefore, their contribution to the oceanic elemental cycles.

Snowball: a novel gene family required for developmental patterning of fruiting bodies of mushroom-forming fungi (Agaricomycetes).

The morphogenesis of sexual fruiting bodies of fungi is a complex process determined by a genetically encoded program. Fruiting bodies reached the highest complexity levels in the Agaricomycetes; yet, the underlying genetics is currently poorly known. In this work, we functionally characterized a highly conserved gene termed snb1, whose expression level increases rapidly during fruiting body initiation. According to phylogenetic analyses, orthologs of snb1 are present in almost all agaricomycetes and may represent a novel conserved gene family that plays a substantial role in fruiting body development. We disrupted snb1 using CRISPR/Cas9 in the agaricomycete model organism Coprinopsis cinerea. snb1 deletion mutants formed unique, snowball-shaped, rudimentary fruiting bodies that could not differentiate caps, stipes, and lamellae. We took advantage of this phenotype to study fruiting body differentiation using RNA-Seq analyses. This revealed differentially regulated genes and gene families that, based on wild-type RNA-Seq data, were upregulated early during development and showed tissue-specific expression, suggesting a potential role in differentiation. Taken together, the novel gene family of snb1 and the differentially expressed genes in the snb1 mutants provide valuable insights into the complex mechanisms underlying developmental patterning in the Agaricomycetes. IMPORTANCE: Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are complex multicellular structures, with a spatially and temporally integrated developmental program that is, however, currently poorly known. In this study, we present a novel, conserved gene family, Snowball (snb), termed after the unique, differentiation-less fruiting body morphology of snb1 knockout strains in the model mushroom Coprinopsis cinerea. snb is a gene of unknown function that is highly conserved among agaricomycetes and encodes a protein of unknown function. A comparative transcriptomic analysis of the early developmental stages of differentiated wild-type and non-differentiated mutant fruiting bodies revealed conserved differentially expressed genes which may be related to tissue differentiation and developmental patterning fruiting body development.

Medium composition optimization and characterization of polysaccharides extracted from Ganoderma boninense along with antioxidant activity.

Ganoderma is a well-known medicinal macrofungal genus, of which several species have been thoroughly studied from the medicinal perspective, but most species are rarely involved in. In this study, we focus on the polysaccharides extracted from Ganoderma boninense and their antioxidant activity. Ganoderma boninense is a serious pathogen of oil palms that are cultivated commercially in Southeast Asia. Response surface methodology was conducted to optimize the liquid medium composition, and the mycelia biomass reached 7.063 g/L, that is, 1.4-fold compared with the seed medium. The crude and purified polysaccharides extracted from the fermentation broth showed well 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid radical scavenging abilities, and the scavenging abilities of purified polysaccharides reached 94.47 % and 99.88 %, respectively. Six fractions of polysaccharides were extracted and purified from fruiting bodies, mycelia and fermentation broth separately with the elution buffers of distilled water and 0.1 M NaCl solution. Generally, the polysaccharides from fruiting bodies showed stronger protective effect on H2O2-induced HepG2 cell oxidative damage than other fractions. A total of five to seven monosaccharides were identified in the six fractions of polysaccharides. The correlation analysis revealed that the content of fucose was significantly correlated with the antioxidant activity of polysaccharides, while xylose showed negative correlation results. In summary, the polysaccharides from G. boninense have a potential to be used as natural antioxidants.

Morphological and phylogenetic analyses reveal a new species of Anthracophyllum (Omphalotaceae, Agaricales) in Zhejiang Province, China.

During the investigations of macrofungi resources in Zhejiang Province, China, an interesting wood rot fungus was collected. Based on morphological and molecular phylogenetic studies, it is described as a new species, Anthracophyllum sinense. A. sinense is characterized by its sessile, charcoal black and pleurotoid pileus, sparse lamellae occasionally branching, clavate basidia with long sterigmata [(3-)6-7(-8) µm], and non-heteromorphous cystidia. A. sinense establishes a separate lineage close to A. archeri and A. lateritium in the phylogenetic tree.

Two new phyllospheric species of Colacogloea (Colacogloeaceae, Pucciniomycotina) identified in China.

During our ongoing survey of basidiomycetous yeasts associated with plant leaves in virgin forest, five Colacogloea strains were isolated in the Baotianman Nature Reserve, Henan Province, central China. Phenotypes from cultures and a phylogeny based on the internal transcribed spacer (ITS) regions and the D1/D2 domains of the large subunit (LSU) rRNA gene were employed to characterize and identify these isolates. As a result, two new species, namely Colacogloeaceltidissp. nov. and C.pararetinophilasp. nov., are introduced herein. In the phylogeny of combined ITS and LSU dataset, the new species C.celtidissp. nov. formed a clade with the unpublished Colacogloea strain (KBP: Y-6832), and together these formed the sister group to C.armeniacae, while C.pararetinophilasp. nov. was retrieved as a sister to C.retinophila. A detailed description and illustration of both new species, as well as the differences between them and their closest relatives in the genus are provided. Results from the present study will add to our knowledge of the biodiversity of Colacogloea in China.

Three New Species of Russulaceae (Russulales, Basidiomycota) from Southern China.

The characterization of natural fungal diversity impacts our understanding of ecological and evolutionary processes and can lead to novel bioproduct discovery. Russula and Lactarius, both in the order Russulales, represent two large genera of ectomycorrhizal fungi that include edible as well as toxic varieties. Based on morphological and phylogenetic analyses, including nucleotide sequences of the internal transcribed spacer (ITS), the 28S large subunit of ribosomal RNA (LSU), the second largest subunit of RNA polymerase II (RPB2), the ribosomal mitochondrial small subunit (mtSSU), and the translation elongation factor 1-α (TEF1-α) gene sequences, we here describe and illustrate two new species of Russula and one new species of Lactarius from southern China. These three new species are: R. junzifengensis (R. subsect. Virescentinae), R. zonatus (R. subsect. Crassotunicatae), and L. jianyangensis (L. subsect. Zonarii).

Why Are There So Few Basidiomycota and Basal Fungi as Endophytes? A Review.

A review of selected studies on fungal endophytes confirms the paucity of Basidiomycota and basal fungi, with almost 90% attributed to Ascomycota. Reasons for the low number of Basidiomycota and basal fungi, including the Chytridiomycota, Mucoromycota, and Mortierellomycota, are advanced, including isolation procedure and media, incubation period and the slow growth of basidiomycetes, the identification of non-sporulating isolates, endophyte competition, and fungus-host interactions. We compare the detection of endophytes through culture-dependent methods and culture-independent methods, the role of fungi on senescence of the host plant, and next-generation studies.

The evolution of ectomycorrhizal symbiosis in the Late Cretaceous is a key driver of explosive diversification in Agaricomycetes.

Ectomycorrhizal (EcM) symbiosis, a ubiquitous plant-fungus interaction in forests, evolved in parallel in fungi. Why the evolution of EcM fungi did not necessarily increase ecological opportunities for explosive diversification remains unclear. This study aimed to reveal the driving mechanism of the evolutionary diversification in the fungal class Agaricomycetes, specifically by testing whether the evolution of EcM symbiosis in the Late Cretaceous increased ecological opportunities. The historical character transitions of trophic state and fruitbody form were estimated based on phylogenies inferred from fragments of 89 single-copy genes. Moreover, five analyses were used to estimate the net diversification rates (speciation rate minus extinction rate). The results indicate that the unidirectional evolution of EcM symbiosis occurred 27 times, ranging in date from the Early Triassic to the Early Paleogene. The increased diversification rates appeared to occur intensively at the stem of EcM fungal clades diverging in the Late Cretaceous, coinciding with the rapid diversification of EcM angiosperms. By contrast, the evolution of fruitbody form was not strongly linked with the increased diversification rates. These findings suggest that the evolution of EcM symbiosis in the Late Cretaceous, supposedly with coevolving EcM angiosperms, was the key drive of the explosive diversification in Agaricomycetes.

Mushrooms Adapted to Seawater: Two New Species of Candolleomyces (Basidiomycota, Agaricales) from China.

Marine fungi have been studied for a long history in many realms, but there are few reports on marine mushrooms. In this study, marine fungi with conspicuous subglobose sequestrate basidioma were discovered from mangrove forests in South China. They grow on the deadwood of mangroves in the intertidal zone, periodically submerging into seawater due to the tide. Some marine animals were observed to nest in their basidiomata or consume them as food. The pileus-gleba-inner veil complex (PGI) of the basidioma was observed to be detached from the stipe and transferred into seawater by external forces, and drifting on sea to spread spores after maturity. The detachment mechanism of their PGIs was revealed through detailed microscopic observations. The contrast culturing experiment using freshwater and seawater potato dextrose agar media showed they have probably obligately adapted to the marine environment. Based on morphological and molecular phylogenetic evidence, two new species of Candolleomyces (Basidiomycota, Agaricales), namely C. brunneovagabundus and C. albovagabundus, were described. They are similar and close to each other, but can be distinguished by the size and color of the basidioma, and the size of the basidiospores.

New Tricholomalides D-G from the Mushroom Tricholoma ustaloides Grown in an Italian Beech Wood.

Four novel seconeodolastane diterpenoids, named tricholomalides D-G, were isolated, together with the known tricholomalide C, from the fruiting bodies of Tricholoma ustaloides Romagn., a species belonging to the large Tricholoma genus of higher mushrooms (Basidiomycota, family Tricholomataceae). They were isolated through multiple chromatographic separations, and the structures, including the absolute configuration, were established through a detailed analysis of MS, NMR, and CD spectral data and comparison with related compounds reported in the literature, which has been thoroughly revised.

Preliminary species diversity and community phylogenetics of wood-inhabiting basidiomycetous fungi in the Dabie Mountains, Central China reveal unexpected richness.

Wood-inhabiting fungi have important economic values as well as playing a major ecological role in forest ecosystem cycles. The Dabie Mountains, at the junction of Henan, Hubei, and Anhui Provinces, Central China, provide an ideal climate and favorable niches for the speciation and diversification of various forms of life including fungi. We studied the species diversity and community phylogenetics of wood-inhabiting basidiomycetous fungi that revealed 175 wood-inhabiting basidiomycetous species, of which 20 represented unidentified species, based on morphological and phylogenetic analyses of 575 specimens collected from ten sampling sites. These species belonged to two classes, 11 orders, 42 families, and 106 genera of Basidiomycota, and included 12 edible species, 28 medicinal species, four poisonous species, and seven forest pathogens. Four types of fungal distribution pattern at the genus level were recognized for 65 genera, while another 41 genera could not be placed in any known distribution pattern. The five sampling sites in the eastern part of the Dabie Mountains had significantly higher species diversity and phylogenetic diversity of wood-inhabiting basidiomycetous fungi than those in the western part, and thus deserve priority in terms of conservation. The community of wood-inhabiting basidiomycetous fungi in the Dabie Mountains is generally affected by a combination of habitat filtering and competitive exclusion. This study provides a basis on which to build actions for the comprehensive recognition, utilization, and conservation of wood-inhabiting basidiomycetous fungi in the region.

Morphological and Phylogenetic Characterization of Three Novel Thaxterogaster (Cortinariaceae) Species from China with an Emphasis on Their Subtropical Distribution.

Three new phlegmaciod species of Thaxterogaster, T. borealicremeolinus, T. rufopurpureus, and T. sinopurpurascens spp. nov., from subtropical China were described based on their morphological characteristics and molecular data. Thaxterogaster borealicremeolinus belongs to the sect. Cremeolinae and differs from the other species in this section in its larger basidiospores and its habitat in the Northern Hemisphere associated with Quercus sp. trees. Thaxterogaster rufopurpureus and T. sinopurpurascens belong to sect. Purpurascentes, in which T. rufopurpureus is characterized by a pileus with a reddish-brown coloration when mature and a clavate stipe, while T. sinopurpurascens is characterized by a violet basidiomata, except for a greyish orange to brown pileus, the distinctly marginate bulb of its stipe, and its distribution in subtropical China. The phylogenetic analyses were performed based on nrITS, and detailed descriptions of the new species are provided herein.