Ultrastructure and development of acanthocytes, specialized cells in Stropharia rugosoannulata, revealed by scanning electron microscopy (SEM) and cryo-SEM.

Acanthocytes are special cells with a distinct spiky shape produced exclusively by the fungi of Stropharia and can be used to defend against nematodes. In the present study, the ultrastructure and development of acanthocytes were revealed by scanning electron microscopy (SEM) and cryo-SEM in S. rugosoannulata, a popular cultivated mushroom both in China and Europe. The acanthocytes were abundant on the surface of rhizomorph, casing soils, and vegetative mycelia of homokaryotic and heterokaryotic strains in S. rugosoannulata. The development of the acanthocyte was investigated with cryo-SEM, which has distinct advantage for observation of the ultrastructure of live, hydrated structures. Three distinct stages, including formation of lateral branch that was covered with patches, spiky structure formation, and maturation of acanthocytes, were identified and described. The irregular patches deposited on the surface of lateral branches and the holes in the spiky branches of the acanthocytes were reported for the first time. The environmental nitrogen level showed impact on acanthocyte production, but it seemed not to be the indispensable factor. Acid medium could delay the initiation of the acanthocyte formation but did not affect the overall morphology and structure, indicating that the central deposit of acanthocytes should be acid soluble. Acanthocytes of S. rugosoannulata have similar hydrophobicity to mycelia. The observation of ultrastructure and development process of acanthocytes provides insights into the ecological function and evolution of this special structure.

Vermiculite as a culture substrate greatly improves the viability of frozen cultures of ectomycorrhizal basidiomycetes.

We assessed a new cryopreservation protocol that uses vermiculite as a culture substrate, called the vermiculite protocol (VP), by assessing the viability, recovery time of hyphae after revival, and colony diameter of cryosensitive ectomycorrhizal basidiomycete strains after storage for 2 weeks or 1 year in a vapour-phase liquid nitrogen tank. Twelve difficult-to-preserve strains of nine species (Amanita citrina, A. pantherina, A. rubescens, A. spissa, Kobayasia nipponica, Lactarius akahatsu, L. hatsudake, Sarcodon aspratus, and Tricholoma flavovirens) that did not achieve good revival after cryopreservation with our previous Homolka's perlite protocol and modified perlite protocol (MPP) experiments were used to assess the new methodology. Vermiculite and liquid medium were put into a cryotube and inoculated with an agar plug containing mycelia. The cryotube was cultured for various incubation times. After adequate mycelial growth, a mixture of cryoprotectants (5% dimethyl sulfoxide and 10% trehalose [5D10T] or 5% glycerol and 10% trehalose [5G10T]) was placed into the cryotube. The cryotube was frozen in a freezing container in a -80 °C freezer and then stored in vapour-phase liquid nitrogen. In the recovery test, 10 of 12 strains showed 100% revival after 2 weeks of storage in the 5G10T cryoprotectant, and all 12 strains showed 100% revival after 2 weeks of storage in the 5D10T cryoprotectant. Furthermore, all strains were viable after 1 year of storage in a vapour-phase liquid nitrogen tank. Thus, the VP is applicable to a wide range of ectomycorrhizal basidiomycete cultures, including highly cryosensitive strains.

Bovistol B, bovistol D and strossmayerin: Sesquiterpene metabolites from the culture filtrate of the basidiomycete Coprinopsis strossmayeri.

Basidiomycete fungi are a rich source of natural products with a diverse array of potentially exploitable bioactivities. Two dimeric sesquiterpenes, bovistol B (1) and D (2), and one monomeric sesquiterpene, strossmayerin (7), were isolated from the culture filtrate of the basidiomycete fungus Coprinopsis strossmayeri. The structures were determined through a combination of MS and 1D/2D NMR spectroscopic techniques. Likely monomeric precursors, identified on the basis of HRMS analysis, allow a plausible biosynthetic pathway to be proposed for the biosynthesis of 1 and 2, involving the dimerisation of the monomer through a hetero-Diels-Alder mechanism. A gene cluster, including a putative sesquiterpene 1-11 cyclase, was identified through phylogenetic and RNA-seq analysis, and is proposed to be responsible for the biosynthesis of 1 and 2.

Taxonomic update of Clitocybula sensu lato with a new generic classification.

The taxonomy and phylogeny of the hydropoid clade (genera Clitocybula s.l., Megacollybia, Leucoinocybe gen. nov., Hydropus, Trogia, Gerronema, Porotheleum and Lignomphalia gen. nov.) in Europe is studied using morphological and molecular approaches; the first three genera in detail including all known European species. Only two European species remain in Clitocybula s.str., Clitocybula lacerata and Clitocybula familia. The European C. lacerata is a species complex which should be treated as C. lacerata agg. at the current state of knowledge. A neotype originating from type area was designated to fix the application of the name. The presence of American species Clitocybula abundans in Europe is insufficiently proved. "Clitocybula dryadicola Ë® belongs to the genus Hydropus, and Clitocybula tilieti has an unclear systematic position. The results showed that Megacollybia and Leucoinocybe represent independent genera separated from Clitocybula. The genus Leucoinocybe is validly published with two European species, Leucoinocybe lenta and Leucoinocybe taniae. "Clitocybula flavoaurantia" proved to be conspecific with the latter species. The genus Lignomphalia is published for "Pseudoomphalina lignicola", a lignicolous omphalinoid species. The Indian "Clitocybula sulcata" is transferred to Leucoinocybe and "Clitocybula atrialba" to Gerronema. The first European records of Megacollybia marginata are published.

Spores of puffball fungus Lycoperdon pyriforme as a reference standard of stable monodisperse aerosol for calibration of optical instruments.

Advanced air quality control requires real-time monitoring of particulate matter size and concentration, which can only be done using optical instruments. However, such techniques need regular calibration with reference samples. In this study, we suggest that puffball fungus (Lycoperdon pyriforme) spores can be utilized as a reference standard having a monodisperse size distribution. We compare the Lycoperdon pyriforme spores with the other commonly used reference samples, such as Al2O3 powder and polystyrene latex (PSL) microspheres. Here we demonstrate that the puffball spores do not coagulate and, thus, maintain the same particle size in the aerosol state for at least 15 minutes, which is enough for instrument calibration. Moreover, the puffball mushrooms can be stored for several years and no agglomeration of the spores occurs. They are also much cheaper than other calibration samples and no additional devices are needed for aerosol generation since the fungal fruiting body acts as an atomizer itself. The aforementioned features make the fungal spores a highly promising substance for calibration and validation of particle size analyzers, which outperforms the existing, artificially produced particles for aerosol sampling. Furthermore, the L. pyriforme spores are convenient for basic research and development of new optical measurement techniques, taking into account their uniform particle size and absent coagulation in the aerosol.

Molecular phylogenetic analysis of new Entoloma rhodopolium-related species in Japan and its identification method using PCR-RFLP.

Poisonous Entoloma rhodopolium and other similar species including edible E. sarcopum are morphologically diverse. People mistake poisonous species for edible species. Classification and the detection method of these species need to be defined. The morphological and phylogenetic studies have been reported in northern Europe. In Japan, the genetic study remains unsolved. Thus, phylogenetic analysis of E. rhodopolium was conducted using ITS and RPB2 sequences, and the result was compared with that of European species. Japanese E. rhodopolium was classified into three clades, none of which belonged to the true European E. rhodopolium and other known species. Three species were defined as new species. Entoloma rhodopolium clade-I (named E. lacus) was genetically close to but morphologically separated from E. majaloides. Clade-II (E. subrhodopolium) was classified to the same group as E. sinuatum and E. subsinuatum, but distinct from these species. Clade-III was segregated from known Entoloma species including E. lupinum, and named E. pseudorhodopolium. Based on the classification, a simple identification method PCR-RFLP was developed to discriminate between poisonous species and edible E. sarcopum, which is very similar in morphology. The study can help to clarify the taxonomy of complex E. rhodopolium-related species, and to prevent food poisoning.

The oldest fossil mushroom.

A new fossil mushroom is described and illustrated from the Lower Cretaceous Crato Formation of northeast Brazil. Gondwanagaricites magnificus gen. et sp. nov. is remarkable for its exceptional preservation as a mineralized replacement in laminated limestone, as all other fossil mushrooms are known from amber inclusions. Gondwanagaricites represents the oldest fossil mushroom to date and the first fossil mushroom from Gondwana.

Mushrooms as Rainmakers: How Spores Act as Nuclei for Raindrops.

Millions of tons of fungal spores are dispersed in the atmosphere every year. These living cells, along with plant spores and pollen grains, may act as nuclei for condensation of water in clouds. Basidiospores released by mushrooms form a significant proportion of these aerosols, particularly above tropical forests. Mushroom spores are discharged from gills by the rapid displacement of a droplet of fluid on the cell surface. This droplet is formed by the condensation of water on the spore surface stimulated by the secretion of mannitol and other hygroscopic sugars. This fluid is carried with the spore during discharge, but evaporates once the spore is airborne. Using environmental electron microscopy, we have demonstrated that droplets reform on spores in humid air. The kinetics of this process suggest that basidiospores are especially effective as nuclei for the formation of large water drops in clouds. Through this mechanism, mushroom spores may promote rainfall in ecosystems that support large populations of ectomycorrhizal and saprotrophic basidiomycetes. Our research heightens interest in the global significance of the fungi and raises additional concerns about the sustainability of forests that depend on heavy precipitation.

Stipe cell wall architecture varies with the stipe elongation of the mushroom Coprinopsis cinerea.

A large amount of granular protrusions overlie the outer cell wall surfaces in both elongating and non-elongating stipe regions but overlie the inner cell wall surfaces only in non-elongating stipe regions. Removal of granular protrusions using alkali, amorphous materials overlying on both the inner and outer cell wall surfaces were explored in the non-elongating stipe regions. ß-1,3-Glucanase treatment not only removed above those granular protrusions and underlying amorphous materials on the wall surfaces but also removed wall matrices embedding chitin microfibrils on the cell walls of most stipe regions, except for the outer cell wall surfaces of the non-elongating stipe regions where most of the wall matrices remained. The chitin microfibrils were closely and transversely arranged on both the inner and outer cell wall surfaces in the elongating apical stipe region, whereas they were loosely and transversely arranged on the inner cell wall surfaces and further became sparser and even randomly arranged on the outer cell wall surface in the non-elongating stipe regions. We propose that the surface deposition of granular protrusions and amorphous materials and the change of microfibril architecture and wall matrices may cause loss of wall plasticity and cessation of stipe elongation.

Antioxidant components of selected indigenous edible mushrooms of the obsolete order Aphyllophorales / Compuestos antioxidantes en setas autóctonas comestibles del antiguo orden Aphyllophorales

Background: Species of phylum Basidiomycota are of greatinterestin many studies as a source of valuable biological compounds. Aims: To estimate the levels of antioxidant compounds (phenolic acids, indole compounds and sterols) in edible species of Aphyllophorales (sensu lato): Hydnum repandum L. and Sparassis crispa (Wulf.) Fr. Methods: A reversed-phase high performance liquid chromatography (RP-HPLC) method was used for the quantitative and qualitative analyses of antioxidant components (phenolic acids, indole compounds, and sterols). Results: Analysis of phenolic acids revealed that 8 out of the 10 analyzed compounds were present. The quantitatively predominant in Hydnum repandum was protocatechuic acid (75.23 mg/100 g DW), and in Sparassis crispa it was p-hydroxybenzoic acid (43.92 mg/100 g DW). In turn, analysis of indole compounds identified 5 out ofthe 12 analyzed compounds: indole, melatonin, serotonin, tryptamine, and l-tryptophan. Moreover, ergosterol was also identified and quantitatively determined (150.37 mg/100 g DW) in Sparassis crispa extracts. Conclusions: The data presented in this paper confirm the significant potential of chemical components with recognized antioxidant activity. The species can be considered as an alternative source of phenolic acids and ergosterol (AU)

Antecedentes: Los hongos del filo Basidiomycota son de gran interés en diversas investigaciones por ser una fuente de compuestos biológicos valiosos. Objetivos: Determinar los niveles de compuestos antioxidantes (ácidos fenólicos, compuestos de indol y esteroles) en las especies comestibles de Aphyllophorales (sensu lato) Hydnum repandum L. y Sparassis crispa (Wulf.) Fr. Métodos: El método RP-HPLC se empleó en el análisis cuantitativo y cualitativo de los siguientes componentes con actividad antioxidante: ácidos fenólicos, compuestos de indol y esteroles. Resultados: El análisis de los ácidos fenólicos reveló que 8 de los 10 compuestos analizados estaban presentes. El ácido cuantitativamente predominante en Hydnum repandum era el ácido protocatéquico (75,23 mg/100 g DW) y en Sparassis crispa el ácido p-hidroxibenzoico (43,92 mg/100 g DW). A su vez, el análisis de compuestos de indol identificó 5 de los 12 compuestos en estudio: indol, melatonina, serotonina, triptamina y l-triptófano. Por otra parte, en los extractos de Sparassis crispa se determinó también cuantitativamente la presencia de ergosterol (150,37 mg/100 g DW). Conclusiones: Los datos presentados en este trabajo confirman el gran potencial de los componentes químicos con actividad antioxidante reconocida. Las especies pueden ser consideradas como una fuente alternativa de ácidos fenólicos y ergosterol (AU)

DNA barcoding of wild edible mushrooms consumed by the ethnic tribes of India.

Wild edible mushrooms are consumed by the tribes of Meghalaya in the North-Eastern region of India, as part of their ethnic cuisine because of their favored organoleptic characteristics and traditionally known health benefits. Majority of these mushrooms have not yet been characterized in detail and are slowly shrinking in their natural habitats owing to anthropogenic factors and climate change. In the present study, representative specimens of ten morphologically distinct groups of wild edible mushrooms available in the traditional markets and their respective forest habitats, were subjected to multi-loci molecular characterization using SSU, ITS, RPB1 and RPB2 markers. The species identities inferred for the ten mushroom types using the SSU marker matched their morphological description in the case of four morphological groups only whereas the ITS marker successfully resolved the species identity for nine out of the ten mushroom groups under study. Both the protein coding gene markers RPB1 and RPB2 successfully resolved the species identity for three out of the ten morphologically distinct groups. Finally the most likely identity of the wild edible mushrooms under study has been suggested by matching their unique morphological characteristics with the generated DNA barcoding data. The present molecular characterization reveals the ten widely consumed wild mushroom types of Meghalaya, India to be Gomphus floccosus, Lactarius deliciosus, Lactarius volemus, Cantharellus cibarius, Tricholoma viridiolivaceum, Inocybe aff. sphaerospora, Laccaria vinaceoavellanea, Albatrellus ellisii, Ramaria maculatipes and Clavulina cristata. The final species identity generated by the ITS marker matched more accurately with the morphological characteristics/appearance of the specimens indicating the ITS region as a reliable barcode for identifying wild edible mushrooms.

Cercopemyces crocodilinus, a new genus and species related to Ripartitella, is described from North America.

Cercopemyces is described as a new genus based on collections of a newly discovered agaricoid species from the arid Cercocarpus forests of Utah and Colorado. The new genus is near Ripartitella and Cystodermella based on nLSU, rpb1 and ITS molecular sequences but distinctly different from these taxa. The ornamented basidiospores and inflated cells in the scales of the pileus surface of Cercopemyces indicate a close relationship to some species of Ripartitella, and the molecular analyses support the sister group relationship. Morphologically Cercopemyces crocodilinus is reminiscent of the genus Amanita in the field, but the inamyloid, cyanophilic, ornamented basidiospores and lack of acrophysalidic hyphae in the trama indicate otherwise. A rare eastern USA species, Ripartitella ponderosa, is transferred to Cercopemyces based on morphological and molecular data.

Inocybe section Rimosae in Utah: phylogenetic affinities and new species.

Results of a study on species of Inocybe section Rimosae sensu lato in Utah are presented. Eight species, seven from the Pseudosperma clade (section Rimosae sensu stricto) and one from the Inosperma clade (section Rimosae pro parte), are documented morphologically and phylogenetically. Five of the eight species, I. aestiva, I. breviterincarnata, I. cercocarpi, I. niveivelata and I. occidentalis-all members of the Pseudosperma clade-are described as new from Utah and other western states. Two European species, I. spuria and I. obsoleta, are confirmed from Utah. Inocybe aurora, originally described from Nova Scotia, is synonymized with I. obsoleta. The only member of the Inosperma clade recorded from Utah is I. lanatodisca, a widely distributed species for which three geographical clusters were detected. The phylogenetic analyses indicate that the Pseudosperma clade includes 53 clusters or species worldwide and that the Inosperma clade includes 47 such clusters. Many of these probably correspond to undescribed species. A key to species of section Rimosae sensu lato from Utah is provided together with illustrations of the eight species found in the state.

Taxonomic revision of true morels (Morchella) in Canada and the United States.

Recent molecular phylogenetic studies have revealed the existence of at least 50 species of Morchella worldwide and demonstrated a high degree of continental endemism within the genus. Here we describe 19 phylogenetic species of Morchella from North America, 14 of which are new (M. diminutiva, M. virginiana, M. esculentoides, M. prava, M. cryptica, M. frustrata, M. populiphila, M. sextelata, M. septimelata, M. capitata, M. importuna, M. snyderi, M. brunnea and M. septentrionalis). Existing species names (M. rufobrunnea, M. tomentosa, M. punctipes and M. angusticeps) are applied to four phylogenetic species, and formal description of one species (M. sp. "Mel-8") is deferred pending study of additional material. Methods for assessing morphological features in Morchella are delineated, and a key to the known phylogenetic species of Morchella in North America is provided. Type studies of M. crassistipa, M. hotsonii, M. angusticeps and M. punctipes are provided. Morchella crassistipa is designated nomen dubium.

A new Neopaxillus species (Agaricomycetes) from the Dominican Republic and the status of Neopaxillus within the Agaricales.

The new species Neopaxillus dominicanus is described on the basis of collections from the Dominican Republic. It is distinguished by having a basidiome with decurrent, distant, white lamellae with evident pink-lilac tinges, a non-depressed pileus at maturity and well developed catenulate cheilocystidia. A description, color photographs of fresh basidiomes and line drawings of relevant microscopic traits are provided. N. dominicanus is morphologically similar to Neopaxillus echinospermus, a type species of the genus. Based on comparative ITS-LSU rDNA gene sequence analyses, Neopaxillus, formerly placed in the Boletales, is considered within the Agaricales where it is sister to Crepidotus (Crepidotaceae), and N. dominicanus is supported as distinct from N. echinospermus. Finally, according to our morphological and molecular analyses, two collections of N. echinospermus from Mexico are referable to N. dominicanus.

A new species of Lactarius (subgenus Gerardii) from two relict Fagus grandifolia var. mexicana populations in Mexican montane cloud forests.

A new milkcap species, Lactarius fuscomarginatus, was found in the subtropical region of central Veracruz (eastern Mexico) associated with two relict populations of Fagus grandifolia var. mexicana. The species is characterized macroscopically by its dark pileus and stipe and by its distant and whitish lamellae with blackish to blackish brown edges. A molecular phylogenetic analyses based on ITS and LSU nucDNA sequences confirms the delimitation of this new taxon and places L. fuscomarginatus in subgenus Gerardii. A detailed morphological comparison is given with similar species.

Phylogeny and species delimitation in the genus Coprinellus with special emphasis on the haired species.

We inferred the phylogenetic structure and species limits within Coprinellus by using a newly generated multigene alignment of LSU, ITS and ß-tubulin sequences. We sampled 154 specimens of 71 species out of ca. 80 known taxa in Coprinellus and inferred phylogenetic relationships by Bayesian MCMC and ML bootstrapping. Morphological and phylogenetic analyses revealed 17 new species, five of which are described herein. Coprinellus was split into three large clades, one comprising species with an initially continuous sheath of veil (Domestici/Micacei clade) and two consisting of primarily setulose (haired) species (eurysporoid and Core Setulosi clades). The separation of veiled species and the clade structure therein corresponds well to the morphology-based sectional classification, although the inclusion of certain setulose taxa (e.g. C. disseminatus, C. verrucispermus, C. curtus etc.) in these clades necessitates a new morphological definition for the sections. Morphological traits are discussed for all clades of Coprinellus. Species limits in the eurysporoid and Core Setulosi clades are scrutinized in detail. Future directions of species-rank research and an artificial key to the recovered setulose species of Coprinellus is presented.

Cryptolepiota, a new sequestrate genus in the Agaricaceae with evidence for adaptive radiation in western North America.

Phylogenetic analyses based on nLSU and ITS sequence data indicate that the sequestrate genus Gigasperma is polyphyletic. Gigasperma cryptica, which is known only from New Zealand, has affinities with the Cortinariaceae whereas G. americanum and two additional undescribed taxa from western North America are derived from Lepiota within the Agaricaceae. The three North American taxa appear to be recently evolved and are closely related. They occur in similar environments and form a well supported clade indicating that adaptive radiation has occurred within this group of fungi. An independent genus with sequestrate fructifications, Cryptolepiota is proposed to accommodate the three species in this clade. Cryptolepiota microspora and C. mengei are described as new, and G. americanum is transferred to Cryptolepiota. Gigasperma cryptica is illustrated and compared with the species of Cryptolepiota.

Revision of taxonomic concept and systematic position of some Clavariaceae species.

A taxonomic and nomenclatural revision of some representatives of Clavariaceae is presented based on extensive collecting in central and western Europe. Five species originally described from Europe are identified, redescribed and delimited: Clavaria fragilis, Ramariopsis crocea, R. corniculata, R. helvola and R. pulchella. Lectotypes, epitypes or neotypes are designated for all these species. Descriptions are based on macro- and micromorphological characters and supplemented with DNA analyses of the nrLSU regions from 20 specimens. The molecular phylogenetic analyses reconstructed a phylogram showing relationships among the discussed species as well as some closely related taxa. The taxonomic value of the ratio of length and width of spores (Q-value) is discussed.

Time series hyperspectral chemical imaging data: challenges, solutions and applications.

Hyperspectral chemical imaging (HCI) integrates imaging and spectroscopy resulting in three-dimensional data structures, hypercubes, with two spatial and one wavelength dimension. Each spatial image pixel in a hypercube contains a spectrum with >100 datapoints. While HCI facilitates enhanced monitoring of multi-component systems; time series HCI offers the possibility of a more comprehensive understanding of the dynamics of such systems and processes. This implies a need for modeling strategies that can cope with the large multivariate data structures generated in time series HCI experiments. The challenges posed by such data include dimensionality reduction, temporal morphological variation of samples and instrumental drift. This article presents potential solutions to these challenges, including multiway analysis, object tracking, multivariate curve resolution and non-linear regression. Several real world examples of time series HCI data are presented to illustrate the proposed solutions.