An overview of Agaricus section Hondenses and Agaricus section Xanthodermatei with description of eight new species from Pakistan.

In a recent revision of the genus Agaricus, A. section Xanthodermatei was split into two sections A. sect. Hondenses and A. sect. Xanthodermatei. Our objectives were to investigate the species diversity of both sections in Pakistan and to give an overview of the major clades. Phylogenetic analyses based on the combined nucLSU, ITS and TEF1 dataset from 35 specimens of both sections revealed three major clades. Analyses based on ITS dataset and 106 specimens, including 33 from Pakistan, reveal eight new species and one new record species. These nine species are described in detail. It is noteworthy that intraspecific variability as well as interspecific variability between closely related species were very low in ITS sequences in many cases. In the case of the two new species A. xanthochromaticus and A. griseovariegatus, TEF1 sequence data were much more efficient than ITS to distinguish these species from each other. The other new species are A. atroumbonatus, A. fumidicolor, A. macropeplus, A. parviniveus, A. swaticus and A. bambusetorum. The latter is the only new species of A. sect. Hondenses in which it is morphologically atypical and also the unique (sub)tropical species. Agaricus gregariomyces is recorded for the first time in Pakistan. In addition, brief descriptions are provided not only for A. bisporiticus, A. endoxanthus and A. punjabensis, which are reported again in Pakistan, but also for A. californicus, which is reported for the first time in Spain and outside North America. In total 12 species of both sections were reported in Pakistan and half of them were from subtropical climatic areas, underlining the contribution of the climatic diversity to the high species richness in this country.

A revision of Agaricus section Arvenses with nine new species from China.

Agaricus sect. Arvenses includes numerous species that are potential candidates for cultivation, and some have high nutritional and medicinal interests. Between 2012 and 2017, 147 specimens of A. sect. Arvenses were collected in China. For this study, nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), nuc 28S rDNA (28S), and translation elongation factor 1-alpha (tef1) sequences were used to assess species boundaries of these samples from China. Combined with morphological examination, we recognize 22 species of A. sect. Arvenses from China, of which 12 are known species, one is new record for China, and nine are proposed as new.

FISHing in fungi: Visualisation of mushroom virus X in the mycelium of Agaricus bisporus by fluorescence in situ hybridisation.

Agaricus bisporus is a commercial mushroom crop susceptible to a disease caused by a complex of viruses known collectively as mushroom virus X (MVX). Symptoms of MVX include bare patches and mushroom cap discolouration (browning) in the fruiting bodies, phenotypes associated with the viruses AbV6 and AbV16, respectively. Limited understanding exists of the localisation and mobilisation of these viruses within the mycelium of A. bisporus. To this end, a non-destructive fluorescence in situ hybridisation (FISH) method was developed for in situ targeting of AbV6 and AbV16 in A. bisporus mycelium. An MVX strain associated with the bare patch disease phenotype revealed predominantly high signal towards the growing edges of cultures when probed for AbV6, with a 'halo-effect' of high signal intensity around putative vacuoles. An MVX strain associated with the browning disease phenotype showed high signal intensities within reticulating networks of hyphae in a highly compartmentalised manner when probed for AbV16. Localisation of the two viruses in MVX-infected cultures appears independent, as both viruses were found in completely discrete areas of the mycelium in differential patterns. FISH detected low level presence of the two viruses, AbV6 and AbV16 in a number of cultures which had tested negative for the viruses by RT-PCR. This suggests that FISH may be more sensitive at detecting viruses at low levels than molecular methods. This study demonstrates that FISH is a powerful tool in the field of mycovirology.

Ultrasound enhanced production of mycelia and exopolysaccharide by Agaricus bitorquis (Quél.) Sacc. Chaidam.

Agaricus bitorquis (Quél.) Sacc. Chaidam (ABSC), is a kind of rare edible macrofungi with a variety of biological ingredients, especially its polysaccharides. However, the low yield limits the popularity and promotion of rare edible macrofungi as well as its macrofungi polysaccharides. Hence, developing a positive and effective cultivation method is of great importance. Herein, an efficient ultrasonic (US) stimulation strategy was developed to improve mycelial growth and exopolysaccharides (EPS) biosynthesis from ABSC in submerged cultivation without light. A time design was employed to illustrate the effect of various process parameters including duration, starting point and times of US irradiation on ABSC productivity. 5 min US treatment for once upon ABSC after fermentation for 48 h could significantly improve EPS production and mycelia growth by above 26% and 15.03%, respectively. Furthermore, six times of 5 min US treatment could make the amount of EPS reach 218.78 ± 17.09 mg/g, which was 2.52-fold higher than that of the control. Moreover, the enhanced effect induced by US was further expounded by fermentation kinetics. Besides, the US treatment could increase mycelia permeability, change structure and reduce mycelial diameter to promote mass transfer, resulting in the improvement of EPS production and mycelia accumulation. The results demonstrated that the present proposed US intensification approach could be useful to boost up the fermentation of ABSC, which possibly applied to yield increase and fermentation product acquisition of macrofungi.

Purification and biochemical characterization of two isolated laccase isoforms from Agaricus bisporus CU13 and their potency in dye decolorization.

Agaricus bisporus CU13 laccase was purified using ammonium sulfate precipitation (40-80%), Sephadex G100, and DEAE Sephadex A50 anion exchange column chromatography, respectively. Two laccase isoenzymes (Lacc1 & Lacc2) with purification folds of 1.40 and 5.81 respectively, were obtained from DEAE Sephadex A50 column. Optimal temperature and pH were recorded at 55 °C and pH 5.0 for both laccase isoenzymes using ABTS as substrate. Lacc1 was more thermostable than Lacc2 with residual activity of 95, 80 and 6%, while Lacc2 only retained 72, 25 and 0.4% of its activity after incubation for 90 min. at 50, 60 and 70 °C, respectively. Lacc2 retained about 93 and 86% of the initial activity at pH 9.0 and 7.0, whereas Lacc1 was stable at pH 7.0 and 5.0 followed by pH 9.0 and retained about 87, 76, and 36% of its activity respectively, after 4 h of incubation. Lacc1 was activated by 40% in the presence of Cu2+ (10 mM). Km and Vmax values found to be 0.394 and 0.158 µM, and 0.1351 and 0.4755 µmol min-1 for Lacc1 and Lacc2, respectively. The efficiency of both isoenzymes to decolorize Acid blue dye, make the enzyme seems to be a prospective for further biotechnological applications.

A phylogenetic and morphological overview of sections Bohusia, Sanguinolenti, and allied sections within Agaricus subg. Pseudochitonia with three new species from France, Iran, and Portugal.

The genus Agaricus was recently rearranged to accommodate numerous tropical taxa. Accordingly, the genus was split into six subgenera and 22 sections of which 12 are included in A. subg. Pseudochitonia. Preliminary data indicated that three putative new species belong to this subgenus. Our objectives were to describe these species, to determine to which sections they belong, and to experience the interest of some traditional traits in this new context. We morphologically described Agaricus coniferarum from France and Portugal, Agaricus iranicus from Iran, and Agaricus lusitanicus from Portugal. Multi-gene phylogenetic analyses based on ITS, LSU, and tef1 sequence data of representatives of the 12 sections clearly indicated that A. coniferarum and A. lusitanicus are placed in Agaricus sect. Bohusia, while A. iranicus is in A. sect. Sanguinolenti. Incidentally, we replaced the illegitimate name Agaricus magnivelaris by Agaricus fiardianus. In a phylogenetic tree, based on all available ITS sequence data and focussing on six related sections, we examined the phylogenetic distribution of various characters. The intensity of red discolouration when the sporocarp is rubbed or cut appeared as a phylogenetically weak informative trait. We propose a determination key leading to a group of three hardly distinguishable sections (Bohusia, Nigrobrunnescentes, and Sanguinolenti).

Morphological and molecular characterization of three Agaricus species from tropical Asia (Pakistan, Thailand) reveals a new group in section Xanthodermatei.

The genus Agaricus is known for its medicinal and edible species but also includes toxic species that belong to section Xanthodermatei. Previous phylogenetic reconstruction for temperate species, based on sequence data of nuc rRNA gene (rDNA) internal transcribed spacers (ITS), has revealed two major groups in this section and a possible third lineage for A. pseudopratensis. Recent research in Agaricus has shown that classifications need improving with the addition of tropical taxa. In this study we add new tropical collections to section Xanthodermatei. We describe three species from collections made in Pakistan and Thailand and include them in a larger analysis using all available ITS data for section Xanthodermatei. Agaricus bisporiticus sp. nov. and A. fuscopunctatus sp. nov. are introduced based on molecular and morphological studies, whereas A. microvolvatulus is recorded for the first time in Asia. Specimens from Thailand however have a much larger pileus than the type specimens from Congo. In maximum likelihood (ML) and maximum parsimony (MP) phylogenetic analyses these three species cluster with A. pseudopratensis from the Mediterranean area and A. murinocephalus recently described from Thailand. In Agaricus section Xanthodermatei this new group is monophyletic and receives low bootstrap support whereas the two previously known groups receive strong support. Within the new group, the most closely related species share some traits, but we did not find any unifying morphological character; however the five species of the group share a unique short nucleotide sequence. Two putatively toxic species of section Xanthodermatei are now recognized in Pakistan and six in Thailand.

Carbohydrate composition of compost during composting and mycelium growth of Agaricus bisporus.

Changes of plant cell wall carbohydrate structures occurring during the process to make suitable compost for growth of Agaricus bisporus are unknown. In this paper, composition and carbohydrate structures in compost samples collected during composting and mycelium growth were analyzed. Furthermore, different extracts of compost samples were prepared with water, 1M and 4M alkali and analyzed. At the beginning of composting, 34% and after 16 days of mycelium growth 27% of dry matter was carbohydrates. Carbohydrate composition analysis showed that mainly cellulose and poorly substituted xylan chains with similar amounts and ratios of xylan building blocks were present in all phases studied. Nevertheless, xylan solubility increased 20% over the period of mycelium growth indicating partial degradation of xylan backbone. Apparently, degradation of carbohydrates occurred over the process studied by both bacteria and fungi, mainly having an effect on xylan-chain length and solubility.

Mitochondrial recombination in natural populations of the button mushroom Agaricus bisporus.

In the majority of sexual eukaryotes, the mitochondrial genomes are inherited uniparentally and have predominantly clonal population structures. In clonally evolving genomes, alleles at different loci will be in significant linkage disequilibrium. In this study, the associations among alleles at nine mitochondrial loci were analyzed for 379 isolates in four natural populations of the button mushroom Agaricus bisporus. The results indicated that the mitochondrial genome in the Desert California population was not significantly different from random recombination. In contrast, the three other populations all showed predominantly clonal mitochondrial population structure. While no evidence of recombination was found in the Alberta, Canada A. bisporus population, signatures of recombination were evident in the Coastal Californian and the French populations. We discuss the potential mechanisms that could have contributed to the observed mitochondrial recombination and to the differences in allelic associations among the geographic populations in this economically important mushroom.

Estrogen-like activity and dual roles in cell signaling of an Agaricus blazei Murrill mycelia-dikaryon extract.

Agaricus blazei (A. blazei) Murrill mycelia-dikaryon has attracted the attention of scientists and clinicians worldwide owing to its potential for the treatment of cancer. However, little is known about its effect on other pathologies. This study sought to extend the potential medical usefulness of A. blazei for preventing vascular damage and to unravel its mechanism of action. The A. blazei extract showed estrogen-like activity in both gene expression profiling and a luciferase assay. Indeed, the extract inhibited oxidized low-density lipoprotein-stimulated activation of Erk1/2, Akt and p38 in HUVECs and macrophage-derived TIB-67 cells. Moreover, the extract enhanced transcription of the glutathione peroxidase 3 (GPX3), α-synuclein (SNCA) and endothelial nitrogen-oxide synthase (eNOS) genes. Furthermore, atherosclerotic lesions in rabbits were reduced by intake of A. blazei powder. Therefore, A. blazei may be useful for preventing atherosclerosis via dual roles in cell signaling, suppression of macrophage development and the recovery of endothelial cells from vascular damage.

Sequestrate species of Agaricus and Macrolepiota from Australia: new species and combinations and their position in a calibrated phylogeny.

Australian collections of sequestrate Agaricaceae were examined with morphological and molecular data (nuclear DNA from ITS and LSU), and the majority were found to belong to the genera Agaricus and Macrolepiota. Previously described Australian species of Endoptychum are transferred to the appropriate agaricoid genera and several new combinations proposed. Descriptions and illustrations are provided for these and eight new species: Agaricus eburneocanus sp. nov., A. chartaceus sp. nov., A. erythrosarx sp. nov., A. inilleasper sp. nov., A. pachydermus sp. nov., Macrolepiota gasteroidea sp. nov., M. vinaceofibrillosa sp. nov. and M. turbinata sp. nov. The sequestrate genus Barcheria is retained as a distinct taxon. Timing of evolution of sequestrate sporocarp forms in Macrolepiota, Chlorophyllum and Agaricus seems to have occurred in the past 15 000 000 y, and a stem age is approximately 65 000 000 y for Barcheria.

Mushroom (Agaricus bisporus).

We have devised an easy and effective genetic transformation method for the preeminent edible mushroom, Agaricus bisporus. Our method exploits the T-DNA transfer mechanism in Agrobacterium tumefaciens and relies on the reproductive fruiting body as the recipient tissue. The use of fruiting body explants, particularly the gill, provided high-frequency transformation, overcoming the inefficacy of Agrobacterium-based methods targeting fungal spores or vegetative mycelium. The protocol entails incubation of A. tumefaciens for 3 h with acetosyringone, a signaling molecule that launches the gene transfer mechanism, co-cultivation of the induced bacterium and gill explants for 3 d, and selection for transformants based on an inherited resistance to the antibiotic hygromycin. Between 7 and 28 d on the selection medium, upwards of 95% of the gill explants generate hygromycin-resistant colonies. About 75% of the mushroom transformants show a single-copy of the hygromycin-resistant gene integrated at random sites in the genome.

Karyological evidence for meiosis in the three different types of life cycles existing in Agaricus bisporus.

In Agaricus bisporus all cytological studies performed until now concerned the pseudohomothallic and bisporic var. bisporus. In the past 12 y two tetrasporic varieties have been described, the heterothallic var. burnettii and the homothallic var. eurotetrasporus. Our aim was to compare the behavior of the nuclei in the vegetative and reproductive cells of the three varieties with light microscopy (Feulgen and DAPI staining) and transmission electron microscopy. Most of the vegetative cells contained 3-5 nuclei in the three varieties. Nuclear migrations through the septum were detected. In the basidia relative locations of nuclei and vacuoles, meiotic spindle alignments, relative content of nuclear DNA and synaptonemal complexes were measured or observed. From the observation of numerous asynchronous second division of meiosis within basidia of var. bisporus and var. burnettii a new hypothesis emerges to explain the nonrandom distribution of the four meiotic products in the two spores of the bisporic basidia. Karyogamy and meiosis similarly occurred in the three varieties. In the case of A. bisporus var. eurotetrasporus this implies that the reproductive mode is sexual and therefore homothallic in the strict sense. The three different types of life cycles are described.

Morphological and molecular characterization of two novel species of Agaricus section Xanthodermatei.

Agaricus specimens collected in France belong to two novel entities resembling small forms of A. moelleri and A. xanthodermus, two common species in section Xanthodermatei. Molecular (IT1+ITS2 DNA sequence) and morphological comparisons between eight presumed similar taxa of the section support the elevation of both entities to species rank. The new entities are described as A. parvitigrinus and A. xanthodermulus. They form a group with A. laskibarii, a rare species also recently described from France, and A. californicus, a North-American species. The well known A. moelleri and A. xanthodermus are the most related species among the studied sample. Like other species of the section, both new species have a phenolic odor and are probably toxic.

[Ultrastructure of the cell wall in vegetative mycelia of Agaricus bisporus (Lange) Imbach]. / Osobennosti ul'trastruktury kletochnoi stenki vegetativnogo mitseliia Agaricus bisporus (Lange) Imbach.

A comparative description of the cell wall ultrastructure was made for vegetative mycelia in Agaricus bisporus, other species of Agaricus, and Pleurotus ostreatus. Apart from well known ultrastructural features of cell walls of the examined strains, inclusions of different kinds were observed. The cell wall thickness varied in different Agaricus strains. Maximum differences between the examined strains were 1.4 times. The cell wall thickness in P. ostreatus strains was by 3 to 4 times less than that in Agaricus strains. Using electron micrographs, a brief description of mycelia and A. bisporus basidia ultrastructure was composed.

Fine-scale genetic analyses reveal unexpected spatial-temporal heterogeneity in two natural populations of the commercial mushroom Agaricus bisporus.

This study examined the fine-scale genetic variation of the commercial mushroom, Agaricus bisporus, over 2 years at two sites in France. One site was a meadow fertilized with horse manure and disturbed regularly by humans; the other was a Monterey cypress forest free of human disturbance. Altogether, 50 mushrooms were collected and analysed for mitochondrial and nuclear genetic variation marked by RFLPs and multilocus enzyme electrophoretic polymorphisms. Population samples from these two sites were genetically different and both sites contained high levels of genetic diversity. No identical genotypes were found at either site between the 2 years and there was little evidence for extensive vegetative clonality for this species. Contrary to expectations, very limited evidence of pseudohomothallic reproduction was found. Results from tests of Hardy-Weinberg equilibrium and genotypic equilibrium showed that outcrossing and recombination have played significant roles in both populations. The results demonstrated spatial-temporal genetic heterogeneity of A. bisporus in natural populations.

Sexuality and genetic identity in the Agaricus section Arvenses.

Twelve wild collections and one commercial strain were used to characterize breeding systems and to develop molecular identities in the Arvenses section of the genus Agaricus, which includes the "horse mushroom" A. arvensis. Two morphotypes were identified based on macro- and micromorphological features. However, not all collections could be delimited by conventional taxonomic characters. Sequencing of the small subunit intergenic spacer (ITS) region (368 to 370 bp) of the rRNA genes clearly resolved the 13 collections into two clusters consistent with the identified morphotypes. Single-spore progenies and mating type testers were established and used to test intra- and interstock compatibility. The two compatibility groups identified were consistent with ITS clusters. Compatibility group I stocks readily interbred within the constraints of a unifactorial heterothallic system with a multiallelic mating type factor. Compatibility group II had a more restricted breeding pattern, and interactions were difficult to predict on the basis of mating type. Morphological data, ITS sequences, and the ability to interbreed suggest that these collections are part of a complex of interrelated species. Single-spore, homokaryotic isolates from both compatibility groups were able to fruit in compost culture, and two of the collections may represent natural homokaryotic fruiting. We conclude that species from the section Arvenses have versatile unifactorial heterothallic life cycles that permit both interbreeding and homokaryotic fruiting.

Quantitative 1H-NMR imaging of water in white button mushrooms (Agaricus bisporus).

MRI represents a valuable tool for studying the amount and physical status of water in plants and agricultural products, for example, mushrooms (Agaricus bisporus). Contrast in NMR images originates from the mixed influence of the fundamental NMR parameters, amongst others, spin-density, T2- and T1 relaxation processes. Maps of these parameters contain valuable anatomical and physiological information. They can, however, be severely distorted, depending on the combination of parameter settings and anatomy of the object under study. The influence of the tissue structure of mushrooms, for example, tissue density (susceptibility inhomogeneity) and cell shape on the amplitude, T2, and T1 images is analyzed. This is achieved by vacuum infiltration of the cavities in the mushroom's spongy structure with Gd-DTPA solutions and acquiring Saturation Recovery-Multispin Echo images. It is demonstrated that the intrinsic long T2 values in the cap and outer stipe tissue strongly relate to the size and geometry of the highly vacuolated cells in these spongy tissues. All observed T2 values are strongly affected by susceptibility effects. The T2 of gill tissue is shorter than T2 of the cap and outer stipe, probably because these cells are less vacuolized and smaller in size. The calculated amplitude images are not directly influenced by susceptibility inhomogeneities as long as the observed relaxation times remained sufficient long. They reflect the water distribution in mushrooms best if short echo times are applied in a multispin echo imaging sequence at low magnetic field strength.

A meiotic DNA polymerase from a mushroom, Agaricus bisporus.

A meiotic DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7], which likely has a role in meiotic DNA repair, was isolated from a mushroom, Agaricus bisporus. The purified fraction displays three bands in SDS/PAGE, at molecular masses of 72 kDa, 65 kDa and 36 kDa. Optimal activity is at pH 7.0-8.0 in the presence of 5 mM Mg2+ and 50 mM KCl and at 28-30 degrees C, which is the temperature for meiosis. This enzyme is resistant to N-ethylmaleimide and sensitive to 2',3'-dideoxythymidine 5'-triphosphate, suggesting that it is a beta-like DNA polymerase. These characteristics are similar to those of Coprinus DNA polymerase beta [Sakaguchi and Lu (1982) Mol. Cell. Biol. 2, 752-757]. In Western-blot analysis, the antiserum against the Coprinus polymerase reacts only with the 65 kDa band, which coincides with the molecular mass of the Coprinus polymerase. Western-blot analysis also showed that the antiserum could react with crude extracts not only from the Agaricales family, to which Agaricus and Coprinus belong, but also from different mushroom families and Saccharomyces. The Agaricus polymerase activity can be found only in the meiotic-cell-rich fraction, but the enzyme is also present in the somatic cells in an inactive state.

Amanita phalloides in Victoria.

Amanita phalloides is found regularly in suburban Melbourne and country areas where old oaks and other deciduous trees are grown as street trees, in parks, and in private gardens. This deadly poisonous mushroom may be mistaken for the edible Agaricus spp.