Death caps (Amanita phalloides) frequently establish from sexual spores, but individuals can grow large and live for more than a decade in invaded forests.

Global change is reshaping Earth's biodiversity, but the changing distributions of nonpathogenic fungi remain largely undocumented, as do mechanisms enabling invasions. The ectomycorrhizal Amanita phalloides is native to Europe and invasive in North America. Using population genetics and genomics, we sought to describe the life history traits of this successfully invading symbiotic fungus. To test whether death caps spread underground using hyphae, or aboveground using sexual spores, we mapped and genotyped mushrooms from European and US sites. Larger genetic individuals (genets) would suggest spread mediated by vegetative growth, while many small genets would suggest dispersal mediated by spores. To test whether genets are ephemeral or persistent, we also sampled from populations over time. At nearly every site and across all time points, mushrooms resolve into small genets. Individuals frequently establish from sexual spores. But at one Californian site, a single individual measuring nearly 10 m across dominated. At two Californian sites, the same genetic individuals were discovered in 2004, 2014, and 2015, suggesting single individuals (both large and small) can reproduce repeatedly over relatively long timescales. A flexible life history strategy combining both mycelial growth and spore dispersal appears to underpin the invasion of this deadly perennial ectomycorrhizal fungus.

Diversity, Dispersal and Mode of Reproduction of Amanita exitialis in Southern China.

Amanita exitialis is a poisonous mushroom and has caused many deaths in southern China. In this study, we collected 118 fruiting bodies of A. exitialis from seven different sites in Guangdong Province in southern China and investigated their genetic relationships using 14 polymorphic molecular markers. These 14 markers grouped the 118 fruiting bodies into 20 multilocus genotypes. Among these 20 genotypes, eight were each found only once while the remaining 12 were each represented by two to 54 fruiting bodies. Interestingly, among the 12 shared genotypes, four were shared between/among local populations that were separated by as far as over 80 km, a result consistent with secondary homothallic reproduction and long-distance spore dispersal. Despite the observed gene flow, significant genetic differentiations were found among the local populations, primarily due to the over-representation of certain genotypes within individual local populations. STRUCTURE analyses revealed that the 118 fruiting bodies belonged to three genetic clusters, consistent with divergence within this species in this geographic region. Interestingly, we found an excess of heterozygous individuals at both the local and the total sample level, suggesting potential inbreeding depression and heterozygous advantage in these populations of A. exitialis. We discuss the implications of our results for understanding the life cycle, dispersal, and evolution of this poisonous mushroom.

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.

In Colombia the Eurasian fungus Amanita muscaria is expanding its range into native, tropical Quercus humboldtii forests.

To meet a global demand for timber, tree plantations were established in South America during the first half of the 20th century. Extensive plantings of non-native species now are found in Brazil, Chile, Argentina, and Uruguay. In Colombia, miscellaneous plantations were established in the 1950s, during a period of intensive local logging, when policies to limit deforestation in native Quercus humboldtii forests were established. One unforeseen consequence of planting non-native trees was the simultaneous introduction and subsequent persistence of ectomycorrhizal fungi. We sought to document the origins and spread of the introduced Amanita muscaria found in Colombian plantations of the Mexican species Pinus patula, North American species P. taeda, and Australian species Acacia melanoxylon and Eucalyptus globulus. In Colombia, Amanita muscaria is establishing a novel association with native Q. humboldtii and has spread to local Q. humboldtii forests. According to a Bayesian phylogeny and haplotype analysis based on the nuclear rDNA internal transcribed spacer region ITS1-5.8-ITS2 (ITS barcode), A. muscaria individuals found in four exotic plant species, and those colonizing Q. humboldtii roots, have a Eurasian origin and belong to two Eurasian haplotypes. This is the first time the spread of an introduced mutualist fungus into native Colombian Q. humboldtii forests is reported. To arrest its spread, we suggest the use of local inocula made up of native fungi, instead of inocula of introduced fungi.

Growth of Amanita caesarea in the presence of Pseudomonas fluorescens and Bacillus cereus.

The ectomycorrhizal (ECM) fungus Amanita caesarea CECT 20127 was tested in vitro with two potentially mycorrhizal-promoting bacterial strains, Pseudomonas fluorescens CECT 844 and Bacillus cereus CECT 148. Although P. fluorescens showed spatial and temporal compatibility with A. caesarea, it did not affect growth of the fungus. Conversely, B. cereus exhibited no such compatibility and also inhibited fungal growth. The expression pattern of the A. caesarea gene AcMST-1 was analysed using real-time quantitative polymerase chain reaction (qPCR) at three time points. This gene displays a high degree of homology with two genes, possible orthologues to AcMST-1, previously described in Amanita muscaria (AmMST-1) and Laccaria bicolor (LbMST-1) and encoding monosaccharide transporter proteins. The transcription levels of AcMST1 increased shortly after initial contact between A. caesarea and B. cereus, but expression of the gene was inhibited in the presence of P. fluorescens. Our results show that A. caesarea may possess orthologous genes of similar ECM fungal species that would allow it to adapt in nature to optimize sugar uptake from the environment depending on the presence of different microorganisms.

The Gondwanan connection - Southern temperate Amanita lineages and the description of the first sequestrate species from the Americas.

Amanita is a diverse and cosmopolitan genus of ectomycorrhizal fungi. We describe Amanita nouhrae sp. nov., a new hypogeous ('truffle-like') species associated with Nothofagus antarctica in northern Patagonia. This constitutes the first report of a sequestrate Amanita from the Americas. Thick-walled basidiospores ornamented on the interior spore wall ('crassospores') were observed consistently in A. nouhrae and its sister epigeous taxon Amanita morenoi, a rarely collected but apparently common species from northern Patagonia that has sometimes been misidentified as the Australian taxon Amanita umbrinella. Nuclear 18S and 28S ribosomal DNA and mitochondrial 16S and 26S DNA placed these two species in a southern temperate clade within subgenus Amanita, together with other South American and Australian species. Based on a dated genus-level phylogeny, we estimate that the southern temperate clade may have originated near the Eocene/Oligocene boundary (ca. 35 Ma ± 10 Ma). This date suggests a broadly distributed ancestor in the Southern Hemisphere, which probably diversified as a result of continental drift, as well as the initiation of the Antarctic glaciation. By comparison, we show that this clade follows an exceptional biogeographic pattern within a genus otherwise seemingly dominated by Northern Hemisphere dispersal.

Microanalysis characterization of bioactive protein-bound polysaccharides produced by Amanita ponderosa cultures.

Different compounds of edible mushrooms are responsible for their bioactivity. The ability to synthesize polysaccharides, namely protein-polysaccharide (PPS) complexes, is related to the antioxidant capacity of these compounds and present great interest in preventing a number of diseases, including cancer, cardiovascular and auto-immune diseases, and accelerated aging. Amanita ponderosa are wild edible mushrooms that grow in Mediterranean "montado" areas [Portuguese name given to cork oak (Quercus suber) and holm oak (Quercus ilex) forests]. The aim of this study was to evaluate the production of PPS complexes obtained from A. ponderosa cultures using a new microanalytical approach to quickly and easily monitor the production process. Microanalysis using Fourier-transform infrared using attenuated total reflection and Raman spectroscopy of PPS samples showed spectra compatible with identification of this type of compound in culture extracts. PPS separated by size-exclusion chromatography showed seven main complexes. Molecular weights of the main PPS complexes isolated from cultures ranged between 1.5 and 20 kDa and did not present toxicity against Artemia salina, demonstrating the potential of A. ponderosa as a source of biologically active compounds with nutraceutical value. Application of this microanalytical approach to monitoring the production of PPS compounds can be successfully applied in biotechnological processes.

Amatoxin and phallotoxin concentration in Amanita phalloides spores and tissues.

Most of the fatal cases of mushroom poisoning are caused by Amanita phalloides. The amount of toxin in mushroom varies according to climate and environmental conditions. The aim of this study is to measure α-, ß-, and γ-amanitin with phalloidin and phallacidin toxin concentrations. Six pieces of A. phalloides mushrooms were gathered from a wooded area of Düzce, Turkey, on November 23, 2011. The mushrooms were broken into pieces as spores, mycelium, pileus, gills, stipe, and volva. α-, ß-, and γ-Amanitin with phalloidin and phallacidin were analyzed using reversed-phase high-performance liquid chromatography. As a mobile phase, 50 mM ammonium acetate + acetonitrile (90 + 10, v/v) was used with a flow rate of 1 mL/min. C18 reverse phase column (150 × 4.6 mm; 5 µm particle) was used. The least amount of γ-amanitin toxins was found at the mycelium. The other toxins found to be in the least amount turned out to be the ones at the spores. The maximum amounts of amatoxins and phallotoxin were found at gills and pileus, respectively. In this study, the amount of toxin in the spores of A. phalloides was published for the first time, and this study is pioneering to deal with the amount of toxin in mushrooms grown in Turkey.

The irreversible loss of a decomposition pathway marks the single origin of an ectomycorrhizal symbiosis.

Microbial symbioses have evolved repeatedly across the tree of life, but the genetic changes underlying transitions to symbiosis are largely unknown, especially for eukaryotic microbial symbionts. We used the genus Amanita, an iconic group of mushroom-forming fungi engaged in ectomycorrhizal symbioses with plants, to identify both the origins and potential genetic changes maintaining the stability of this mutualism. A multi-gene phylogeny reveals one origin of the symbiosis within Amanita, with a single transition from saprotrophic decomposition of dead organic matter to biotrophic dependence on host plants for carbon. Associated with this transition are the losses of two cellulase genes, each of which plays a critical role in extracellular decomposition of organic matter. However a third gene, which acts at later stages in cellulose decomposition, is retained by many, but not all, ectomycorrhizal species. Experiments confirm that symbiotic Amanita species have lost the ability to grow on complex organic matter and have therefore lost the capacity to live in forest soils without carbon supplied by a host plant. Irreversible losses of decomposition pathways are likely to play key roles in the evolutionary stability of these ubiquitous mutualisms.

Mercury in European Blushers, Amanita rubescens, mushrooms and topsoils: bioconcentration potential and intake assessment.

Total mercury content has been determined in fruiting bodies of European Blushers and topsoils collected from 11 sites across Poland in 2006-2008. Mercury analysis was carried out using a validated analytical method and cold-vapour atomic absorption (CV-AAS). The European Blusher effectively accumulated mercury in fruiting bodies. The mean values of total mercury in caps of European Blushers from background (uncontaminated) areas were from 0.22 to 1.0 (0.067-3.2) and in stipes from 0.16 to 0.65 (0.071-2.7) µg/g dry weight. In topsoil beneath to fruiting bodies, the median Hg concentration at 10 sites in Northern Poland varied between 0.030 and 0.072 (0.0096-0.19) µg/g dw, and in one site in Southern Poland was 0.20 (0.079-0.34) µg/g dw. Data on Hg in European Blushers from different countries were reviewed. The mean concentrations of total Hg in caps of European Blushers from two "pristine" sites in northern part of Poland were ∼1.0 µg/g dw. A meal made with 300-500 g of fresh caps of European Blushers collected at such sites (assuming 90% water content in caps) can result in Hg intake of 0.0003-0.0005 mg Hg/kg bm (assuming a 60 kg bm), which is a dose equipotent to a new provisional tolerable weekly intake (PTWI) value set for inorganic Hg.

Amanita thiersii is a saprotrophic fungus expanding its range in the United States.

Although most species in the genus Amanita form ectomycorrhizal associations, a few are reported to be saprotrophs living in grassland habitats. Little is known about the ecology and distribution of these free-living Amanita species. We describe the ecology of Amanita thiersii, a species commonly collected in lawns throughout the Mississippi River Basin. Stable isotopes of carbon, transcriptomic sequences and patterns of growth on complex carbon sources provide evidence for A. thiersii as a saprotrophic species. Sporocarps of A. thiersii are less depleted in (13)C compared to published data for ectomycorrhizal fungi, supporting a saprotrophic mode of carbon acquisition in the field. Orthologs of cellulase genes known to play key roles in the decomposition of cellulose in other basidiomycetes were identified in a transcriptome of A. thiersii, establishing that this species has the genetic potential to degrade cellulose. Amanita thiersii also can use artificial cellulose or sterile grass litter as a sole carbon source. DNA sequences of three nuclear gene regions and banding patterns from four inter-simple sequence repeat markers were identical across 31 populations from throughout the known range of the species, which suggests the genetic diversity of A. thiersii populations is low. Maps of A. thiersii collections made from the 1950s until present suggest this species is experiencing a range expansion. It was reported first in 1952 in Texas and now occurs in nine states north to Illinois. These data provide an ecological context for interpreting the genome of A. thiersii, currently being sequenced at the United States Department of Energy's Joint Genome Institute.

Characterisation of ectomycorrhizal formation by the exotic fungus Amanita muscaria with Nothofagus cunninghamii in Victoria, Australia.

The occurrence of the exotic ectomycorrhizal fungus Amanita muscaria in a mixed Nothofagus-Eucalyptus native forest was investigated to determine if A. muscaria has switched hosts to form a successful association with a native tree species in a natural environment. A mycorrhizal morphotype consistently found beneath A. muscaria sporocarps was examined, and a range of morphological and anatomical characteristics in common with those described for ectomycorrhizae formed by A. muscaria on a broad range of hosts were observed. A full description is provided. The likely plant associate was determined to be Nothofagus cunninghamii based upon anatomy of the roots. Analysis of ITS-1 and ITS-2 regions of nuclear ribosomal DNA sequences confirmed the identities of both fungal and plant associates. These findings represent conclusive evidence of the invasion of a non-indigenous ectomycorrhizal fungus into native forest and highlight the ecological implications of this discovery.

Changes in hyphal morphology and activity of phenoloxidases during interactions between selected ectomycorrhizal fungi and two species of Trichoderma.

Patterns of phenoloxidase activity can be used to characterize fungi of different life styles, and changes in phenoloxidase synthesis were suspected to play a role in the interaction between ectomycorrhizal and two species of Trichoderma. Confrontation between the ectomycorrhizal fungi Amanita muscaria and Laccaria laccata with species of Trichoderma resulted in induction of laccase synthesis, and the laccase enzyme was bound to mycelia of ectomycorrhizal fungi. Tyrosinase release was noted only during interaction of L. laccata strains with Trichoderma harzianum and T. virens. Ectomycorrhizal fungi, especially strains of Suillus bovinus and S. luteus, inhibited growth of Trichoderma species and caused morphological changes in its colonies in the zone of interaction. In contrast, hyphal changes occurred less often in the ectomycorrhizal fungi tested. Species of Suillus are suggested to present a different mechanism in their interaction with other fungi than A. muscaria and L. laccata.

Distribution and abundance of the introduced ectomycorrhizal fungus Amanita phalloides in North America.

Despite a growing awareness of the global reach of ectomycorrhizal (EM) fungal introductions, little is known about the fate of introduced EM fungi in novel ranges. Using herbarium specimens, species distribution models, and field collections of sporocarps, root tips and extramatrical mycelia, we assessed the distribution and abundance of the European species Amanita phalloides in North America. There are two distinct ranges of the fungus, one along the West Coast (California to British Columbia) and the second on the East Coast (Maryland to Maine). As predicted by a species distribution model, the West Coast range is larger. Amanita phalloides is more frequently found in native forests on the West Coast than on the East Coast. At Point Reyes Peninsula in California, A. phalloides dominates community sporocarp biomass, and is frequent as root tips. In individual soil cores at Point Reyes, root tips of A. phalloides make up 50% of total root tip biomass. Hyphae of A. phalloides are frequent, but make up only 2% of total hyphal biomass. The contrasting patterns of the distribution and abundance of A. phalloides on the East and West Coasts of North America may influence both its future spread and its impacts.

Ectomycorrhizal fungi and their enzymes in soils: is there enough evidence for their role as facultative soil saprotrophs?

Although ectomycorrhizal (ECM) fungi are generally regarded as dependent upon the supply of carbon from their plant hosts, some recent papers have postulated a role for these fungi in the saprotrophic acquisition of carbon from soil. This theory was mainly based on the increase in enzymatic activity during periods of low photosynthate supply from tree hosts and emergence of the theory has led to a question about the overall influence of saprotrophy by ECM fungi on soil carbon turnover. However, I argue here that there is still not enough evidence to confirm this proposed function. My argument is based on inference from several lines of observation and concern over several aspects of the past studies. First, ECM fungi mainly inhabit deeper soil horizons, in which the availability of carbon compounds with positive energetic value is low. Second, the ability of ECM fungi to produce ligninolytic enzymes and cellulases is much weaker than that of saprotrophic basidiomycetes. This is most apparent in the low copy abundance of corresponding genes in the sequenced genomes of ECM species Laccaria bicolor and Amanita bisporigenes compared to the saprotrophic species Galerina marginata. I offer alternative hypotheses to explain the past observations of increased enzyme activity during starvation periods. These include, the induction of autolytic processes in ECM fungal mycelia or an attack on the host tissues to support escape from a dying root and to allow for a search for new hosts.

The ectomycorrhizal fungus Amanita phalloides was introduced and is expanding its range on the west coast of North America.

The deadly poisonous Amanita phalloides is common along the west coast of North America. Death cap mushrooms are especially abundant in habitats around the San Francisco Bay, California, but the species grows as far south as Los Angeles County and north to Vancouver Island, Canada. At different times, various authors have considered the species as either native or introduced, and the question of whether A. phalloides is an invasive species remains unanswered. We developed four novel loci and used these in combination with the EF1α and IGS loci to explore the phylogeography of the species. The data provide strong evidence for a European origin of North American populations. Genetic diversity is generally greater in European vs. North American populations, suggestive of a genetic bottleneck; polymorphic sites of at least two loci are only polymorphic within Europe although the number of individuals sampled from Europe was half the number sampled from North America. Endemic alleles are not a feature of North American populations, although alleles unique to different parts of Europe were common and were discovered in Scandinavian, mainland French, and Corsican individuals. Many of these endemic European haplotypes were found together at single sites in California. Early collections of A. phalloides dated prior to 1963 and annotated using sequences of the ITS locus proved to be different species of Amanita. The first Californian collections that we confirmed as A. phalloides were made from the Del Monte Hotel (now the Naval Postgraduate School) in Monterey, and on the campus of the University of California, Berkeley, in 1938 and in 1945. These historical data are used in combination with data on A. phalloides' current distribution to estimate a rate of spread for A. phalloides in California. Many species of ectomycorrhizal (EM) fungi have been introduced across and among continents, but with this evidence, the death cap becomes the only known invasive EM fungus in North America.

Hyperaccumulation of silver by Amanita strobiliformis and related species of the section Lepidella.

Two ectomycorrhizal macrofungal Amanita species of the section Lepidella, A. strobiliformis and A. solitaria, were found to hyperaccumulate silver (Ag). All samples were collected from non-argentiferous areas with background Ag content in soils (0.07-1.01 mgkg(-1) Ag). The Ag contents of both Amanita species were mostly in the range of 200-700 mgkg(-1)D.W. with the highest Ag content of 1253 mgkg(-1) in one sample of A. strobiliformis. Silver concentrations in macrofungal fruit bodies were commonly 800-2500 times higher than in underlying soils. A. strobiliformis and A. solitaria are the first eukaryotic organisms known to hyperaccumulate Ag.

Interaction with mycorrhiza helper bacterium Streptomyces sp. AcH 505 modifies organisation of actin cytoskeleton in the ectomycorrhizal fungus Amanita muscaria (fly agaric).

The actin cytoskeleton (AC) of fungal hyphae is a major determinant of hyphal shape and morphogenesis, implicated in controlling tip structure and secretory vesicle delivery. Hyphal growth of the ectomycorrhizal fungus Amanita muscaria and symbiosis formation with spruce are promoted by the mycorrhiza helper bacterium Streptomyces sp. AcH 505 (AcH 505). To investigate structural requirements of growth promotion, the effect of AcH 505 on A. muscaria hyphal morphology, AC and actin gene expression were studied. Hyphal diameter and mycelial density decreased during dual culture (DC), and indirect immunofluorescence microscopy revealed that the dense and polarised actin cap in hyphal tips of axenic A. muscaria changes to a loosened and dispersed structure in DC. Supplementation of growth medium with cell-free bacterial supernatant confirmed that reduction in hyphal diameter and AC changes occurred at the same stage of growth. Transcript levels of both actin genes isolated from A. muscaria remained unaltered, indicating that AC changes are regulated by reorganisation of the existing actin pool. In conclusion, the AC reorganisation appears to result in altered hyphal morphology and faster apical extension. The thus improved spreading of hyphae and increased probability to encounter plant roots highlights a mechanism behind the mycorrhiza helper effect.

[Culture conditions and analysis of amanitins on Amanita spissa].

Isolate of Amanita spissa was obtained from basidiome stipe material collected from environment. It could utilize a broad range of carbon and nitrogen resources. Study on the influence of different conditions for solid culture was carried out. Optimal culture conditions were at 28 degrees C, pH6, in the dark. A. spissa was then fermentated in liquid culture for more mycelia. In flask and Airlift/ff bioreactor, maximum dry mycelia weight of A. spissa reached 0.893 g/L and 2.33 g/L, respectively. Mycelia obtained from solid culture and Airlift/ff bioreactor were then analyzed by HPLC. The results showed that mycelia from both cultures contained amatoxins but no phallotoxins. alpha-Amanitin in mycelia reached 26.02 microg/DWg under solid culture condition, and 15.25 microg/DWg under liquid culture condition. The amanitins were also confirmed by bud-inhibited assay. The results revealed that the effect of amanitin on mung bean cell was identical to that of authentic amanitins. This work suggests that it is possible to produce amatoxin by liquid culturing of A. spissa.