Amanita in the Guineo-Congolian rainforest: Epitypes and new species from the Dja Biosphere Reserve, Cameroon.

Four epitypes and three new species of Amanita (Amanitaceae, Agaricales, Agaricomycetes, Basidiomycota) are described from Guineo-Congolian rainforests of Cameroon. Amanita echinulata, A. fulvopulverulenta, A. robusta, and A. bingensis are epitypified based on collections that are the first since the species were described nearly a century ago. Morphological features of the epitypes are described and enumerated. Amanita minima, Amanita luteolamellata, and A. goossensfontanae are described as new and added to the known macromycota of tropical Africa. Habit, habitat, and known distribution are provided for each species. Sequence data for the internal transcribed spacer (ITS) locus are provided for types and other collections of all taxa, and a molecular phylogenetic analysis across the genus Amanita corroborates morphology-based infrageneric placement for each.

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.

Fatal Amanita muscaria poisoning in a dog confirmed by PCR identification of mushrooms.

Diagnosing mushroom poisoning in dogs can be difficult and often includes identification of suspect mushrooms. Visual identification may be hindered by mastication, oral medications, or poor quality of environmental mushroom samples. Other analytical techniques may thus be necessary to aid in mushroom identification. A 5-y-old neutered male Labrador Retriever dog developed acute onset of vomiting, diarrhea, tremors, seizures, and somnolence. The dog was treated at a veterinary clinic and was briefly stabilized, but died during transport to an emergency clinic. On postmortem examination at the University of Kentucky Veterinary Diagnostic Laboratory, the dog's stomach was full of mushrooms covered with activated charcoal. Mushrooms were damaged, fragmented, and discolored, precluding accurate visual identification. Mushroom pieces were sent to the Department of Plant Pathology at the University of California-Davis for PCR identification; the neurotoxic mushroom Amanita muscaria was identified. A qualitative liquid chromatography-mass spectrometry (LC-MS) method was developed to detect ibotenic acid and muscimol, the toxic compounds present in A. muscaria. Mushrooms, stomach contents, and urine were analyzed by LC-MS; ibotenic acid and muscimol were detected in all samples. Because identification of ingested mushrooms is sometimes necessary to confirm mushroom poisoning, PCR can identify ingested mushrooms when visual identification is unreliable.

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.

Defining the phylogenetic position of Amanita species from Andean Colombia.

Amanita is a worldwide-distributed fungal genus, with approximately 600 known species. Most species within the genus are ectomycorrhizal (ECM), with some saprotrophic representatives. In this study, we constructed the first comprehensive phylogeny including ECM species from Colombia collected in native Quercus humboldtii forests and in introduced Pinus patula plantations. We included 8 species (A. brunneolocularis, A. colombiana, A. flavoconia, A. fuligineodisca, A. muscaria, A. rubescens, A. sororcula, and A. xylinivolva) out of 16 species reported for the country, two new reports: A. citrina and A. virosa, and a new variety A. brunneolocularis var. pallida. Morphological taxonomic keys together with a phylogenetic approach using three nuclear gene regions: partial nuc rDNA 28S nuc rDNA internal transcribed spacers ITS1 and ITS2 and partial translation elongation factor 1-α gene (TEF1), were used to classify the specimens. Several highly supported clades were obtained from the phylogenetic hypotheses obtained by Bayesian inference and maximum likelihood approaches, allowing us to position the Colombian collections in a coherent infrageneric level and to contribute to the knowledge of local Amanita diversity.

Lethal Amanita species in China.

Lethal amanitas (Amanita sect. Phalloideae) cause many casualties worldwide. Recent molecular phylogenetic studies revealed diverse lethal Amanita spp. in China. Here a 5-gene phylogeny (nuc rDNA region encompassing the internal transcribed spacers 1 and 2 with the 5.8S rDNA, the D1-D3 domains of nuc 28S rDNA, and partial RNA polymerase II second largest subunit, translation elongation factor 1-α and ß-tubulin genes) is used to investigate the phylogenetic lineages and species delimitation in this section. Thirteen species are recognized, including four new species, namely A. griseorosea, A. molliuscula, A. parviexitialis, and A. subfuliginea They are documented with morphological, multigene phylogenetic, and ecological evidence, line drawings, and photographs and compared with similar species. A key to the Chinese lethal Amanita species is provided.

Molecular identification of poisonous mushrooms using nuclear ITS region and peptide toxins: a retrospective study on fatal cases in Thailand.

Cases of mushroom poisoning in Thailand have increased annually. During 2008 to 2014, the cases reported to the National Institute of Health included 57 deaths; at least 15 died after ingestion of amanitas, the most common lethal wild mushrooms inhabited. Hence, the aims of this study were to identify mushroom samples from nine clinically reported cases during the 7-year study period based on nuclear ITS sequence data and diagnose lethal peptide toxins using a reversed phase LC-MS method. Nucleotide similarity was identified using BLAST search of the NCBI database and the Barcode of Life Database (BOLD). Clade characterization was performed by maximum likelihood and Bayesian phylogenetic approaches. Based on BLAST and BOLD reference databases our results yielded high nucleotide similarities of poisonous mushroom samples to A. exitialis and A. fuliginea. Detailed phylogenetic analyses showed that all mushroom samples fall into their current classification. Detection of the peptide toxins revealed the presence of amatoxins and phallotoxins in A. exitialis and A. fuliginea. In addition, toxic α-amanitin was identified in a new provisional species, Amanita sp.1, with the highest toxin quantity. Molecular identification confirmed that the mushrooms ingested by the patients were members of the lethal amanitas in the sections Amanita and Phalloideae. In Thailand, the presence of A. exitialis was reported here for the first time and all three poisonous mushroom species provided new and informative data for clinical studies.

Laboratory confirmation of Amanita smithiana mushroom poisoning.

CONTEXT: Here we present a case of Amanita smithiana poisoning resulting in acute kidney injury requiring dialysis, and highlight laboratory methods used to confirm the diagnosis. Identification of Amanita smithiana toxin using thin-layer chromatography can provide greater diagnostic certainty than history and renal function tests alone. CASE DETAILS: A 63-year-old male presented to hospital with anuria and gastrointestinal symptoms, two days after consuming a soup of wild mushrooms he had picked. He was found to be in acute renal failure, requiring hemodialysis. After nine days of supportive treatment, he recovered renal function, and was discharged in good health 15 days post-ingestion. The patient provided a sample of leftover soup, and examination of cooked mushroom fragments by a mycologist provided preliminary identification of A. smithiana. Thin-layer chromatography revealed the presence of A. smithiana toxin in the soup, confirming this identification. DISCUSSION: A. smithiana is a nephrotoxic mushroom that can be easily mistaken for the edible and highly prized Pine mushroom (Tricholoma magnivelare). It causes initial gastrointestinal symptoms, followed by acute renal failure. Treatment includes dialysis and supportive care until the patient recovers renal function. The chemical structure of the A. smithiana toxin is unknown, but it can be identified as a characteristic spot on thin-layer chromatography.

In vitro mycorrhization and acclimatization of Amanita caesareoides and its relatives on Pinus densiflora.

Amanita caesareoides is a sister species of Amanita caesarea, also known as Caesar's mushroom and one of the most desirable edible mycorrhizal mushrooms. However, cultivation of Caesar's mushrooms has not yet been successful due to the difficulties involved in establishing pure cultures. In this study, we established pure cultures of four Asian Caesar's mushroom species, i.e., A. caesareoides, Amanita javanica, Amanita esculenta, and Amanita similis, which were identified by sequence analysis of their rDNA internal transcribed spacer (ITS) region. Five selected isolates in A. caesareoides, A. javanica, and A. esculenta were tested for ectomycorrhizal syntheses with axenic Pinus densiflora seedlings in vitro. Ectomycorrhizal tips of each fungal isolate tested were observed on pine lateral roots within 5 months of inoculation. Seventeen pine seedlings that formed ectomycorrhizas in vitro with these three Amanita species were acclimatized under non-sterile conditions. Seven months following acclimatization, ectomycorrhizal colonization by A. caesareoides was observed on newly grown root tips, which was confirmed by polymerase chain reaction restriction fragment length polymorphism analysis of the fungal rDNA ITS region. Two other Amanita species also survived during ectomycorrhizal acclimatization. These results suggest that the cultivation of A. caesareoides and its relatives can be attempted through mycorrhizal synthesis using P. densiflora as a host. This is the first report of in vitro mycorrhization of Asian Caesar's mushrooms and their acclimatization under non-sterile conditions.

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.

Molecular detection of poisonous mushrooms in different matrices.

Amanita phalloides, Lepiota cristata, Lepiota brunneoincarnata and Inocybe asterospora are among the most important species responsible of mushroom poisoning in northern Italy. A real time PCR method for the identification of samples containing DNA from each of these species was developed. To test specificity all protocols were applied on DNA extracted from various mushroom species; sensitivity was assessed performing serial dilutions on all samples; versatility of the protocols was evaluated performing tests on DNA extracted from different matrices. The protocols showed high sensitivity (32 ng dried mushroom), high specificity and sensitive detection of DNA extracted from difficult samples, including pasta with mushroom, cooked mushrooms and gastric aspirates.

Amanita phalloides poisoning-induced end-stage renal failure.

Fungi poisoning is quite frequent: in particular, Amanita phalloides has life-threatening toxicity. It is responsible for fulminant hepatitis, and also has renal toxicity. Herein, we report on a patient who developed acute renal failure after ingesting A. phalloides, which required definitive renal replacement therapy, despite rapid liver injury recovery. A kidney biopsy showed massive acute tubular necrosis, mainly in the proximal convoluted tubule, and mild interstitial infiltration by mononuclear cells.

[Amanita pantherina poisoning or brain stroke?]. / Zatrucie muchomorem plamistym czy udar mózgu?

A case of a 72-year-old female who ingested panther cap (Amanita pantherina) was presented. During the ingestion of mushrooms she developed short-time diarrhea and severe transient neurological disorders; short-term hallucination followed by deep coma, skeletal muscle flaccidity with hyporeflexia. Also significant facial asymmetry was noted. A head CT showed a small ischaemic focus in capsula interna. After a neurological consult it was concluded that this small malformation was not the cause of severe patient's condition. The sporological test revealed numerous spores of panther cap in patient's vomit. After the next 6 hours complete resolution of neurological symptoms was observed. Finally it was assumed that patient's symptoms and signs were most probably a result of panther cap poisoning complicated with transient ischaemia of the brain.

Molecular phylogeny and biogeography of the widely distributed Amanita species, A. muscaria and A. pantherina.

The molecular phylogeny and biogeography of two widely distributed Amanita species, A. muscaria and A. pantherina, were studied based on specimens from diverse localities. Analyses of both a partial sequence of the ITS region of nuclear DNA and a partial sequence of the beta-tubulin gene were able to resolve specimens of each species. Analyses revealed a greater divergence of the beta-tubulin region than the ITS region. Based on molecular phylogeny of the combination of the ITS and beta-tubulin regions, A. muscaria could be separated into at least three groups (Eurasian, Eurasian subalpine, and North American), and A. pantherina could be separated into at least two groups (North American and Eurasian). We hypothesize that the speciation of A. muscaria occurred in Eurasia with subsequent migration to North America via land bridges. However, it is impossible to determine whether A. pantherina moved from Eurasia to North America or vice versa. For both A. muscaria and A. pantherina, the intracontinental relationships of both Eurasia and North America were closer than the relationships between eastern Asia and eastern North America.

Distribution of Amanita spp. genotypes under eastern Australian sclerophyll vegetation.

Basidiomes of Amanita alboverrucosa, A. ochrophylla, and A. pyramidifera were collected from native mixed sclerophyll forest sites and of A. conicoverrucosa and A. punctata from planted stands of Eucalyptus maculata in New South Wales, Australia. DNA was extracted from stipe tissue and subjected to inter-simple sequence repeat (ISSR) PCR analysis conducted using the primers (GTG)5 and (GACA)4 in order to determine genotype distribution at each site. Two to nine genotypes of one of the species were identified at each field site. Genotypes of A. ochrophylla, A. conicoverrucosa and A. punctata were spread over areas of ca 10-60 m diam, suggesting vegetative spread via large below-ground mycelial genets. In contrast, genotypes of A. alboverrucosa were more spatially restricted, suggesting recent establishment via basidiospores and more limited below-ground vegetative spread. Two groups of A. pyramidifera basidiomes that were separated by ca 600 m were found to be of the same genotype. While this might reflect long distance spread of below-ground mycelium in this taxon, the proximity of the basidiomes to a roadway makes movement of vegetative basidiome tissue via vehicular activity and subsequent establishment equally plausible.

The examination of mushroom poisonings at Akita University.

In the past 10 years from 1991 to 2000, the number of consultations to the Japan Poison Information Center were 947 concerning mushroom poisonings. However, those from the hospital cases were not analyzed toxicologically. We examined toxicologically 20 cases (35 patients) of mushroom poisonings from 1993 to 2001. Investigation of amanita toxin poisoning was requested in 19 cases. We could detect the amanita toxin, amanitin, and phalloidin, in two cases, which resulted in concluding the cause of death. A fatal case by the magic mushroom poisoning was analyzed in the blood, urine, and mushroom, and we detected the hallucinogenic substances from the body fluids and ingested mushrooms. We report the results of our examinations, and point out the usefulness of the examination of the mushroom itself and biological samples toxicologically for forensic practice.

Utilisation of inorganic and organic nitrogen sources by Amanita species native to temperate eastern Australia.

The abilities of isolates of Amanita alboverrucosa, A. conicoverrucosa, A. fuscosquamosa, A. nauseosa, A. ochrophylla, A. pyramidifera, A. roseolamellata, A. xanthocephala and six unidentified Amanita species from eastern Australian temperate sclerophyll forests to utilise a range of inorganic and organic nitrogen sources for growth was examined in axenic liquid cultures. All taxa utilised NH4+ and at least some amino acids readily, while biomass yields on NO3- and histidine were generally low. All taxa were able to utilise bovine serum albumin, but for most taxa biomass yields on this substrate were significantly lower than on NH4+. Significant intraspecific variation in biomass yield was observed on all substrates for taxa for which multiple isolates were screened. As a group, eastern Australian Amanita species thus have the potential to utilise nitrogen from a broad range of organic substrates and this might be important in the nitrogen nutrition of their ectomycorrhizal tree hosts in sclerophyll forests.

[Analysis of the main amatoxins and phallotoxins in Amanita exitialis--a new species in China].

The content and distribution of the main Amatoxins (alpha-amanitin, beta-amanitin) and Phallotoxins (Phallacidin, Phallisin, Phalloin, Phalloidin) in the three tissues (cap, stipe and volva) of Amanita exitialis were evaluated by means of high-performance liquid chromatography. The results showed that Amanita exitialis was a lethal mushrooms, the cap had the highest content of total toxins, it reached 8152.6 microg/g dry weight, the toxins content in stipe reached 3742.3 microg/g dry weight, whereas the volva had the lowest content of total toxins,it had only 1142.5 microg/g dry weight. The distribution of Amatoxins and Phallotoxins in the tissues were revealed and it displayed that the content of Amatoxins (alpha-amanitin and beta-amanitins especially alpha-amanitin) in the cap, stipe or volva of A. exitialis was higher than that of Phallotoxins (Phallacidin, Phallisin, Phalloidin and Phalloin). But the content of Phallotoxins especially Phallacidin was gradationally higher from cap to stipe and to volva.