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Amanita is one of the most salient mushroom genera due to its cultural, economic, and medical importance. Recently, many new Amanita species have been described worldwide, increasing the genus richness. However, several clades have cryptic diversity, and many species complexes have not yet been resolved. This is the case of the rubescent species in the Validae section, which have been widely cited under the name Amanita rubescens s.l. We used a four-locus matrix (nuc rDNA internal transcribed spacer [ITS] and 28S regions and genes for RNA polymerase II subunit 2 [ rpb2 ], translation elongation factor 1-α [ tef1-α ], and β-tubulin [ tub2 ]) to solve the phylogenetic relationships within the Amanita section Validae . To analyze the diversity and distribution patterns of species, we used an extensive ITS sequence sampling including environmental DNA databases. The phylogenetic analyses demonstrated that the Validae section is divided into three monophyletic and highly supported major clades: Mappae, Validae , and Rubescentes . At least 11 species-level clades within the Rubescentes clade were highly supported: A. cruentilemurum nom. prov. A. brunneolocularis, A. rubescens s.s. (European clade), A. rubescens s.s. (Asiatic clade), A. orsonii s.s. A . ' orsonii ,' A. aureosubucula nom. prov., A. novinupta, A. flavorubens , and two undescribed North American species. We proved that A. rubescens s.s. has two segregated populations (European and Asiatic) and it is not naturally distributed in America. Furthermore, we found that America has more cryptic species within the Rubescentes clade than Eurasia.

Amanita exitialis, a deadly mushroom found in eastern Asia, causes the highest death rates among all poisonous mushrooms in China. The aim of the present study was to develop an efficient, accurate, and user-friendly PCR-based method for identifying A. exitialis that could facilitate the prevention, diagnosis, and treatment of associated food poisoning. A. exitialis-specific primers and probes were designed based on the internal transcribed spacer region variations of 27 mushroom species. Specificity was confirmed using conventional and real-time PCR for 23 non-target mushroom species, including morphologically similar and closely related species. Compared to conventional PCR, real-time PCR was more sensitive (detectable DNA concentration: 1.36 × 10 -2 ng/μL vs. 1.36 × 10 -3 ) and efficient (analysis time: 1 h vs. 40 min). Furthermore, the real-time PCR results could be immediately visualized using amplification curve analysis. The results present two robust PCR-based methods for A. exitialis identification that can facilitate food safety., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning. KEY POINTS: • Development of a DNA mini-barcoding method for Amanita species identification without fresh samples. • The ITS-a primer set was optimized for robust universality in Amanita samples. • The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases., (© 2024. The Author(s).)

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., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

The genus Amanita sect. Amanita harbors approximately 150 species in the world, and 27 species have been recognized in China. Some of the species in China have continuously caused poisoning. The responsible toxins should be ibotenic acid (IBO) and muscimol (MUS). However, species of the section Amanita containing IBO and MUS and their systematic positions are unclear. In this study, the contents of IBO and MUS in 84 samples of 24 species in section Amanita were detected using UPLC‒MS/MS, and the distribution of toxin-containing species in the molecular phylogeny was analyzed by the combined (ITS, nrLSU, RPB2, TUB2 and TEF1-α) dataset using maximum likelihood (ML) analysis and Bayesian inference (BI). Our results indicated that 10 of the 24 species contained IBO and MUS ranging from 0.6125 to 32.0932 and 0.0056-5.8685 g/kg dry weight, respectively. Among these 10 species, the toxins of eight species, including Amanita altipes, A. concentrica, A. flavopantherina, A. griseopantherina, A. pseudopantherina, A. rubrovolvata, A. subglobosa and A. sychnopyramis, were detected for the first time. In addition, the IBO and MUS contents of A. subglobosa in different growth stages showed that both toxins decreased in the mature stage. The phylogenetic analysis showed that all species of sect. Amanita from China were divided into 5 groups. And IBO- and MUS-containing species were gathered in clades Ⅰ and Ⅳ, but not all of the species in the two clades contain the toxins. No presence of IBO and MUS in the species of clades Ⅱ, Ⅲ and Ⅴ were confirmed., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zuohong Chen reports financial support was provided by The National Natural Science Foundation of China (Grant No. 31872616). Yuting Su reports financial support was provided by The Postgraduate Scientific Research Innovation Project of Hunan Province (Grant No. CX20210429)., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The poisonous European mushroom Amanita phalloides (the "death cap") is invading California. Whether the death caps' toxic secondary metabolites are evolving as it invades is unknown. We developed a bioinformatic pipeline to identify the MSDIN genes underpinning toxicity and probed 88 death cap genomes from an invasive Californian population and from the European range, discovering a previously unsuspected diversity of MSDINs made up of both core and accessory elements. Each death cap individual possesses a unique suite of MSDINs, and toxin genes are significantly differentiated between Californian and European samples. MSDIN genes are maintained by strong natural selection, and chemical profiling confirms MSDIN genes are expressed and result in distinct phenotypes; our chemical profiling also identified a new MSDIN peptide. Toxin genes are physically clustered within genomes. We contextualize our discoveries by probing for MSDINs in genomes from across the order Agaricales, revealing MSDIN diversity originated in independent gene family expansions among genera. We also report the discovery of an MSDIN in an Amanita outside the "lethal Amanitas" clade. Finally, the identification of an MSDIN gene and its associated processing gene (POPB) in Clavaria fumosa suggest the origin of MSDINs is older than previously suspected. The dynamic evolution of MSDINs underscores their potential to mediate ecological interactions, implicating MSDINs in the ongoing invasion. Our data change the understanding of the evolutionary history of poisonous mushrooms, emphasizing striking parallels to convergently evolved animal toxins. Our pipeline provides a roadmap for exploring secondary metabolites in other basidiomycetes and will enable drug prospecting., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Amanita muscaria is an ectomycorrhizal mushroom that commonly grows at metal-polluted sites. Sporocarps from the lead smelter-polluted area near Příbram (Central Bohemia, Czech Republic) showed elevated concentrations of Cd and Zn. Size exclusion chromatography of the cell extracts of the sporocarps from both polluted and unpolluted sites indicated that substantial part of intracellular Cd and Zn was sequestered in 6-kDa complexes, presumably with metallothionein(s) (MT). When the cultured mycelial isolates were compared, those from Příbram were more Cd-tolerant and accumulated slightly less Cd and Zn than those from the unpolluted site. The analysis of the available A.muscaria sequence data returned a 67-amino acid (AA) MT encoded by the AmMT1 gene. Weak Cd and Zn responsiveness of AmMT1 in the mycelia suggested its metal homeostasis function in A.muscaria, rather than a major role in detoxification. The AmMT1 belongs to a ubiquitous peptide group in the Agaricomycetes consisting of 60-70-AA MTs containing seven cysteinyl domains and a conserved histidyl, features observed also in a newly predicted, atypical 45-AA RaMT1 of the Zn-accumulator Russula bresadolae in which the C-terminal cysteinyl domains VI and VII are missing. Heterologous expression in metal-sensitive yeast mutants indicated that AmMT1 and RaMT1 encode functional peptides that can protect cells against Cd, Zn, and Cu toxicity. The metal protection phenotype observed in yeasts with mutant variants of AmMT1 and RaMT1 further indicated that the conserved histidyl seems to play a structural, not metal binding role, and the cysteinyls of the C-terminal domains VI and VII are important for Cu binding. The data provide an important insight into the metal handling of site-associated ectomycorrhizal species disturbed by excess metals and the properties of MTs common in Agaricomycetes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The deadly toxin α-amanitin is a bicyclic octapeptide biosynthesized on ribosomes. A phylogenetically disjunct group of mushrooms in Agaricales (Amanita, Lepiota, and Galerina) synthesizes α-amanitin. This distribution of the toxin biosynthetic pathway is possibly related to the horizontal transfer of metabolic gene clusters among taxonomically unrelated mushrooms with overlapping habitats. Here, our work confirms that two biosynthetic genes, P450-29 and FMO1, are oxygenases important for amanitin biosynthesis. Phylogenetic and genetic analyses of these genes strongly support their origin through horizontal transfer, as is the case for the previously characterized biosynthetic genes MSDIN and POPB. Our analysis of multiple genomes showed that the evolution of the α-amanitin biosynthetic pathways in the poisonous agarics in the Amanita, Lepiota, and Galerina clades entailed distinct evolutionary pathways including gene family expansion, biosynthetic genes, and genomic rearrangements. Unrelated poisonous fungi produce the same deadly amanitin toxins using variations of the same pathway. Furthermore, the evolution of the amanitin biosynthetic pathway(s) in Amanita species generates a much wider range of toxic cyclic peptides. The results reported here expand our understanding of the genetics, diversity, and evolution of the toxin biosynthetic pathway in fungi.

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.

Food poisoning caused by toxic mushrooms, such as species in the Amanita genus, occurs frequently around the world. To properly treat these patients, it is important to rapidly and accurately identify the causal species. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry is a rapid technique that has been used in medical laboratories for the past three decades to identify bacteria, yeasts, and filamentous fungi.Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-Tof MS) is a rapid method used for the past three decades to identify microorganisms. In this study, we created and internally validated a MALDI-Tof MS reference database comprising 15 Amanita species frequently encountered in France, and we challenged this database with 38 Amanita specimens from four French locations, using a free online application for MALDI-ToF spectra identifications.Assessment of the database showed that mass spectra can be obtained by analyzing any portion of a carpophore and that all portions enabled identification of the carpophore at the species level. Most carpophores were correctly identified using our database, with the exception of specimens from the Vaginatae section. Decay tests also demonstrated that decayed portions (like those found in the kitchen garbage can) of Amanita phalloides mushrooms could be properly identified using MALDI-ToF MS.Our findings provide important insight for toxicology laboratories that often rely on DNA sequencing to identify meal leftovers implicated in food poisoning. In future developments, this technique could also be used to detect counterfeit mushrooms by including other genera in the reference database., Lay Summary: MALDI-ToF MS is a powerful identification tool for microorganisms. We demonstrate that the technique can be applied to Amanita specimens. This will prevent food intoxications as a rapid and definite identification can be obtained, and it can also be used for food remnants., (© The Author(s) 2021. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

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.

By introducing novel capacities and functions, new genes and gene families may play a crucial role in ecological transitions. Mechanisms generating new gene families include de novo gene birth, horizontal gene transfer, and neofunctionalization following a duplication event. The ectomycorrhizal (ECM) symbiosis is a ubiquitous mutualism and the association has evolved repeatedly and independently many times among the fungi, but the evolutionary dynamics enabling its emergence remain elusive. We developed a phylogenetic workflow to first understand if gene families unique to ECM Amanita fungi and absent from closely related asymbiotic species are functionally relevant to the symbiosis, and then to systematically infer their origins. We identified 109 gene families unique to ECM Amanita species. Genes belonging to unique gene families are under strong purifying selection and are upregulated during symbiosis, compared with genes of conserved or orphan gene families. The origins of seven of the unique gene families are strongly supported as either de novo gene birth (two gene families), horizontal gene transfer (four), or gene duplication (one). An additional 34 families appear new because of their selective retention within symbiotic species. Among the 109 unique gene families, the most upregulated gene in symbiotic cultures encodes a 1-aminocyclopropane-1-carboxylate deaminase, an enzyme capable of downregulating the synthesis of the plant hormone ethylene, a common negative regulator of plant-microbial mutualisms., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Amanita fuligineoides, a lethal mushroom discovered in China, contains abundant cyclic peptide toxins that can cause fatal poisoning. However, the MSDIN gene family encoding for these cyclic peptides in A. fuligineoides has not been systematically studied. In this research, the transcriptome sequencing of A. fuligineoides was performed and its MSDIN family members were analyzed. A total of 4.41 Gb data containing 30833 unigenes was obtained; sequence alignments throughout several databases were done to obtain their functional annotations. Based on these annotations, MSDIN genes were found and verified by RT-PCR. A total of 29 different core peptides were obtained: 3 toxin genes, encoding β-amanitin (β-AMA), phalloidin (PHD), and phallacidin (PCD), and 26 genes encoding unknown cyclic peptides, 20 of which are reported for the first time and may encode for novel cyclic peptides. Analysis of the predicted precursor peptides indicated that octocyclic peptides were the main MSDIN peptides synthesized by A. fuligineoides, accounting for the 45%. A phylogenetic analysis suggested that studied precursor peptides could be clustered into 7 clades, which might represent different functionalities. Results suggested that A. fuligineoides might have a strong capacity to synthesize cyclopeptides, laying the foundation for their excavation and utilization., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Hyperbranched rolling circle amplification (HRCA) with a padlock probe (PLP) targeting the α-amanitin (α-AMA) gene, as a screening tool for the universal identification of lethal amanitas, was established in this study. With the isothermal HRCA assay, all of the lethal Amanita species tested from Phalloideae (10) were positive, while the non-Phalloideae Amanita species (15) and three amanitin-containing Lepiota and Galerina species were negative. Furthermore, the PLP based on α-AMA sequences from lethal Amanita species was effective for Amanita α-AMA, but not Amanita β-AMA or non-Amanita α-AMA. HRCA sensitivity was 100-fold higher than conventional PCR with a detection limit of 100 copies (recombinant plasmid containing α-AMA), and 0.2% lethal amanitas could be detected in dry mushroom blends. The HRCA method presented provided a rapid, specific, sensitive and low-cost identification tool for lethal amanitas., (Copyright © 2019. Published by Elsevier Ltd.)

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.

Ectomycorrhizal (ECM) fungi, symbiotically associated with woody plants, markedly improve the uptake of mineral nutrients such as potassium (K) and phosphorus (P) by their host trees. Although it is well known that ECM fungi can obtain K and P from soil minerals through biological weathering, the mechanisms regulating this process are still poorly understood at the molecular level. Here, we investigated the transcriptional regulation of the ECM fungus Amanita pantherina in weathering K-containing feldspar and apatite using transcriptome sequencing (RNA-seq) and validated these results for differentially expressed genes using real-time quantitative PCR. The results showed that A. pantherina was able to improve relevant metabolic processes, such as promoting the biosynthesis of unsaturated fatty acids and steroids in the weathering of K-containing feldspar and apatite. The expression of genes encoding ion transporters was markedly enhanced during exposure to solid K-containing feldspar and apatite, and transcripts of the high-affinity K transporter ApHAK1, belonging to the HAK family, were significantly upregulated. The results also demonstrated that there was no upregulation of organic acid biosynthesis, reflecting the weak weathering capacity of the A. pantherina isolate used in this study, especially its inability to utilize P in apatite. Our findings suggest that under natural conditions in forests, some ECM fungi with low weathering potential of their own may instead enhance the uptake of mineral nutrients using their high-affinity ion transporter systems. IMPORTANCE In this study, we revealed the molecular mechanism and possible strategies of A. pantherina with weak weathering potential in the uptake of insoluble mineral nutrients by using transcriptome sequencing (RNA-seq) technology and found that ApHAK1 , a K transporter gene of this fungus, plays a very important role in the acquisition of K and P. Ectomycorrhizal (ECM) fungi play critical roles in the uptake of woody plant nutrients in forests that are usually characterized by nutrient limitation and in maintaining the stability of forest ecosystems. However, the regulatory mechanisms of ECM fungi in acquiring nutrients from minerals/rocks are poorly understood. This study investigated the transcriptional regulation of A. pantherina weathering K-containing feldspar and apatite and improves the understanding of fungal-plant interactions in promoting plant nutrition enabling increased productivity in sustainable forestry., (Copyright © 2019 American Society for Microbiology.)

Fungi are evolutionary shape shifters and adapt quickly to new environments. Ectomycorrhizal (EM) symbioses are mutualistic associations between fungi and plants and have evolved repeatedly and independently across the fungal tree of life, suggesting lineages frequently reconfigure genome content to take advantage of open ecological niches. To date analyses of genomic mechanisms facilitating EM symbioses have involved comparisons of distantly related species, but here, we use the genomes of three EM and two asymbiotic (AS) fungi from the genus Amanita as well as an AS outgroup to study genome evolution following a single origin of symbiosis. Our aim was to identify the defining features of EM genomes, but our analyses suggest no clear differentiation of genome size, gene repertoire size, or transposable element content between EM and AS species. Phylogenetic inference of gene gains and losses suggests the transition to symbiosis was dominated by the loss of plant cell wall decomposition genes, a confirmation of previous findings. However, the same dynamic defines the AS species A. inopinata, suggesting loss is not strictly associated with origin of symbiosis. Gene expansions in the common ancestor of EM Amanita were modest, but lineage specific and large gene family expansions are found in two of the three EM extant species. Even closely related EM genomes appear to share few common features. The genetic toolkit required for symbiosis appears already encoded in the genomes of saprotrophic species, and this dynamic may explain the pervasive, recurrent evolution of ectomycorrhizal associations.

Amanita exitialis Zhu L. Yang & T. H. Li is the species responsible for the largest number of mushroom-associated human poisonings and fatalities in South China due to its lethal cyclic peptide toxins. Prolyl oligopeptidase B (POPB) is considered a key enzyme in the production of the highly toxic cyclic peptide α-amanitin. However, the POPB gene of A. exitialis has not been studied. In the present study we cloned and sequenced the full-length A. exitialis POPB (AePOPB) gene. The aim was to verify the gene structure and functions of AePOPB. The full-length sequence of AePOPB is 3144 bp, including 18 exons encoding 730 aa, and the advanced structure is very similar to that of the previously reported POPB in Galerina marginata (GmPOPB). The amino acid sequence of AePOPB is highly homologous with those from other amanitin-producing lethal mushrooms, implying that AePOPB may have a similar role in the biosynthesis of cyclic peptide toxins. Expression levels of AePOPB were detectable in all parts and developmental stages of the fruiting bodies, and AePOPB was expressed more strongly at early development stages (early and late elongation stages). At early and late elongation stages, the expression peaks occurred in the stipe, whereas at early and late mature stages, the expression peaks occurred in the pileus. The expression patterns of AePOPB in different stages and different parts of the fruiting bodies were highly consistent with those of Aeα-AMA, which is required for α-amanitin accumulation. These results indicate that AePOPB should be involved in the α-amanitin biosynthesis in A. exitialis., (Copyright © 2018. Published by Elsevier B.V.)

Amanita exitialis is a lethal mushroom found in China. Knowledge regarding taxonomic characterization, toxin detection, general poisoning conditions, clinical manifestations, laboratory examinations, and clinical treatments for this species is currently lacking. We investigated three A. exitialis mushroom poisoning cohorts in Yunnan Province in 2014 and 2015, involving 10 patients. Mushroom samples were identified by morphological and molecular studies. Ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry was used to detect the peptide toxins in the mushroom samples. Epidemiological information, clinical data, and results of laboratory examinations were collected and analyzed. The mushroom samples were all identified as A. exitialis. The average toxin concentration decreased from the cap to the stipe to the volva, and the average concentration of the peptide toxins decreased in the order of α-amanitin > phallacidin > β-amanitin > γ-amanitin. The latency period between ingestion and the onset of symptoms was 13.9 ± 2.1 h, and the time from ingestion to hospitalization was 49.6 ± 8.5 h. The most common symptoms were nausea and vomiting (100%). Four patients died from fulminant hepatic failure. Laboratory examinations showed that the alanine transaminase, aspartate transaminase, prothrombin time, and activated partial thromboplastin time levels peaked on the third day post-ingestion. Total bilirubin and direct bilirubin values peaked on day 7. The death group and the survival group had a similar variation trend of serological indexes, but the death group had a greater change. A. exitialis is an extremely dangerous mushroom and there is a need to educate the public to avoid picking and eating wild mushrooms that have not been definitively identified.

Amanita cf. lavendula collections in eastern North America, Mexico, and Costa Rica were found to consist of four cryptic taxa, one of which exhibited consistently unreadable nuclear rDNA ITS1-5.8S-ITS2 (fungal barcode) sequences after ITS1 base 130. This taxon is designated here as Amanita cf. lavendula taxon 1. ITS sequences from dikaryotic basidiomata were cloned, but sequences recovered from cloning did not segregate into distinct haplotypes. Rather, there was a mix of haplotypes that varied among themselves predominantly at 28 ITS positions. Analysis of each of these 28 variable bases showed predominantly two alternate bases at each position. Based on these findings and additional sequence data from the nuclear rDNA 28S, RNA polymerase II subunit 2 (RPB2) and mitochondrial rDNA small subunit (SSU) and 23S genes, we speculate that taxon 1 represents an initial hybridization event between two divergent taxa followed by failure of the ribosomal repeat to homogenize. Homogenization failure may be a result of repeated hybridization between divergent internal transcribed spacer (ITS) types with inadequate time for concerted evolution of the ribosomal repeat or, alternately, a complete failure of the ribosomal homogenization process. To our knowledge, this finding represents the first report of a geographically widespread taxon (Canada, eastern USA, Costa Rica) with apparent homogenization failure across all collections. Findings such as these have implications for fungal barcoding efforts and the application of fungal barcodes in identifying environmental sequences.

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., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Amanita ballerina and A. brunneitoxicaria spp. nov. are introduced from Thailand. Amanita fuligineoides is also reported for the first time from Thailand, increasing the known distribution of this taxon. Together, those findings support our view that many taxa are yet to be discovered in the region. While both morphological characters and a multiple-gene phylogeny clearly place A. brunneitoxicaria and A. fuligineoides in sect. Phalloideae (Fr.) Quél., the placement of A. ballerina is problematic. On the one hand, the morphology of A. ballerina shows clear affinities with stirps Limbatula of sect. Lepidella. On the other hand, in a multiple-gene phylogeny including taxa of all sections in subg. Lepidella, A. ballerina and two other species, including A. zangii, form a well-supported clade sister to the Phalloideae sensu Bas 1969, which include the lethal "death caps" and "destroying angels". Together, the A. ballerina-A. zangii clade and the Phalloideae sensu Bas 1969 also form a well-supported clade. We therefore screened for two of the most notorious toxins by HPLC-MS analysis of methanolic extracts from the basidiomata. Interestingly, neither α-amanitin nor phalloidin was found in A. ballerina, whereas Amanita fuligineoides was confirmed to contain both α-amanitin and phalloidin, and A. brunneitoxicaria contained only α-amanitin. Together with unique morphological characteristics, the position in the phylogeny indicates that A. ballerina is either an important link in the evolution of the deadly Amanita sect. Phalloideae species, or a member of a new section also including A. zangii.

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., (Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

Some Amanita specimens collected from Malaysia are critically investigated by morphological examination and molecular analysis of two gene fragments, the nuc rDNA partial 28S (28S) gene and the internal transcriber spacer (ITS1-5.8S-ITS2 = ITS) regions. Six phylogenetic species of Amanita section Caesareae are recognized among the studied collections. One of them is described as new, A. malayensis. Four of the phylogenetic species correspond with existing morphology-based taxa: A. aporema, A. javanica, A. princeps, and A. similis. The remaining species is not described because of the paucity of material. Detailed descriptions and the distribution of these southeastern Asian species are provided, along with a key to the species of section Caesareae from Malaysia.

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 β-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., (© 2016 by The Mycological Society of America.)

Macrofungi can accumulate in their sporocarps remarkably high concentrations of Cu and Ag. We have previously demonstrated that the non-essential Ag is in the ectomycorrhizal, Ag-hyperaccumulating Amanita strobiliformis sequestered by 3.4-kDa metallothioneins (MTs) produced as AsMT1a, 1b and 1c isoforms. Here, we describe two populations of wild-grown A. strobiliformis sporocarps, which showed certain correlation between the concentrations of accumulated Ag (284 ± 64 and 67 ± 15 mg kg(-1)) and Cu (76 ± 13 and 30 ± 12 mg kg(-1)), suggesting that an overlap may exist in the cell biology of Ag and Cu in this species. Metal speciation analysis revealed that the intracellular Cu in the sporocarps of both populations was, like Ag, associated with the 3.4-kDa MTs. A search of A. strobiliformis transcriptome for sequences encoding proteins of the Cu transporter (CTR) family identified four AsCTR cDNAs, which were, like AsMT1s, confirmed in both populations. The predicted AsCTR proteins showed homology to vacuolar (AsCTR1 and AsCTR4) and plasma membrane (AsCTR2 and AsCTR3) CTRs. Heterologous expression of AsCTR2, AsCTR3 and their translational fusions with green fluorescent protein (GFP) in Cu uptake-deficient S. cerevisiae indicated that both AsCTRs are functional Cu and Ag uptake transporters: recombinant genes complemented growth defects and increased Cu and Ag uptake rates in yeasts and the GFP-tagged protein localized to the cell periphery. Site directed mutagenesis revealed the importance of the conserved-among-CTRs M-X3-M motif for the AsCTR2- and AsCTR3-mediated transport of both Cu and Ag. These results provide the first evidence that fungal CTRs can recognize Ag for transport.

Mechanisms evolved in eukaryotes to handle heavy metals involve cytosolic, metal-binding metallothioneins (MTs). We have previously documented that the sequestration of silver (Ag) in the Ag-hyperaccumulating Amanita strobiliformis is dominated by 34-amino-acid (AA) AsMT1a, 1b, and 1c isoforms. Here we show that in addition to AsMT1a, 1b, and 1c isogenes, the fungus has two other MT genes: AsMT2 encoding a 34-AA AsMT2 similar to MTs known from other species, but unrelated to AsMT1s; AsMT3 coding for a 62-AA AsMT3 that shares substantial identity with as-yet-uncharacterized conserved peptides predicted in agaricomycetes. Transcription of AsMT1s and AsMT3 in the A. strobiliformis mycelium was specifically inducible by treatments with Ag or copper (Cu) and zinc (Zn) or cadmium (Cd), respectively; AsMT2 showed a moderate upregulation in the presence of Cd. Expression of AsMTs in the metal-sensitive Saccharomyces cerevisiae revealed that all AsMTs confer increased Cd tolerance (AsMT3 proved the most effective) and that, unlike AsMT1 and AsMT2, AsMT3 can protect the yeasts against Zn toxicity. The highest level of Cu tolerance was observed with yeasts expressing AsMT1a. Our data indicate that A. strobiliformis can specifically employ different MT genes for functions in the cellular handling of Ag and Cu (AsMT1s) and Zn (AsMT3)., (Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

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.

Some of the effects of past climate dynamics on plant and animal diversity make-up have been relatively well studied, but to less extent in fungi. Pleistocene refugia are thought to harbour high biological diversity (i.e. phylogenetic lineages and genetic diversity), mainly as a product of increased reproductive isolation and allele conservation. In addition, high extinction rates and genetic erosion are expected in previously glaciated regions. Some of the consequences of past climate dynamics might involve changes in range and population size that can result in divergence and incipient or cryptic speciation. Many of these dynamic processes and patterns can be inferred through phylogenetic and coalescent methods. In this study, we first delimit species within a group of closely related edible ectomycorrhizal Amanita from North America (the American Caesar's mushrooms species complex) using multilocus coalescent-based approaches; and then address questions related to effects of Pleistocene climate change on the diversity and genetics of the group. Our study includes extensive geographical sampling throughout the distribution range, and DNA sequences from three nuclear protein-coding genes. Results reveal cryptic diversity and high speciation rates in refugia. Population sizes and expansions seem to be larger at midrange latitudes (Mexican highlands and SE USA). Range shifts are proportional to population size expansions, which were overall more common during the Pleistocene. This study documents responses to past climate change in fungi and also highlights the applicability of the multispecies coalescent in comparative phylogeographical analyses and diversity assessments that include ancestral species., (© 2015 John Wiley & Sons Ltd.)

Understanding the patterns of biodiversity through time and space is a challenging task. However, phylogeny-based macroevolutionary models allow us to account and measure many of the processes responsible for diversity buildup, namely speciation and extinction. The general latitudinal diversity gradient (LDG) is a well-recognized pattern describing a decline in species richness from the equator polewards. Recent macroecological studies in ectomycorrhizal (EM) fungi have shown that their LDG is shifted, peaking at temperate rather than tropical latitudes. Here we investigate this phenomenon from a macroevolutionary perspective, focusing on a well-sampled group of edible EM mushrooms from the genus Amanita-the Caesar's mushrooms, which follow similar diversity patterns. Our approach consisted in applying a suite of models including (1) nontrait-dependent time-varying diversification (Bayesian analysis of macroevolutionary mixtures [BAMM]), (2) continuous trait-dependent diversification (quantitative-state speciation and extinction [QuaSSE]), and (3) diversity-dependent diversification. In short, results give strong support for high speciation rates at temperate latitudes (BAMM and QuaSSE). We also find some evidence for different diversity-dependence thresholds in "temperate" and "tropical" subclades, and little differences in diversity due to extinction. We conclude that our analyses on the Caesar's mushrooms give further evidence of a temperate-peaking LDG in EM fungi, highlighting the importance and the implications of macroevolutionary processes in explaining diversity gradients in microorganisms., (© 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.)

Intact, growing cells of strongly acidophilic fungi Acidea extrema and Acidothrix acidophila have been successfully transformed by introduction of heterologous DNA fragment (composed of the glyceraldehyde-phosphate-dehydrogenase gene promoter from Emericella nidulans, a metallothionein-coding gene AsMt1 from Amanita strobiliformis and glyceraldehyde-phosphate-dehydrogenase gene terminator from Colletotrichum gloeosporioides) with the length of 1690 bp. The transformation procedure was based on the DNA transfer mediated by Agrobacterium tumefaciens bearing disarmed helper plasmid pMP90 and binary vector pCambia1300 with inserted DNA fragment of interest. The transformants proved to be mitotically stable, and the introduced gene was expressed at least at the level of transcription. Our work confirms that metabolic adaptations of strongly acidophilic fungi do not represent an obstacle for genetic transformation using conventional methods and can be potentially used for production of heterologous proteins. A promising role of the fast growing A. acidophila as active biomass in biotechnological processes is suggested not only by the low susceptibility of the culture grown at low pH to contaminations but also by reduced risk of accidental leaks of genetically modified microorganisms into the environment because highly specialized extremophilic fungi can poorly compete with common microflora under moderate conditions.

The genus Amanita encompasses both symbiotic, ectomycorrhizal fungi and asymbiotic litter decomposers; all species are derived from asymbiotic ancestors. Symbiotic species are no longer able to degrade plant cell walls. The carbohydrate esterases family 1 (CE1s) is a diverse group of enzymes involved in carbon metabolism, including decomposition and carbon storage. CE1 genes of the ectomycorrhizal A. muscaria appear diverged from all other fungal homologues, and more similar to CE1s of bacteria, suggesting a horizontal gene transfer (HGT) event. In order to test whether AmanitaCE1s were acquired horizontally, we built a phylogeny of CE1s collected from across the tree of life, and describe the evolution of CE1 genes among Amanita and relevant lineages of bacteria. CE1s of symbiotic Amanita were very different from CE1s of asymbiotic Amanita, and are more similar to bacterial CE1s. The protein structure of one CE1 gene of A. muscaria matched a depolymerase that degrades the carbon storage molecule poly((R)-3-hydroxybutyrate) (PHB). Asymbiotic Amanita do not carry sequence or structural homologues of these genes. The CE1s acquired through HGT may enable novel metabolisms, or play roles in signaling or defense. This is the first evidence for the horizontal transfer of carbohydrate metabolism genes into ectomycorrhizal fungi., (© 2014 The Authors New Phytologist © 2014 New Phytologist Trust.)

Background: Lethal amanitas (Amanita section Phalloideae) are a group of wild, fatal mushrooms causing many poisoning cases worldwide. However, the diversity and evolutionary history of these lethal mushrooms remain poorly known due to the limited sampling and insufficient gene fragments employed for phylogenetic analyses. In this study, five gene loci (nrLSU, ITS, rpb2, ef1-α and β-tubulin) with a widely geographic sampling from East and South Asia, Europe, North and Central America, South Africa and Australia were analysed with maximum-likelihood, maximum-parsimony and Bayesian inference methods. Biochemical analyses were also conducted with intention to detect amatoxins and phalloidin in 14 representative samples., Result: Lethal amanitas were robustly supported to be a monophyletic group after excluding five species that were provisionally defined as lethal amanitas based on morphological studies. In lethal amanitas, 28 phylogenetic species were recognised by integrating molecular phylogenetic analyses with morphological studies, and 14 of them represented putatively new species. The biochemical analyses indicated a single origin of cyclic peptide toxins (amatoxins and phalloidin) within Amanita and suggested that this kind of toxins seemed to be a synapomorphy of lethal amanitas. Molecular dating through BEAST and biogeographic analyses with LAGRANGE and RASP indicated that lethal amanitas most likely originated in the Palaeotropics with the present crown group dated around 64.92 Mya in the early Paleocene, and the East Asia-eastern North America or Eurasia-North America-Central America disjunct distribution patterns were primarily established during the middle Oligocene to Miocene., Conclusion: The cryptic diversity found in this study indicates that the species diversity of lethal amanitas is strongly underestimated under the current taxonomy. The intercontinental sister species or sister groups relationships among East Asia and eastern North America or Eurasia-North America-Central America within lethal amanitas are best explained by the diversification model of Palaeotropical origin, dispersal via the Bering Land Bridge, followed by regional vicariance speciation resulting from climate change during the middle Oligocene to the present. These findings indicate the importance of both dispersal and vicariance in shaping the intercontinental distributions of these ectomycorrhizal fungi.

Transposable elements (TEs) are ubiquitous inhabitants of eukaryotic genomes and their proliferation and dispersal shape genome architectures and diversity. Nevertheless, TE dynamics are often explored for one species at a time and are rarely considered in ecological contexts. Recent work with plant pathogens suggests a link between symbiosis and TE abundance. The genomes of pathogenic fungi appear to house an increased abundance of TEs, and TEs are frequently associated with the genes involved in symbiosis. To investigate whether this pattern is general, and relevant to mutualistic plant-fungal symbioses, we sequenced the genomes of related asymbiotic (AS) and ectomycorrhizal (ECM) Amanita fungi. Using methods developed to interrogate both assembled and unassembled sequences, we characterized and quantified TEs across three AS and three ECM species, including the AS outgroup Volvariella volvacea. The ECM genomes are characterized by abundant numbers of TEs, an especially prominent feature of unassembled sequencing libraries. Increased TE activity in ECM species is also supported by phylogenetic analysis of the three most abundant TE superfamilies; phylogenies revealed many radiations within contemporary ECM species. However, the AS species Amanita thiersii also houses extensive amplifications of elements, highlighting the influence of additional evolutionary parameters on TE abundance. Our analyses provide further evidence for a link between symbiotic associations among plants and fungi, and increased TE activity, while highlighting the importance individual species' natural histories may have in shaping genome architecture., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Mushrooms in lethal Amanita species are responsible for a large number of food poisoning cases and deaths. However, the diversity of the toxins in these mushrooms remains largely unknown. This study analyzed the gene families of toxins found in 6 lethal Amanitae from Asia and Europe. Fifty-four gene sequences were obtained, accounting for 70.1% of the known MSDIN family members. Of the 54 gene sequences, 20 encode α-amanitin, 5 encode β-amanitin, 16 encode phallacidin, and 13 encode peptides of unknown functions. Bayesian analysis of MSDIN family members identified differences in the number of toxin genes in different toxin families among the Amanita species. Ten of the 13 peptides of unknown functions were closely related to known phallotoxins, while the remaining 3 were similar to amatoxins. The α-AMA tree indicated that there were significant differences between the Amanita and Galerina species. However, both the α-AMA and PHA trees showed that these toxin genes have similar upstream and downstream motif sequences among the Amanita species. This study greatly enriches the available diversity information regarding toxin gene families in lethal Amanita mushrooms, and could lay a strong foundation for further research about the evolution of Amanita toxin genes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The capability of Fluorescent Random Amplified Microsatellites (F-RAMS) to profile hallucinogenic mushrooms to species and sub-species level was assessed. Fifteen samples of Amanita rubescens and 22 samples of other hallucinogenic and non-hallucinogenic mushrooms of the genera Amanita and Psilocybe were profiled using two fluorescently-labeled, 5'degenerate primers, 5'-6FAM-SpC3-DD (CCA)5 and 5'-6FAM-SpC3-DHB (CGA)5, which target different microsatellite repeat regions. Among the two primers, 5'-6FAM-SpC3-DHB (CGA)5 provided more reliable data for identification purposes, by grouping samples of the same species and clustering closely related species together in a dendrogram based on amplicon similarities. A high degree of intra-specific variation between the 15 A. rubescens samples was shown with both primers and the amplicons generated for all A. rubescens samples were organized into three classes of amplicons (discriminant, private, and marker) based on their individualizing potential., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Mushrooms are often poorly digested by humans. Thus, their remains (tissues, spores) may persist in the gastrointestinal tract and can be detected in feces several days after mushroom consumption. In this report, we present protocols for the rapid PCR-based detection of fungal traces in a variety of complex samples. Novel primers were designed to amplify portions of ribosomal DNA from deadly poisonous European members of the genus Amanita, namely the death cap (A. phalloides), the destroying angel (A. virosa) and the fool's mushroom (A. verna), respectively. Assay sensitivity was sufficient to discover diluted DNA traces in amounts below the genomic content of a single target mushroom cell. Specificity testing was performed with DNA extracts from a variety of mushroom species. Template amplification was exclusively observed with intended targets and it was not compromised by a vast excess of non-target DNA (i.e. DNA from human and human fecal origin, respectively). A series of experiments was conducted with prepared specimens in order to follow the course of mushroom food processing and digestion. Amplification by direct PCR was successful with raw, fried and digested mixed mushrooms. To improve assay performance with fecal samples, a rapid protocol for sample pre-processing (including water-ether sedimentation and bead beating) and a modified PCR reaction mix were applied. Thereby, it was possible to detect the presence of A. phalloides DNA in spiked feces as well as in clinical samples (vomit, stool) from two independent cases of suspected mushroom poisoning., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Although rarely seen, Amanita phalloides var. alba, a variety of A. phalloides type mushrooms, causes mushroom poisoning resulting in death. Since it is frequently confused with some edible mushrooms due to its white colored cap and macroscopic appearance, it becomes important in toxicological terms. Knowledge of the toxin amount contained in this mushroom type is invaluable in the treatment of cases involving poisoning. In this study, we examined the toxin levels of various parts of the A. phalloides var. alba mushroom growing Duzce region of Turkey. Toxin analyses were carried out for A. phalloides var. alba, which were collected from the forests Duzce region of Turkey in 2011, as a whole and also separately in its spore, pileus, gills, stipe and volva parts. The alpha amanitin, beta amanitin, gamma amanitin, phalloidin and phallacidine analyses of the mushrooms were carried out using the RP-HPLC method. A genetic analysis of the mushroom showed that it had similar genetic characteristics as A. phalloides and was a variety of it. The lowest toxins quantity was detected in spores, volva and stipe among all parts of the mushroom. The maximum amount of amatoxins was measured in the gills. The pileus also contained a high amount of amatoxins. Generally, amatoxins and phallotoxin concentrations were lower as compared to A. phalloides, but interestingly all toxins other than gamma toxin were higher in the spores of A. phalloides var. alba. The amount of toxin in all of its parts had sufficient concentrations to cause death. With this study, the amatoxin and phallotoxin concentrations in A. phalloides var. alba mushroom and in its parts have been revealed in detail for the first time., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Amanita exitialis is a lethal mushroom that was first discovered in Guangdong Province, China. The high content of amanitin in its basidiocarps makes it lethal to humans. To comprehensively characterize the A. exitialis transcriptome and analyze the Amanita toxins as well as their related gene family, transcriptome sequencing of A. exitialis was performed using Illumina HiSeq 2000 technology. A total of 25,563,688 clean reads were collected and assembled into 62,137 cDNA contigs with an average length of 481 bp and N50 length of 788 bp. A total of 27,826 proteins and 39,661 unigenes were identified among the assembled contigs. All of the unigenes were classified into 166 functional categories for understanding the gene functions and regulation pathways. The genes contributing to toxic peptide biosynthesis were analyzed. From this set, eleven gene sequences encoding the toxins or related cyclic peptides were discovered in the transcriptome. Three of these sequences matched the peptide toxins α-amanitin, β-amanitin, and phallacidin, while others matched amanexitide and seven matched unknown peptides. All of the genes encoding peptide toxins were confirmed by polymerase chain reaction (PCR) in A. exitialis, and the phylogenetic relationships among these proprotein sequences were discussed. The gene polymorphism and degeneracy of the toxin encoding sequences were found and analyzed. This study provides the first primary transcriptome of A. exitialis, which provided comprehensive gene expression information on the lethal amanitas at the transcriptional level, and could lay a strong foundation for functional genomics studies in those fungi., (© 2013.)

Background: Small peptides encoded as one- or two-exon genes in plants have recently been shown to affect multiple aspects of plant development, reproduction and defense responses. However, popular similarity search tools and gene prediction techniques generally fail to identify most members belonging to this class of genes. This is largely due to the high sequence divergence among family members and the limited availability of experimentally verified small peptides to use as training sets for homology search and ab initio prediction. Consequently, there is an urgent need for both experimental and computational studies in order to further advance the accurate prediction of small peptides., Results: We present here a homology-based gene prediction program to accurately predict small peptides at the genome level. Given a high-quality profile alignment, SPADA identifies and annotates nearly all family members in tested genomes with better performance than all general-purpose gene prediction programs surveyed. We find numerous mis-annotations in the current Arabidopsis thaliana and Medicago truncatula genome databases using SPADA, most of which have RNA-Seq expression support. We also show that SPADA works well on other classes of small secreted peptides in plants (e.g., self-incompatibility protein homologues) as well as non-secreted peptides outside the plant kingdom (e.g., the alpha-amanitin toxin gene family in the mushroom, Amanita bisporigera)., Conclusions: SPADA is a free software tool that accurately identifies and predicts the gene structure for short peptides with one or two exons. SPADA is able to incorporate information from profile alignments into the model prediction process and makes use of it to score different candidate models. SPADA achieves high sensitivity and specificity in predicting small plant peptides such as the cysteine-rich peptide families. A systematic application of SPADA to other classes of small peptides by research communities will greatly improve the genome annotation of different protein families in public genome databases.

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.

Amatoxins, including α-amanitin, are bicyclic octapeptides found in mushrooms (Agaricomycetes, Agaricales) of certain species in the genera Amanita, Galerina, Lepiota, and Conocybe. Amatoxins and the chemically similar phallotoxins are synthesized on ribosomes in Amanita bisporigera, Amanita phalloides, and Amanita ocreata. In order to determine if amatoxins are synthesized by a similar mechanism in another, distantly related mushroom, we obtained genome survey sequence data from a monokaryotic isolate of Galerinamarginata, which produces α-amanitin. The genome of G. marginata contains two copies of the α-amanitin gene (GmAMA1-1 and GmAMA1-2). The α-amanitin proprotein sequences of G. marginata (35 amino acids) are highly divergent from AMA1 of A. bisporigera except for the toxin region itself (IWGIGCNP in single-letter amino acid code) and the amino acids immediately upstream (N[A/S]TRLP). G. marginata does not contain any related toxin-encoding sequences besides GmAMA1-1 and GmAMA1-2. DNA from two other α-amanitin-producing isolates of Galerina (G. badipes and G. venenata) hybridized to GmAMA1, whereas DNA from the toxin non-producing species Galerinahybrida did not. Expression of the GmAMA1 genes was induced by growth on low carbon. RNASeq evidence indicates that both copies of GmAMA1 are expressed approximately equally. A prolyl oligopeptidase (POP) is strongly implicated in processing of the cyclic peptide toxins of A. bisporigera and Conocybe apala. G. marginata has two predicted POP genes; one, like AbPOPB of A. bisporigera, is present only in the toxin-producing isolates of Galerina and the other, like AbPOPA of A. bisporigera, is present in all species. Our results indicate that G.marginata biosynthesizes amatoxins on ribosomes by a pathway similar to Amanita species, involving a genetically encoded proprotein of 35 amino acids that is post-translationally processed by a POP. However, due to the high degree of divergence, the evolutionary relationship between AMA1 in the genera Amanita and Galerina is unclear., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

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.

The cyclic peptide toxins of poisonous Amanita mushrooms are chemically unique among known natural products. Furthermore, they differ from other fungal cyclic peptides in being synthesized on ribosomes instead of by nonribosomal peptide synthetases. Because of their novel structures and biogenic origins, elucidation of the biosynthetic pathway of the Amanita cyclic peptides presents both challenges and opportunities. In particular, a full understanding of the pathway should lead to the ability to direct synthesis of a large number of novel cyclic peptides based on the Amanita toxin scaffold by genetic engineering of the encoding genes. Here, we highlight some of the principal methods for working with the Amanita cyclic peptides and the known steps in their biosynthesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

A DNA-based macroarray was designed to quickly and accurately identify certain Amanita mushroom specimens at the species level. The macroarray included probes for Amanita phalloides and Amanita ocreata, toxic species responsible for most mushroom poisonings, and Amanita lanei and Amanita velosa, edible species sometimes confused with toxic species, based on sequences of the highly variable internal transcribed spacer (ITS) region of rDNA. A cryptic species related to A. ocreata and one related to A. lanei, identifiable by ITS sequences, were also included. Specific multiple oligonucleotide probes were spotted onto nylon membranes and the optimal hybridization temperatures were determined. The Amanita DNA array was highly specific, sensitive (0.5 ng DNA/μL and higher were detected), and reproducible. In two case studies, the method proved useful when only small amounts of mushroom tissue remained after a suspected poisoning. An identification could be completed in 12 h., (© 2011 American Academy of Forensic Sciences.)

Metallothioneins (MTs) are cysteine-rich peptides involved in heavy metal tolerance of many eukaryotes. Here, we examined their involvement in intracellular binding of silver (Ag) in the ectomycorrhizal fungus Amanita strobiliformis. The Ag complexes and their peptide ligands were characterized using chromatography and mass spectrometry. The full-length coding sequences obtained from a cDNA library were used for complementation assays in yeast mutant strains. Abundance of respective transcripts in A. strobiliformis was measured by quantitative real-time reverse-transcribed polymerase chain reaction (qRT-PCR). Ag-speciation analyses showed that intracellular Ag was in wild-grown fruit bodies and cultured extraradical mycelia of A. strobiliformis sequestered by metallothioneins. The determined sequence of the peptide facilitated isolation of three cDNA clones, AsMT1a, AsMT1b and AsMT1c. These encode isomorphic MTs consisting of 34 amino acid residues and sharing 82% identity. In mycelia the expression of AsMT1s is induced by Ag. All AsMT1s expressed in yeasts complemented hypersensitivity of mutants to cadmium (Cd) and copper (Cu) and formed Ag complexes. Only the Ag-AsMT1a complex was detected in the A. strobiliformis fruit body in which AsMT1a was the prevailing transcript. The present study identified the existence of metallothionein isoforms in ectomycorrhizal fungi. We demonstrated that intracellular sequestration of Ag in fruit bodies and mycelia of hyperaccumulating A. strobiliformis is dominated by metallothioneins., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

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.