Three coralloid species of the genus Trechispora (Trechisporales, Basidiomycota) in China: two newly discovered taxa and one reported for the first time.

Two new species of Trechispora indigenous to southern China, T.laxa and T.tongdaoensis, are described and illustrated, and the first record of T.khokpasiensis in China is reported. Molecular phylogenetic analyses of the concatenated nuclear rDNA ITS1-5.8S-ITS2 and nuclear large subunit sequences supported the inclusion of the three species within the Trechispora clade, together with species formerly classified in Scytinopogon. The new species are similar in micromorphology to species of Trechispora (as traditionally circumscribed) but are distinguished by having coralloid basidiomata. A key to the known coralloid Trechispora species in China is provided.

A contribution to the knowledge of the genus Infundibulicybe (Tricholomatineae, Agaricales) in China: Two new species and five redescribed taxa.

In China, species of the genus Infundibulicybe are often confused with other taxa and misdetermined. Here we describe two newly discovered species, I. bispora and I. ellipsospora, and redescribe five known taxa of this genus present in China. These identifications are supported by both morphological and DNA-based phylogenetic evidence. Additionally, a key to all known species of Infundibulicybe is provided.

Development of a loop-mediated isothermal amplification assay for the rapid detection of Russula subnigricans and Russula japonica.

Russula subnigricans is the only deadly species in the genus Russula with a mortality rate of more than 50%, and Russula japonica is the most common poisonous species, making rapid species identification in mushroom poisoning incidents extremely important. The main objective of this study was to develop a rapid, specific, sensitive, and simple loop-mediated isothermal amplification (LAMP) assay for the detection of R. subnigricans and R. japonica. Two sets of species-specific LAMP primers targeting internal transcribed spacer (ITS) regions were designed to identify R. subnigricans and R. japonica. The results demonstrated that while LAMP could specifically detect R. subnigricans and R. japonica, the polymerase chain reaction (PCR) could not distinguish R. subnigricans from Russula nigricans. In addition, the results demonstrated that, compared to electrophoresis-LAMP and real-time quantitative LAMP (RT-qLAMP), the detection sensitivity of HNB-LAMP (a mixture of LAMP with hydroxy naphthol blue (HNB) dye) for R. subnigricans could reach 0.5 pg/µl and was 100-fold higher than that of PCR. The LAMP reaction could be completed in 45 min, which is much faster than the conventional PCR. In the future, LAMP can be used a quick, specific, and sensitive detection tool in various fields.

A new clitocyboid genus Spodocybe and a new subfamily Cuphophylloideae in the family Hygrophoraceae (Agaricales).

Phylogenetically, the genera Cuphophyllus, Ampulloclitocybe and Cantharocybe are treated as basal in the family Hygrophoraceae, despite weak support. However, the exact phylogenetic positions of the three genera have remained unresolved, and taxa related to these genera are poorly known. In this study, a new clitocyboid genus Spodocybe was proposed based on multigenic phylogenetic inference datasets and morphological evidence. The analyses of ITS as well as two combined datasets ITS-nrLSU-rpb2 and ITS-nrLSU-rpb1-rpb2-tef1-α-atp6 supported that (1) Spodocybe formed a well-supported monophyletic clade; and (2) sisters Spodocybe and Ampulloclitocybe, along with Cantharocybe and Cuphophyllus also formed a monophyletic lineage, as sister to the rest of the Hygrophoraceae. Meanwhile, two new species, namely S. rugosiceps and S. bispora, from southwestern China, were documented and illustrated. These results support the new proposed genus Spodocybe, and that Spodocybe, Ampulloclitocybe, Cantharocybe and Cuphophyllus should be retained in the Hygrophoraceae as a new subfamily Cuphophylloideae.

Diversity of MSDIN family members in amanitin-producing mushrooms and the phylogeny of the MSDIN and prolyl oligopeptidase genes.

BACKGROUND: Amanitin-producing mushrooms, mainly distributed in the genera Amanita, Galerina and Lepiota, possess MSDIN gene family for the biosynthesis of many cyclopeptides catalysed by prolyl oligopeptidase (POP). Recently, transcriptome sequencing has proven to be an efficient way to mine MSDIN and POP genes in these lethal mushrooms. Thus far, only A. palloides and A. bisporigera from North America and A. exitialis and A. rimosa from Asia have been studied based on transcriptome analysis. However, the MSDIN and POP genes of many amanitin-producing mushrooms in China remain unstudied; hence, the transcriptomes of these speices deserve to be analysed. RESULTS: In this study, the MSDIN and POP genes from ten Amanita species, two Galerina species and Lepiota venenata were studied and the phylogenetic relationships of their MSDIN and POP genes were analysed. Through transcriptome sequencing and PCR cloning, 19 POP genes and 151 MSDIN genes predicted to encode 98 non-duplicated cyclopeptides, including α-amanitin, ß-amanitin, phallacidin, phalloidin and 94 unknown peptides, were found in these species. Phylogenetic analysis showed that (1) MSDIN genes generally clustered depending on the taxonomy of the genus, while Amanita MSDIN genes clustered depending on the chemical substance; and (2) the POPA genes of Amanita, Galerina and Lepiota clustered and were separated into three different groups, but the POPB genes of the three distinct genera were clustered in a highly supported monophyletic group. CONCLUSIONS: These results indicate that lethal Amanita species have the genetic capacity to produce numerous cyclopeptides, most of which are unknown, while lethal Galerina and Lepiota species seem to only have the genetic capacity to produce α-amanitin. Additionally, the POPB phylogeny of Amanita, Galerina and Lepiota conflicts with the taxonomic status of the three genera, suggesting that underlying horizontal gene transfer has occurred among these three genera.

Determination of Amatoxins in Lepiota brunneoincarnata and Lepiota venenata by High-Performance Liquid Chromatography Coupled with Mass Spectrometry.

Three hepatic failure poisoning incidents caused by Lepiota brunneoincarnata and Lepiota venenata mushrooms have been occurred in China in 2017, L. venenata has been described as a new species. However, the cyclopeptide toxins of these lethal mushrooms remain poorly understood. In this study, the composition and content of amatoxins in L. brunneoincarnata and L. venenata are analyzed and compared, the analysis of composition and content of amatoxins in L. venenata are reported for the first time. The results showed that ß-amanitin (ß-AMA), α-amanitin (α-AMA), amanin, and amaninamide were identified in L. brunneoincarnata, and α-AMA, amanin II (an analog of amanin), and an unknown compound were identified in L. venenata. The differences between L. brunneoincarnata and L. venenata in the identified compounds provide chemical evidence for L. venenata as a new species. Quantitative analysis shows that α-AMA concentrations in L. brunneoincarnata and L. venenata were 0.72-1.97 mg/g dry weight, ß-AMA concentrations in L. brunneoincarnata were 0.57-0.94 mg/g dry weight, and ß-AMA was absent in L. venenata.

Development and Evaluation of Isothermal Amplification Methods for Rapid Detection of Lethal Amanita Species.

In the present work, loop-mediated isothermal amplification (LAMP) and hyperbranched rolling circle amplification (HRCA) methods were developed to detect and distinguish different lethal Amanita species. Specific LAMP primers and HRCA padlock probes for species-specific identification and a set of universal LAMP primers for lethal Amanita species were designed and tested. The results indicated that the LAMP-based assay was able to discriminate introclade lethal Amanita species but was not able to discriminate intraclade species perfectly, while the HRCA-based assay could discriminate whether introclade or intraclade species. The universal LAMP primers were positive for 10 lethal species of Amanita section Phalloideae and negative for 16 species of Amanita outside section Phalloideae. The detection limits of LMAP and HRCA were 10 and 1 pg of genomic DNA per reaction, respectively. In conclusion, the two methods could be rapid, specific, sensitive and low-cost tools for the identification of lethal Amanita species.

Universal identification of lethal amanitas by using Hyperbranched rolling circle amplification based on α-amanitin gene sequences.

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 ß-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.

Cloning and analysis of Ophiocordyceps xuefengensis mating type (MAT) loci.

The entomopathogenic fungus Ophiocordyceps xuefengensis, a recently described species and identified as the sister taxon of Ophiocordyceps sinensis, is a desirable alternative to O. sinensis. The mating systems of fungi play a vitally important role in the regulation of sexual reproduction and evolution, but the mating type loci of O. xuefengensis were completely unknown. In this study, the mating systems of O. xuefengensis were analyzed. The conserved α-box region of the MAT1-1-1 and HMG-box of MAT1-2-1 were successfully obtained by PCR amplification. The distribution of both mating types in different tissues of wild and cultivated O. xuefengensis growth was detected and analyzed. The results showed that the asci always harbored both mating types, whereas the sclerotium, the stipe and each isolated strain of wild O. xuefengensis always had only one idiomorph, either MAT1-1 or MAT1-2, which confirmed that O. xuefengensis is heterothallic. The MAT1-1 locus of O. xuefengensis harbors MAT1-1-1, MAT1-1-2 and MAT1-1-3, and MAT1-2 contains the MAT1-2-1 gene. Southern blot analysis showed the MAT-1-1-1 and MAT-1-2-1 genes were single-copy in O. xuefengensis. These results will help to understand its life cycle and support artificial cultivation of O. xuefengensis.

Determination of the Main Nucleosides and Nucleobases in Natural and Cultured Ophiocordyceps xuefengensis.

Ophiocordyceps xuefengensis, a recently described species of Ophiocordycepsthat is associated with the larvae of Phassusnodus (Hepialidae) in the living root or trunk of the medicinal plant Clerodendrumcyrtophyllum, isthe largest known Cordycepsspecies and is recognized as a desirable alternative for natural Ophiocordycepssinensis. This study investigated the main nucleosides and nucleobases in natural and cultured Ophiocordycepsxuefengensis. The contents of the nucleosides and nucleobases in the natural and cultured samples were determined by reverse phase HPLC. The highest concentration of adenosine was found in the natural fruit body and the cultured stroma, with almost no adenosine in the cadaver of Phassusnodus. The contents of adenine, guanosine, uridine and uracil in the cultured mycelium were significantly higher than those in the natural sample. Inosine was only detected in the natural samples. Thymidine and 2-deoxyadenosine were only found in the cadaver of Phassusnodus. Cordycepin was not detected in the five samples examined. These results suggested that the cultured mycelium and cultured stroma of Ophiocordycepsxuefengensis might be a promising substitute for natural O. xuefengensis.

Determination of cyclopeptide toxins in Amanita subpallidorosea and Amanita virosa by high-performance liquid chromatography coupled with high-resolution mass spectrometry.

Amanita subpallidorosea is a recently discovered lethal Amanita sect. Phalloideae species found in China that is clustered with A. virosa in the same clade based on molecular phylogenetic analysis. However, the cyclopeptide toxin contents of these lethal mushrooms remain poorly studied. In this study, the cyclopeptide toxins in A. subpallidorosea were reported for the first time and the cyclopeptide compositions of A. subpallidorosea and A. virosa species were systematically analyzed. Thirteen cyclopeptides and two unknown compounds were identified or observed from these two lethal mushrooms by high-performance liquid chromatography coupled with high-resolution mass spectrometry. Of the known cyclopeptides, the virotoxins alaviroidin, viroisin, and viroidin, which were previously thought to be restricted to A. virosa, were identified in A. subpallidorosea. The cyclopeptide compositions showed that there are diversities in the kinds and levels of amatoxins, phallotoxins, and virotoxins between A. subpallidorosea and A. virosa species, and that the amount of total toxins in the tested A. subpallidorosea is significantly higher than that in the tested A. virosa. Furthermore, consistency of the cyclopeptide toxins with the molecular phylogenetic relationships was demonstrated.

Cyclopeptide toxins of lethal amanitas: Compositions, distribution and phylogenetic implication.

Lethal amanitas (Amanita sect. Phalloideae) are responsible for 90% of all fatal mushroom poisonings. Since 2000, more than ten new lethal Amanita species have been discovered and some of them had caused severe mushroom poisonings in China. However, the contents and distribution of cyclopeptides in these lethal mushrooms remain poorly known. In this study, the diversity of major cyclopeptide toxins in seven Amanita species from Eastern Asia and three species from Europe and North America were systematically analyzed, and a new approach to inferring phylogenetic relationships using cyclopeptide profile was evaluated for the first time. The results showed that there were diversities of the cyclopeptides among lethal Amanita species, and cyclopeptides from Amanita rimosa and Amanita fuligineoides were reported for the first time. The amounts of amatoxins in East Asian Amanita species were significantly higher than those in European and North American species. The analysis of distribution of amatoxins and phallotoxins in various Amanita species demonstrated that the content of phallotoxins was higher than that of amatoxins in Amanita phalloides and Amanita virosa. In contrast, the content of phallotoxins was significantly lower than that of amatoxins in all East Asian lethal Amanita species tested. However, the distribution of amatoxins and phallotoxins in different tissues showed the same tendency. Eight cyclopeptides and three unknown compounds were identified using cyclopeptide standards and high-resolution MS. Based on the cyclopeptide profiles, phylogenetic relationships of lethal amanitas were inferred through a dendrogram generated by UPGMA method. The results showed high similarity to the phylogeny established previously based on the multi-locus DNA sequences.