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.

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 nonduplicated cyclopeptides, including α-amanitin, β-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.

Many species of Inosperma cause neurotoxic poisoning in humans after consumption around the world. However, the toxic species of Inosperma and its toxin content remain unclear. In the present study, we proposed five new Inosperma species from China, namely, I. longisporum, I. nivalellum, I. sphaerobulbosum, I. squamulosobrunneum, and I. squamulosohinnuleum. Morphological and molecular phylogenetic analyses based on three genes (ITS, nrLSU, rpb2) revealed that these taxa are independent species. A key to 17 species of Inosperma in China is provided. In addition, targeted screening for the most notorious mushroom neurotoxins, muscarine, psilocybin, ibotenic acid, and muscimol, in these five new species was performed by using ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Our results show that the neurotoxin contents in these five species varied: I. sphaerobulbosum contains none of the tested neurotoxins; I. nivalellum is muscarine positive; I. longisporum and I. squamulosohinnuleum contain both ibotenic acid and muscimol, and I. squamulosobrunneum only contains muscimol; psilocybin was not detected in these five new species.

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 gened to encode 98 non-duplicated cyclopeptides, including α- amanitin, β-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.

No abstract

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.

Background Amanitin-producing mushrooms, mainly distributed in the genera Amanita , Galerina and Lepiota , possess MSDIN gene family for the biosynthesis of many cyclopeptides catalyzed by prolyl oligopeptidase (POP). Recently, transcriptome sequencing has proven to be a efficient way to mine MSDIN and POP genes in these lethal mushrooms. Until now, only A . palloides and A. bisporigera from North America and A . exitialis from Asia have been studied based on transcriptome analysis. However, MSDIN and POP genes of many amanitin-producing mushrooms in China remain unstudied, and 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 analyzed. Through transcriptome sequencing and PCR cloning, 19 POP genes and 151 MSDIN genes predicted to encode 98 non-duplicated cyclopeptides, including α-amanitin, β-amanitin, phallacidin, phalloidin and 94 unknown peptides, were found in these species. Phylogenetic analysis showed that toxin peptide genes were clustered depending on the chemical substance within genus while depending on the taxonomy between genus and that the POPA genes of Amanita , Galerina and Lepiota were clustered and separated in three different groups, but the POPB genes of the three distinct genera were clustered in a highly monophyletic group. Conclusions These results above 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 horizontal gene transfer might occur among the three genera.

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 β-amanitin (β-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, β-AMA concentrations in L. brunneoincarnata were 0.57–0.94 mg/g dry weight, and β-AMA was absent in L. venenata .