Phylogenetic and taxonomic updates of Agaricales, with an emphasis on Tricholomopsis.

The order Agaricales was divided into eight suborders. However, the phylogenetic relationships among some suborders are largely unresolved, and the phylogenetic positions and delimitations of some taxa, such as Sarcomyxaceae and Tricholomopsis, remain unsettled. In this study, sequence data of 38 genomes were generated through genome skimming on an Illumina sequencing system. To anchor the systematic position of Sarcomyxaceae and Tricholomopsis, a phylogenetic analysis based on 555 single-copy orthologous genes from the aforementioned genomes and 126 publicly accessible genomes was performed. The results fully supported the clustering of Tricholomopsis with Phyllotopsis and Pleurocybella within Phyllotopsidaceae, which formed a divergent monophyletic major lineage together with Pterulaceae, Radulomycetaceae, and Macrotyphula in Agaricales. The analysis also revealed that Sarcomyxaceae formed a unique major clade. Therefore, two new suborders, Phyllotopsidineae and Sarcomyxineae, are proposed for the two major lineages. Analyses of 450 single-copy orthologous genes and four loci suggested that Tricholomopsis consisted of at least four clades. Tricholomopsis is subsequently subdivided into four distinct sections. Seventeen Tricholomopsis species in China, including six new species, are reported. Conoloma is established to accommodate T. mucronata. The substrate preference of Tricholomopsis species and the transitions of the pileate ornamentations among the species within the genus are discussed.

The phosphate starvation response regulator PHR2 antagonizes arbuscule maintenance in Medicago.

Phosphate starvation response (PHR) transcription factors play essential roles in regulating phosphate uptake in plants through binding to the P1BS cis-element in the promoter of phosphate starvation response genes. Recently, PHRs were also shown to positively regulate arbuscular mycorrhizal colonization in rice and lotus by controlling the expression of many symbiotic genes. However, their role in arbuscule development has remained unclear. In Medicago, we previously showed that arbuscule degradation is controlled by two SPX proteins that are highly expressed in arbuscule-containing cells. Since SPX proteins bind to PHRs and repress their activity in a phosphate-dependent manner, we investigated whether arbuscule maintenance is also regulated by PHR. Here, we show that PHR2 is a major regulator of the phosphate starvation response in Medicago. Knockout of phr2 showed reduced phosphate starvation response, symbiotic gene expression, and fungal colonization levels. However, the arbuscules that formed showed less degradation, suggesting a negative role for PHR2 in arbuscule maintenance. This was supported by the observation that overexpression of PHR2 led to enhanced degradation of arbuscules. Although many arbuscule-induced genes contain P1BS elements in their promoters, we found that the P1BS cis-elements in the promoter of the symbiotic phosphate transporter PT4 are not required for arbuscule-containing cell expression. Since both PHR2 and SPX1/3 negatively affect arbuscule maintenance, our results indicate that they control arbuscule maintenance partly via different mechanisms. While PHR2 potentiates symbiotic gene expression and colonization, its activity in arbuscule-containing cells needs to be tightly controlled to maintain a successful symbiosis in Medicago.

Mushroom Poisoning Outbreaks - China, 2023.

What is already known about this topic?: Mushroom poisoning poses a significant food safety concern in China, with a total of 196 species identified in poisoning incidents by the end of 2022. What is added by this report?: In 2023, the China CDC conducted an investigation into 505 cases of mushroom poisoning spanning 24 provincial-level administrative divisions. This investigation resulted in 1,303 patients and 16 deaths, yielding a case fatality rate of 1.23%. A total of 97 mushrooms were identified as the cause of 6 distinct clinical disease types, with 12 species newly documented as poisonous mushrooms in China. What are the implications for public health practice?: Close collaboration among CDC staff, physicians, and mycologists remains crucial for the control and prevention of mushroom poisoning in the future.

Three new species of Cortinarius section Delibuti (Cortinariaceae, Agaricales) from China.

Three new species of CortinariussectionDelibuti, namely C.fibrillososalor, C.pseudosalor, and C.subtropicus are described as new to science based on morphological and phylogenetic evidences. Cortinariuspseudosalor is extremely morphologically similar to C.salor, but it differs from the latter by smaller coarsely verrucose basidiospores. Cortinariusfibrillososalor can be easily differentiated by its fibrillose pileus. The pileus of C.subtropicus becomes brown without lilac tint at maturity comparing with other members of section Delibuti. A combined dataset of ITS and LSU sequences was used for phylogenetic analysis. The phylogenetic reconstruction of section Delibuti revealed that these three new species clustered and formed independent lineages with full support respectively. A key to the three new species and related species of section Delibuti is provided in this work.

Genome-centric metagenomics provides new insights into metabolic pathways of polyhydroxyalkanoates biosynthesis and functional microorganisms subsisting on municipal organic wastes.

The microbial community of a sequencing batch reactor operated under feast and famine conditions for production of polyhydroxyalkanoates (PHAs) was characterized through high-throughput sequencing and metagenomic analysis. The fermented food waste and chemically-enhanced primary sludge was fed in this bioreactor. After acclimation, the PHA yield achieved as high as 0.60-0.69 g CODPHA/g CODS. The complete changes of microbial community structure were found during shifts of feedstock. A synthesis of SCL/MCL-PHAs pathway was established for PHA-producing bioreactor in this mixed-culture system. The structure-performance relationship of PHA-producing microbial community and feedstock composition was investigated. The results showed that microbial community tends to be decentralized and prefer team work for PHA synthesis to consume the multiple substrates and digest inevitable non-VFA contents in fermented liquor. This study also discovered unreported potential PHA producers (e.g., genera Tabrizicola, Nannocystis, Ga0077539, Ga0077559, JOSHI-001, SNC69-320 and UBA2334) subsisting on municipal organic wastes and expands the current knowledge about mixed-culture system that the PHA synthesis pathway is widely existed in activated sludge.

Determination of ibotenic acid and muscimol in species of the genus Amanita section Amanita from China.

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.

Large-scale phage cultivation for commensal human gut bacteria.

Phages are highly abundant in the human gut, yet most of them remain uncultured. Here, we present a gut phage isolate collection (GPIC) containing 209 phages for 42 commensal human gut bacterial species. Genome analysis of the phages identified 34 undescribed genera. We discovered 22 phages from the Salasmaviridae family that have small genomes (∼10-20 kbp) and infect Gram-positive bacteria. Two phages from a candidate family, Paboviridae, with high prevalence in the human gut were also identified. Infection assays showed that Bacteroides and Parabacteroides phages are specific to a bacterial species, and strains of the same species also exhibit substantial variations in phage susceptibility. A cocktail of 8 phages with a broad host range for Bacteroides fragilis strains effectively reduced their abundance in complex host-derived communities in vitro. Our study expands the diversity of cultured human gut bacterial phages and provides a valuable resource for human microbiome engineering.

Updated taxonomy of Chinese Cantharellus subgenera Afrocantharellus and Magni (Hydnaceae, Cantharellales): Three new taxa and amended descriptions of one previous species.

Cantharellus, one of the main genera of Hydnaceae (Cantharellales), is both ecologically and economically important. Although many studies have focused on this genus in China, the taxonomy should be further updated. In the present study, Cantharellus subgenera Afrocantharellus and Magni were investigated based on morphology and molecular phylogenetic analyses with new collections from China. Five phylogenetic species were recognized among the studied collections, three of which were described as new: C. bellus, C. cineraceus, and C. laevigatus; one was previously described taxon: C. hygrophoroides; and the remaining species was not defined due to the paucity of the materials. Among the four described species, both C. bellus and C. laevigatus are members of subgen. Magni, whereas C. cineraceus and C. hygrophoroides belong to subgen. Afrocantharellus.

Five new species of Inosperma from China: Morphological characteristics, phylogenetic analyses, and toxin detection.

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.

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 Contribution to Knowledge of Craterellus (Hydnaceae, Cantharellales) in China: Three New Taxa and Amended Descriptions of Two Previous Species.

Species of Craterellus (Hydnaceae, Cantharellales) in China are investigated on the basis of morphological and molecular phylogenetic analyses of DNA sequences from nuc 28S rDNA D1-D2 domains (28S) and nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 region. Five species are recognized in China, of which three of them are described as new, viz. C. fulviceps, C. minor, and C. parvopullus, while two of them are previously described taxa, viz. C. aureus, and C. lutescens. A key to the known Chinese taxa of the genus is also provided.

Morphological and Phylogenetic Evidences Reveal Four New Species of Cantharellus Subgenus Cantharellus (Hydnaceae, Cantharellales) From China.

Species of Cantharellus subgenus Cantharellus are interesting and important for their mycorrhizal properties, medicinal values, and edibility. In China, there are many undescribed species of the subgenus. In this study, four new species of subg. Cantharellus, viz. Cantharellus albopileatus, Cantharellus chuiweifanii, Cantharellus pinetorus, and Cantharellus ravus from Hainan and Hunan Provinces, respectively, were described based on morphological and phylogenetic evidence as a contribution to the knowledge of the species diversity in China. Detailed descriptions, color photographs of fresh basidiomata, and line drawings of microstructures of these four new species are presented as well as comparisons with related species.

Differential Expression of Amanitin Biosynthetic Genes and Novel Cyclic Peptides in Amanita molliuscula.

Amanita molliuscula is a basal species of lethal Amanita and intrigues the field because it does not produce discernable α-amanitin when inspected by High Performance Liquid Chromatography (HPLC), which sets it apart from all known amanitin-producing (lethal) Amanita species. In order to study the underlining genetic basis of the phenotype, we sequenced this species through PacBio and Illumina RNA-Seq platforms. In total, 17 genes of the "MSDIN" family (named after the first five amino acid residues of the precursor peptides) were found in the genome and 11 of them were expressed at the transcription level. The expression pattern was not even but in a differential fashion: two of the MSDINs were highly expressed (FPKM value > 100), while the majority were expressed at low levels (FPKM value < 1). Prolyl oligopeptidease B (POPB) is the key enzyme in the amanitin biosynthetic pathway, and high expression of this enzyme was also discovered (FPKM value > 100). The two MSDINs with highest transcription further translated into two novel cyclic peptides, the structure of which is distinctive from all known cyclic peptides. The result illustrates the correlation between the expression and the final peptide products. In contrast to previous HPLC result, the genome of A. molliuscula harbors α-amanitin genes (three copies), but the product was in trace amount indicated by MS. Overall, transcription of MSDINs encoding major toxins (α-amanitin, ß-amanitin, phallacidin and phalloidin) were low, showing that these toxins were not actively synthesized at the stage. Collectively, our results indicate that the amanitin biosynthetic pathway is highly active at the mature fruiting body stage in A. molliuscula, and due to the differential expression of MSDIN genes, the pathway produces only a few cyclic peptides at the time.

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.

Behavioural and chronic toxicity of fullerene to Daphnia magna: Mechanisms revealed by transcriptomic analysis.

Extensive application of fullerene nanoparticles (nC60) leads to potential environmental pollution. The acute toxic effects of nC60 have been largely investigated, but studies of behavioural and chronic toxicity at sublethal doses are still rare and the underlying molecular mechanisms remain unknown. The present study investigated behavioural and chronic toxicity of nC60 to Daphnia magna. The results showed that, in response to nC60 exposure, hopping, heartbeat frequencies and feeding ability of D. magna decreased significantly, displaying negative relationship with exposure time and dose. Chronic treatments with 0.1 mg/L or 1 mg/L nC60 for 21 days significantly reduced survival and reproduction of D. magna. These harmful effects suggested negative impacts of nC60 on aquatic ecosystems. Moreover, transcriptome analysis showed that the behavioural and chronic toxicity of nC60 to D. magna might be related to physiological functions such as cell structural repair, protein degradation, energy metabolism and reproduction. We found that nC60 accumulated in guts of D. magna, which should be responsible for the decrease of food ingestion and consequently inhibiting energy intake. Deficiency of energy not only affects behaviours but also declines reproduction in D. magna. Overall, this is the first study comprehensively considered the behavioral and chronic toxicity of nC60 to aquatic organism. The results should be helpful to better understand the ecological consequences of C60 released into water environments.

Alleviative Effects of C60 on the Trophic Transfer of Cadmium along the Food Chain in Aquatic Environment.

C60 could enhance the accumulation of pollutants in organisms, but their effects on higher trophic levels remain unknown. In the present study, the transfer of C60 from Daphnia magna to zebrafish (Danio rerio) and its effects on Cd transfer were investigated. The results showed that C60 could be transferred from D. magna to zebrafish through dietary exposure and accumulate mainly in the intestines, but biomagnification was not observed. The presence of C60 promoted accumulation of Cd in D. magna. However, it decreased Cd burden in the higher trophic level (zebrafish), displaying an alleviative effect on the trophic transfer of Cd along the food chain. To explore the underlying mechanisms, the release of Cd from D. magna in digestive fluids and changes in zebrafish digestive physiology were further investigated. The results showed that C60 did not inhibit Cd release from D. magna, but stimulated the digestive tracts of zebrafish to excrete Cd earlier and in a greater amount, which consequently lowered assimilation efficiency of Cd in zebrafish. Overall, the present study showed the trophic transfer of C60 in the aquatic food chain and revealed the effects of C60 on trophic transfer of Cd along the food chain in aquatic environment.

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.