Tai-Hui Li

Illumina-based de novo transcriptome sequencing and analysis of Amanita exitialis basidiocarps.

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.

Peptide toxin components of Amanita exitialis basidiocarps

Eight peptide toxins were isolated and purified from basidiocarps of Amanita exitialis with high performance liquid chromatography and were subjected to ultraviolet, nuclear magnetic resonance and mass spectrometry. We identified seven peptide toxins, α-amanitin, β-amanitin, amaninamide, phallacin, phallacidin, phallisacin and desoxoviroidin. The molecular weight (729.5 Da) of the eighth compound did not match that of any reported Amanita toxins and, although the UV absorption spectrum indicated it to be a phallotoxin, further studies are required to identify this component. This is the first report of amaninamide, phallacin, phallisacin and desoxoviroidin in this lethal mushroom species.

The molecular diversity of toxin gene families in lethal Amanita mushrooms.

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.

Anti-fatigue property of Cordyceps guangdongensis and the underlying mechanisms

Abstract Context: Cordyceps guangdongensis T.H. Li, Q.Y. Lin & B. Song (Cordycipitaceae) is a nontoxic folk medicine and can be cultivated, with noticeable effects of anti-H9N2, life-prolonging and treating chronic renal failure. Objective: The anti-fatigue effect of C. guangdongensis, possible mechanism and active constituent were investigated. Materials and methods: Treatment mice were treated with C. guangdongensis powder (0.455, 0.91 and 1.82 g/kg bw daily for low, middle and high doses, respectively); treatment rats were fed, respectively, with ethanol, petroleum ether, ethyl acetate, n-butanol, aqueous phase and hot water extract fractions, for 30 d. Forced swimming time to exhaustion, blood urea nitrogen (BUN) and hepatic glycogen (HG) levels of mice and blood lactic acid (BLC) levels of rats were determined. Results: The swimming times to exhaustion of mice were very significantly (p < 0.01) longer in low-, middle- and high-dose groups (respectively 1.87-, 1.94- and 1.88-times), and significantly (p < 0.05) longer in the n-butanol fraction group (1.52-times), hot water extract group (1.88- times) and refined polysaccharide group (2.66-times) than in blank control; the BLC levels of rats were significantly (p < 0.05) lower in the ethyl alcohol partition group (84.8%), the n-butanol fraction group (84.0%) and the hot water extract group (84.4 %) than in blank. The BUN and HG levels were not significantly different. Discussion and conclusion: Cordyceps guangdongensis can potently alleviate fatigue through reducing the accumulation of BLC; a functional constituent was the refined polysaccharide. This might become a new functional food for fatigue resistance.

Phylogeny of Entoloma s.l. subgenus Pouzarella, with descriptions of five new species from China

Taxonomy and phylogeny of Entoloma s.l. subgenus Pouzarella in China are carried out based on morphological characters and molecular data. Seven species in subg. Pouzarella are recognized in China and a key to these species is provided. Five species, Entoloma changchunense, E. crassicystidiatum, E. furfuraceum, E. subaraneosum and E. tenuissimum, are described as new; a new Chinese record E. dysthaloides is also presented in this paper. Among them, E. crassicystidiatum was discovered from southern China, and the others were found in northeastern China. Molecular phylogeny of subg. Pouzarella were conducted based on nLSU, mtSSU and RPB2, and the phylogenetic positions of the five new species are discussed. The placement of subg. Pouzarella in Entoloma s.l. clade is well resolved in all analyses presented here, and the monophyly of subg. Pouzarella is strongly supported by the morphological and molecular phylogeny. The infrageneric classifications in this group were not well recovered in the present study; and further studies based on more samples and gene loci are needed to a better understanding of the infrageneric taxa in subg. Pouzarella in the future.

Safety assessment of Cordyceps guangdongensis

Cordyceps guangdongensis as a kind of fungus, has been discovered and cultivated successfully in recent years. However, its safety assessments have not been studied. In this report, a serial of tests for toxicological safety assessments were depicted in details. These tests included bacterial reverse mutation (Ames) study, bone marrow cell micronucleus test in mice, sperm aberration test in mice, teratogenicaction test in rats, acute toxicity test and 13-week oral toxicity study in rats. After a profound analysis of these tests, it clearly demonstrated that C. guangdongensis did not have any mutagenic, clastogenic nor genotoxic effects; the oral LD50 of the biomass in rats was greater than 15 g/kg body weight; the no-observed adverse-effect-levels (NOAEL) was 5.33 g/kg body weight according to the 13-week oral toxicity analysis. Therefore, a conclusion can be drawn that C. guangdongensis is considered safe for long term consumption.

A new species of Amanita section Lepidella from South China

Amanita macrocarpa from Guangdong Province of China is described. The new species is a large, brownish orange to light brown mushroom, characterized by numerous pyramidal warts on the pileus, yellowish lamellae, a large annulus at the middle of the stipe, and amyloid ellipsoidal basidiospores. Phylogenetic analyses of the new species and related taxa based on the large subunit of nuclear ribosomal DNA (LSU) and the internal transcribed spacer (ITS) sequences are provided. Morphological and molecular data show that A. macrocarpa is a member of Amanita, section Lepidella, and clearly different from any known taxon of the section.

Four new species of Entoloma s.l. (Agaricales) from southern China

Four new Entoloma s.l. species (E. azureosquamulosum, E. caeruleoflavum, E. hainanense, and E. subtenuicystidiatum) are described from southern China. E. azureosquamulosum fits well within the section Rhamphocystotae, E. caeruleoflavum belongs to the section Entoloma, E. hainanense is placed in the section Calliderma, and E. subtenuicystidiatum is a species in the section Cyanula. Preliminary phylogenetic analyses for the four new species are provided based on ITS and LSU sequences in this paper.

Amanita cinereovelata , a new species of Amanita section Lepidella from Bangladesh

Amanita cinereovelata is described and illustrated as a new species of Amanita sect. Lepidella from Bangladesh. The species is characterized by its small to medium-sized basidiomata, gray to lilac-gray pulverulent-floccose volval remnants on the pileus, a bulbous stipe base, globose to subglobose amyloid basidiospores, the presence of clamps at the bases of basidia, and an association with Shorea robusta. Molecular phylogenetic analysis indicates that A. cinereovelata is a species sister to A. eriophora, A. longipes, A. tephrea and A. vestita. Comparisons with morphologically similar taxa are provided.

Molecular cloning of α-amanitin and characterization of its expression pattern in different parts and development stages of Amanita exitialis fruitbody

Amanita exitialis is a lethal mushroom in East Asia. α-Amanitin is one kind of the leading peptide toxins responsible for mushroom poisoning, which are mainly from the fatal amanitae. In the study, the full-length cDNA sequences of α-amanitin (α-AMA) and phallacidin (PHA) from A. exitialis were cloned and analyzed; in addition, the expression patterns of α-AMA in three parts as well as four developmental stages of the fruitbody were first characterized by quantitative real-time PCR. Results demonstrate that α-AMA and PHA were highly homologous to the toxin genes reported from A. bisporigera; the α-AMA could be expressed in all parts and stages of the fruitbody, from late elongation stage to late mature stage, α-AMA showed the highest expression level in the pileus, followed by the stipe and volva in sequence, while at the early elongation stage, the highest expression level was found in the stipe, followed by the pileus and volva. Further analysis revealed that the higher expression level of α-AMA was closely associated with the development stages, and the expression of α-AMA was accumulated in the more vigorous growth parts and stages, from which could be inferred that α-AMA might play an important role in the development of A. exitialis.