Microorganisms play important role in the ecosystem. Recent studies have shown that microbial communities have core groups (taxa) that have important implications for host health, growth, and production. Based on MetaCoMET and network co-occurrence methods, the core microbiome analysis of fungi in medicinal Eucommia ulmoides bark collected from Hunan, Sichuan and Guizhou was carried out. The results of MetaCoMET analysis showed that the core microbiome of fungi included 16 taxa at the OTU level. An unclassified fungus of Nectriaceae was dominant, followed by Fusarium pseudensiforme, Cephalothecaceae sp. and Fusarium sp. and so on. Network co-occurrence analysis revealed 11 hub fungal taxa. Although the analysis results of the two methods were not completely coincident, good consistency was displayed for 11 hub fungi. Overall, certain core fungal biome has stability to some extent.
To analyze the yeast composition and obtain the core yeast strains in Maotai fermented grain, the diversity of yeasts and the dominant genera were investigated by high-throughput sequencing method, and the separation of yeast strains were operated by culture method. In total, 59 genera and 129 species of yeasts were detected in fermented grain of Maotai liquor fermentation from the Xiasha (the first deposits of grain) to the fifth rounds of fermentation, and 41 species of yeast strains were isolated. The amount of detected and obtained yeast species in Maotai liquor is the highest in all types of Chinese liquor. There were significant differences in yeast species and quantities from different fermented rounds. Pichia kudriavzevii was the dominant yeast in Xiasha and Zaosha (the second deposits of grain). With the advance of fermented rounds, the dominant yeast species in fermented grain increased, including Pichia kudriavzevii, Pichia manshurica, Zygosaccharomyces bailii, Saccharomyces cerevisiae, and Candida apicola. The analysis of yeast community structure is helpful to understand the mechanism of liquor production and flavor metabolism in Maotai fermented grain, which could help to control the liquor solid-state fermentation processes effectively.
Shiraia bambusicola is an important medicinal fungus in China, which has a wide applications in medicine, agriculture, food and other fields. The technique of widely-targeted metabolomics was used to detect metabolites produced by Shiraia bambusicola at various developmental stages for identifying differential metabolites and performing metabolic pathway analysis. A total of 612 metabolites were identified from Shiraia bambusicola, of which 27 unique metabolites were found in the early and medium developmental stages. Flavonoids, quinic acid, coumarin and other compounds with notable biological activity were detected for the first time. The differential metabolites selected mainly include lipid, amino acids, nucleotide, flavonoids, terpenoids, organic acids, etc. Among them, flavonoids and amino acids were prior to the others. Six significant metabolic pathways were obtained through enrichment analysis. Flavonoids are considered to be important compounds that are related to the medicinal value of Shiraia bambusicola in addition to hypocrellin. This result provides a basis for further study of the medicinal mechanism and active ingredients of Shiraia bambusicola.
A new fungal disease was found in several Lentinula edodes mushroom growing sheds in Qingyuan, Zhejiang Province. Observation of morphological characteristics, phylogenetic analysis using rDNA sequences of ITS and partial 28S rDNA, and confirmation of pathogenicity using Koch’s postulates revealed that the causal agent of the cobweb disease was Cladobotryum dendroides. Biological characterization showed that the optimum growth condition for the pathogen was growing temperature of 25°C, pH value of 5-6 and culture medium using soluble starch as carbon source and beef extract as nitrogen source. PDA was also suitable medium for growth. Light showed certain inhibitory effect on pathogen growth. This is the first report of the pathogen responsible for cobweb disease on Lentinula edodes in China.
Twenty-one characteristics were evaluated and graded for 45 cultivated varieties of Flammulina filiformis, a famous edible and medicinal fungus in China. Gradation of 12 quantitative characteristics was carried out. Of which interrelationship of ten quantitative characteristics and influence of different measuring positions on two quantitative characteristics were further studied. The results showed that all quantitative characteristics are suitable to DUS (distinctness, uniformity and stability) testing traits. Twelve quantitative characteristics could be classified into 3-5 grades separately. Ten quantitative characteristics were significantly correlated with at least one of the other characteristics. The measuring position had obvious effect on the quantitative characteristics of fruit bodies. The stipe becomes thicker from top to bottom and thickened degree varies with different variety. The measurement of stipe diameter should be made at one-third the upper part of stipe to reduce the error. The number of fruit body decreases gradually from bottom to top and decrement degree varies with different variety. The measurement of fruit body quantity should be made at one-third the bottom part of fruit bodies in a single cultivated bottle. The result of clustering analysis based on the morphological characters supported that colony surface pigment, pileus longitudinal section morphology and pileus surface color could be used as grouping characteristics.
In 2018, the alleged cobweb disease was found in Ganoderma lingzhi-growing farm at Jiaohe, Jilin Province. Morphological identification and molecular analysis of ITS DNA sequence proved that the pathogen was Cladobotryum mycophilum. Artificial inoculation confirmed that the pathogen infected fruitbodies of Ganoderma lingzhi. Its mycelia grew favorably under the conditions of 25°C and pH 5 on medium using glucose as carbon source and yeast extract as nitrogen source. Light might inhibit mycelial growth to some extent, while dark stimulated mycelial growth. The results provide a useful reference for effective control of the cobweb disease.
The internal transcribed spacer (ITS), translation elongation factor 1-α (EF1-α) and second largest subunit of RNA polymerase II (RPB2) are commonly used for taxonomic or phylogenetic studies of fungi. Intragenomic variation of ITS has been detected in many fungal groups but few cases on EF1-α and RPB2 have been reported. In this study, the intragenomic variation of ITS, EF1-α and RPB2 in Ganoderma australe was detected using PCR cloning to determine whether intragenomic variation affects accuracy of molecular identification. The results showed that intragenomic variation of ITS, EF1-α and RPB2 was present in different specimens and their heterogeneity level was different. Heterogeneity level of the same gene also varied between individuals. Based on the examined samples and cloned sequences, the intragenomic variation of ITS was up to 2.3%, while that of RPB2 only 0.5%. Intragenomic variation of EF1-α could reach 1.8%. The highest intragenomic variation of ITS and EF1-α might exceed the interspecific difference, and therefore the use of these sequences for phylogenetic analyses and molecular identification of Ganoderma species could be problematic.
The heterologous expression of a dipeptidase (idp)-encoding gene from medicinal basidiomycete Inonotus obliquus using Pichia pastoris expression system is described. The gene contains an encoding region of 1 814bp nucleotides, which contains six introns and encodes 465 amino acids (aa). Bioinformatic analysis indicates that the predicted amino sequence does not contain signal peptide but harbors a transmembrane region (TR) located between 55-77aa. The coding sequences with (idp) and without TR (didp) were amplified and respectively cloned into the secreting expression vector pPICZαA. The constructed plasmids were transformed into P. pastoris strain X-33 which was induced by 1% (V/V) methanol for iDP expression. SDS-PAGE and Western-blot analysis were used to detect expressed proteins. The gene idp either harboring TR region or not could be successfully expressed in Pichia pastoris. However, the expressed iDP encoded by whole idp cannot be secreted from yeast cells but presented in the sediments of cell debris with no catalytic activity. In contrast, the DP with the removal of TR (diDP) could be secreted from P. pastoris with the capacity to hydrolyze dipeptides. Expressed diDP was purified by Ni-NTA sepharose column chromatography with the protein concentration reaching to 0.12mg/mL. Its specific activity was 433U/mg when detected under the condition of pH 7.3 and 50°C for 2h using Gly-Gly as substrate, which also displayed high activity to hydrolyze dipeptides Ile-Leu, Trp-Trp and Phe-Phe.
The influence of arbuscular mycorrhizal fungi (AMF) Funneliformis mosseae, Glomus versiforme , and F. mosseae+G. versiforme on heat tolerance of Lavandula angustifolia plants was determined under the conditions of different temperatures (25°C/20°C, 35°C/30°C and 40°C/35°C). Results showed that the tested AMF could colonize L. angustifolia roots, and the mixed inoculation treatment gave the highest colonization percentage (68%). As compared with the non-inoculation control, the content of soluble sugar, soluble protein, proline and the chlorophyll in leaves and the root activity of plants treated with F. mosseae+G. versiforme under 40°C/35°C were increased by 46%, 68%, 65%, 29%, and 70%, respectively. Under the conditions of three temperature treatments, the activity of superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, and nitrate reductase in leaves of mycorrhizal plants were higher than that in control, while the content of malondialdehyde and relative conductivity were significantly lower than that of the control. The results showed that AMF could enhance the activity of antioxidant enzymes in L. angustifolia plants, alleviate the damage caused by high temperature and enhance the heat tolerance. It was considered that the mixed inoculation with F. mosseae+G. versiforme gave the most significant effect of increasing heat tolerance towards L. angustifolia plants.
This study aims at revealing characteristics and functional activity of Sparassis latifolia polysaccharides (SCPs), and exploring the relationship between the characteristics and antioxidant and immune activities of SCPs. SCPs were isolated and purified from Sparassis latifolia fruiting bodies by employing a procedure involving water extraction and alcohol precipitation assisted energy-gathered ultrasonic, DEAE-52 and Sephadex G-100. The structure of SCP was detected by using high performance gel permeation chromatography (HP-GPC), ion chromatography (IC), Fourier transformed infrared spectrometry (FT-IR), scanning electron microscope (SEM) and atomic force microscope (AFM). The antioxidant activities of SCPs were assayed through determining the DPPH, ·OH,$O_{2}^{-}$·free radical scavenging activity and total reducing power. The MTT assay was used to measure the effect of SCPs on proliferation of macrophages RAW264.7. The results showed that the molecular weight of SCPs was approximately 215Da-393kDa. SCPs were composed of glucose, mannose, galactose, xylose and fructose at the molar ratio of 13:4:1:2:3. The apparent morphology of SCPs was cluster accumulation, interweaving, irregular structure, smooth surface and a certain network structure. SCPs exhibited chain conformation with a highly branched structure, forming the small loops between chains with certain spherical particles. SCPs had certain reducing ability and showed effective DPPH, ·OH,$O_{2}^{-}$·free radical scavenging activities. SCPs could promote the proliferation of macrophages RAW264.7. The antioxidant and immune activities of SCPs might be related to their molecular weight, monosaccharide composition, carbohydrate branching and molecular conformation.
AlamarBlue? method, flow cytometry (Annxin V-FITC)/PI double staining method and HPLC method were used to evaluate anti-tumor activity of ganoderic acid A (GA-A) against prostate cancer in vitro. Flow cytometry 2’,7’-dichlorofluorescindiacetate (H2DCFDA) staining, quantitative real-time PCR (qPCR) and western blot (WB) were used to explore the mechanism of anti-tumor action. The results indicated that ganoderic acid A (GA-A) could exert its anti-tumor effect by reducing 5-reductase activity, inhibiting the proliferation and inducing early apoptosis of prostate cancer LNCaP cells. The results further showed that GA-A could reduce the expression of androgen receptor and induce apoptosis by triggering mitochondrial dysfunction to release excessive reactive oxygen species (ROS). Realtime PCR and WB data indicated that cell mitochondrial dysfunction was closely related to the high expression of tumor suppressor genes caspase-3, bad and aifm1.
Ganoderma lingzhi is a precious and famous traditional Chinese medicinal fungus. Polysaccharides and triterpenoids are the primary bioactive components of this fungus. In the present study, ethanol-water mixture was adopted for extraction of polysaccarides and triterpenoids from G. lingzhi. Based on the extraction rate, four extraction factors including enthanol concentration, extraction temperature, extraction duration and solid to liquid ratio were optimized using orthogonal array design coupled with desirability function. The optimal extraction conditions were obtained viz. ethanol concentration of 40%, extraction temperature of 95°C, extraction duration of 4h, and solid to liquid ratio of 1:40. Under such an optimization, the extraction rate of polysaccharides and triterpeonids reached 1.13% and 0.46%, respectively. DPPH radical scavenging activity of the polysaccharides obtained under the optimized conditions was higher than that obtained by hot water extraction. There was no difference between triterpenoids isolated from the optimized co-extraction and those from ethanol extraction.
The content of ergocalciferol in Lentinula edodes fruiting body dried in the sun and in the shade was determined. The conversion rate of ergosterol to ergocalciferol was determined under different experimental conditions. Under simulated physiological conditions, the mechanism and conformational changes of the interaction between ergocalciferol and human serum albumin were investigated by using fluorescence spectroscopy, synchronous fluorescence spectroscopy, three-dimensional fluorescence spectroscopy, site marker competitive experiments, UV-visible absorption spectroscopy and molecular docking approach for the purpose of revealing transport mechanism of ergocalciferol in the human body. The results indicated that sunlight could increase the content of ergocalciferol in L. edodes. Under irradiation of ultraviolet for 4h, the conversion rate of ergosterol to ergocalciferol was 21%. Fluorescence spectroscopy showed that ergocalciferol quenched the endogenous fluorescence of human serum albumin through static quenching mechanism. As for binding ergocalciferol and human serum albumin, temperature of 288K was favorable while higher temperature was unfavorable. Thermodynamic parameter analysis and molecular docking results revealed that the hydrophobic interactions, hydrogen bonding and vander Waals force were the primary forces during the binding process. The site marker competitive experiments and synchronous fluorescence spectroscopy demonstrated that site I was the major binding site for the interaction between ergocalciferol and human serum albumin. The energy transfer could occur during the binding process, and the binding distance was 3.46nm. The binding process slightly altered the structure and microenvironment of human serum albumin.
