Macrofungal diversity was explored based on investigation in fifteen sampling plots of five sites in coniferous forests and grassland of Xinjiang during the surveys in 2019. The high-throughput sequencing technology was used to analyze the macrofungal community composition in the sampling plots. Redundancy analyses (RDA) and canonical correspondence analyses (CCA) were employed to reveal the relationship between diversity of macrofungi and habitat types as well as other factors related. The results showed that macrofungi in coniferous forests and grassland of Xinjiang were mainly species of Cortinariaceae, Inocybaceae, Clavariaceae, Hygrophoraceae, and so on. Tricholoma, Hebeloma, Suillus, Russula, Cortinarius, Hygrophorus and Inocybe were dominant genera.
Cycads are the only known gymnosperms with coralloid root up to the present, and they are used as ideal materials to explore the interaction between plants and microorganisms. The diversity and composition of endophytic fungi in coralloid roots of cultivated Cycas revoluta collected from thirteen regions of China were analyzed through next-generation sequencing. High diversity of endophytic fungi presented itself in coralloid roots of C. revoluta, but there was no significant difference in composition and diversity among different regions. The results indicated that the symbiosis between C. revoluta and endophytic fungi within coralloid roots was irrelevant to cultivation area.
Traditional fungal genetic modification methods require resistance markers, but few marker genes are available. The genetic modification of Cordyceps militaris is faced with the problem of insufficient number of resistance genes, while continuous knock-in or knock-out of multiple target genes has not been achieved, therefore, it is particularly important to establish an efficient non-resistant marker transformation technology for C. militaris. In this study, the CRISPR/Cas9 technique was adopted to edit the Cmura5 gene of C. militaris and to transcribe gRNA under the control of endogenous 5S-1, 5S-2, and U6 promoters. The result showed that the editing efficiency of the Cmura5 gene using U6 promoters reached 100%. In the uracil auxotroph strain Cmura5-, the rescued wild Cmura5 gene enables forward selection, i.e. wild strains can grow on the basal medium. The Cmura5 gene can be recycled using the designed homologous arm, which allows reverse selection, i.e. the growth of wild type strains are inhibited in mediums containing 5-fluoroorotic acid. Based on the uracil auxotroph strain Cmura5-, a recombinant plasmid was imported by non-resistant marker transformation, and the efficiency of import reached 75%; the efficiency of continuous import of two recombinant plasmids reached 80%; the efficiency of continuous import of three recombinant plasmids reached 100%; the efficiency of continuous import of four recombinant plasmids reached 50%, the average conversion efficiency was 75.7%, and the recovery rate of each round was 100%, accomplishing the simultaneous expression of four exogenous genes in the C. militaris.
Low phosphorus availability and toxic level of exchangeable aluminum are the main factors impairing plant growth in acidic soil. Several studies have shown that ectomycorrhiza (ECM) can alleviate aluminum toxicity and low phosphorus stress of its host plants. However, the adaptation mechanisms of ECM fungi to these adverse environments are largely unknown. The physiological adaptations of Suillus bovinus, a widely distributed ECM fungus in acidic soil environment in South China, to low phosphorus and acidic-aluminum stresses were investigated under axenic condition. The results showed that S. bovinus was an aluminum-tolerant fungus, and the acidic aluminum stress (1 mmol/L) did not affect its mycelium growth. By contraries, low phosphorus stress significantly limited the mycelial growth (P<0.05). It is astonishing that the inhibitory effects of low phosphorus stress could be alleviated by acid aluminum stress. Low phosphorus stress significantly hampered the phosphorus absorption of S. bovinus (P<0.05), while aluminum stress promoted the absorption of potassium. Low phosphorus and acidic-aluminum stress also significantly changed the mycelial exudate composition of S. bovinus. Compared with the experimental control, a great quantity of phenolic acids, organic acids and lipids decreased under low phosphorus stress; while under acidic aluminum stress, a large number of phenolic acids were up-regulated, and the quantities of organic acids and lipids were also increased. Phenolic acids and organic acids were significantly up-regulated under combining stresses of low phosphate and acidic-aluminum. Besides, the accumulation of IAA were all significantly up-regulated under these stress conditions. These results provided theoretical basis for further interpretation of ECM adaptation mechanisms under these stresses.
The enzyme activity of polysaccharide monooxygenase CtPMO1 from Chaetomium thermophilum was detected by a series of methods like thin-layer chromatography (TLC), high-performance liquid chromatography-refractive index detector (HPLC-RID) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). Two residues (His1 and Gln166) of wild-type CtPMO1 enzyme (WT) were mutated by site- directed mutagenesis to form three mutated enzymes (H1A, Q166A and Q166E), and whether the three mutants of CtPMO1 are involved in catalytic activity of C1 and C4 oxidation was clarified. The synergism of CtPMO1 with cellulase (EGⅡ, BGLⅠand CBHⅠ) was detected by 3,5-dinitrosalicylic acid (DNS). It was found that treatment of phosphoric acid-swollen cellulase (PASC) with CtPMO1 at 50 °C and pH 5.0 mainly produced cello-oligosaccharides with a degree of polymerization (DP) from DP2 to DP5, and C1- and C4-oxidized oligosaccharides in the enzymatic hydrolysis products. It was also found that H1A completely lost its activity, Q166A completely lost activity of C1 and C4 oxidation, and Q166E retained partial activity of C1 oxidation. According to the results of the synergism, the reducing sugar yield increased by 2.10, 2.08 and 2.16 times, respectively, when PASC was catalyzed by CtPMO1 and added EGⅡ, BGLⅠand CBHⅠ. The corresponding synergism degrees were 1.022, 0.799 and 0.875, respectively. We concluded that CtPMO1 could oxidized C1 and C4 position on cellulose, and when CtPMO1 was incubated with insoluble substrate phosphoric acid-swollen cellulase (PASC), the highest activity was exhibited at optimal reaction conditions of 50 °C and pH 5.0. CtPMO1 active center amino acid His1 and flat surface amino acid Gln166 are key sites. After CtPMO1 pretreated PASC, the degradation efficiency of cellulase was improved in different degrees.
Pleurotus ostreatus with high edible and medicinal value is widely cultivated in the world and China. The production of P. ostreatus is always affected by many environmental factors among which temperature is the most important one. The effect of high temperature on P. ostreatus physiology and resistance to mould infection were investigated. Three mould species, Mucor circinelloides, Neurospora tetraspora, and Trichoderma longibrachiatum, were isolated and identified. It was found that high temperature strongly inhibited the mycelial growth of P. ostreatus. After high-temperature treatment, the anti-fungal infection ability of P. ostreatus decreased, the microstructure was damaged, and the extracellular polysaccharide content of mycelia increased significantly, resulting in the increase of attractive and adhesive ability of mycelia to mould spore. The mechanism of high temperature reducing the resistance of P. ostreatus to mould infection needs further study.
The effect of cordycepin on apoptosis and migration of human bronchoalveolar carcinoma cell line H1781 cells and its mechanism of action were investigated. The H1781 cells were cultured and grouped, and the blank control group was treated with drug-free medium, while the cordycepin treatment group was treated with cordycepin of 10, 20, 30 and 40 μmol/L. Cell viability was measured after 24 h of treatment; cell morphology was observed by microscopy; the effect of cordycepin on the whole cells was observed by HE staining, and apoptosis was detected by MMP-9 and DAPI nuclear staining using cell immunofluorescence method. The apoptosis- related protein expression was detected by Western blotting. Compared with the blank control, the viability of H1781 cell line was significantly reduced after 24 h of cordycepin treatment; the number of cells was significantly reduced. HE staining revealed that the number of cells and cell groups became significantly less with the increase of cordycepin concentration, and the immunofluorescence detection revealed that apoptosis was significantly promoted after drug treatment; the scratching experiment revealed that cordycepin significantly reduced cell migration ability; Western blotting assay showed the expression of Bax and cleaved caspase-3 proteins were significantly up-regulated and the expression of MMP-9 and Bcl-2 proteins were significantly down-regulated. The results come to the conclusion that cordycepin can inhibit the migration of lung cancer H1781 cells and promote apoptosis, and these probably result from up-regulation of pro-apoptotic protein expression and down-regulation of anti-apoptotic protein expression.
The purpose of this study was to explore the effect of Auricularia heimuer fruiting body powder (AH) replacing diet in part on preventing nutritional obesity of high-fat model mice. Forty eight five-week-old ICR male mice were randomly divided into normal control group (CK), high fat treatment group (HFD), positive drug control group (PD) and AH treatment group. After nine weeks of feeding, the physiological and biochemical indicators of mice were measured. Compared with HFD group, results showed that the body weight and epididymis fat index of AH group were significantly decreased (P<0.05), and the Lee’s index, the serum glucos (GLU), triglyceride (TG) content and the liver index were significantly decreased (P<0.01). Meanwhile, the serum high-density lipoprotein cholesterol (HDL-C) and the liver HDL-C contents were significantly increased (P<0.01). HE staining indicated that the lipid content and the adipocyte size in liver were decreased, the fat volume of epididymis was also decreased, and the shape of epididymis cells was regular in the AH group. High-flux sequencing analysis showed that the relative abundance of Proteobacteria, Helicobacter and Bacteroides in the intestinal tract of mice were improved after the intervention of AH powder. The results indicated that AH had a significant effect on preventing nutritional obesity of mice, and providing a basis for the development and utilization of AH.
Pholiota adiposa is a valuable edible and medicinal fungus. The anti-diabetic effects of the polysaccharides extracted from Pholiota adiposa (PAP) on streptozotocin-induced diabetic ICR male mice were investigated. The results revealed that PAP treatment significantly reducted fasting blood glucose level of mice and increased the body weight. The levels of triacylglycerol (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were found to be decreased, while the levels of superoxide dismutase (SOD), high-density lipoprotein cholesterol (HDL-C), catalase (CAT), and glutathione peroxidase (GSH-Px) increased. The pancreatic and liver sections of diabetic model control (DC) exhibited notable histopathological changes, but the PAP treatment groups exhibited improvement effect. PAP treatment led to significant restoration of islet morphology and function. The results of Western blotting analysis indicate that the expression of PI3K, p-IRS1, p-GSK3β, and p-AKT increases after PAP treatment. This study comes to the conclusion that PAP exerts an anti-diabetic effect on the diabetic mice.
Pisolithus tinctorius can extensively establish ectomycorrhizas with forest trees, and the symbiosis can promote plant growth and improve plant tolerance to heavy metals. However, the physiological response of P. tinctorius to heavy metal zinc (Zn) stress is not completely clear. In this study, the effects of Zn stress on the biomass of P. tinctorius, the active form and subcellular distribution of Zn, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity, glutathione (GSH), ascorbic acid (AsA) and melanin content under pure culture were analyzed. The results showed that Zn stress inhibited the growth of P. tinctorius, reaching the half maximum inhibitory concentration (IC50) at a concentration of 563 μmol/L, and when the concentration was 600 μmol/L, the biomass decreased by 42.6%. Zn in P. tinctorius is mainly in the form of low-activity oxalate, insoluble phosphate and pectinate, and the form of binding or sorption with protein. Zn stress significantly increased the activity of CAT, POD and SOD in P. tinctorius hyphae, increased the content of GSH and melanin, but had no significant effect on the content of AsA. This study shows that P. tinctorius mainly regulates the distribution of Zn and the antioxidant system to enhance the tolerance to Zn, and it provides theoretical support for remediation of heavy metal contaminated soil with the help of ectomycorrhizal fungi.
Lentinula edodes ‘Shenxiang 1644’ was a new variety bred by multispore inbreeding technique using the traditional cultivated strain ‘Shenxiang 215’ as the parent. The pilei of the fruiting bodies are convex, thick and compact, with diameter of (6.15±0.38) cm and thickness of (2.27±0.42) cm. The strain is high yielding with more than 95% biological transformation rate. ‘Shenxiang 1644’ has clear differences in molecular patterns and cultivation characters as compared with the parent. Its pileus is light yellow-brown, being lighter than the pileus color of parent. Its growth period is 100-105 days, being 5-10 days shorter than that of parent. The suitable temperature for mycelial growth is 22-26 °C and for primordium development 16-22 °C. This strain can be cultivated throughout the country.
‘Yuxiang 2’ is a new cultivar of Lentinula edodes, bred and domesticated from a Henan local wild strain with excellent character. The fruit body is medium-sized, single or occasionally grouped. The new cultivar is high yielding, and suitable for durable fresh storage. It is suitable for cultivation at medium and high altitudes in Henan Province.
YL1701-2 was obtained by sexual hybridization between domesticated cultivar HZ14080 and wild strain HZ16009 of Phlebopus portentosus. The optimum temperature for mycelium growth was 25-32 °C, and for fruiting body development was 25-32 °C. The variety has the advantages of thick stipe and thick, large-sized and fleshy pelei, strong resistance to adverse circumstances, short growth cycle, early and uniform fruiting, high yield, good appearance quality, and high degree of commercialization. It can be cultivated in bags or in bottles, but mainly in bottles. At present, the strain has been cultivated on a large scale and it is protected by patent.
