The taxonomy, breeding, cultivation and omics of Wolfiporia cocos, one of the traditional Chinese medicines, are reviewed in this paper. The patents and applications concerning this fungus are analyzed. Research aspects of mating type, fruiting body induction and life cycle are also highlighted. The major challenges existing in the current study are discussed and recommendations for future development are proposed.
Contaminated samples or fruiting bodies of Hypsizygus marmoreus were collected from 3 provincial producing bases subordinated to a large-scale mushroom production enterprise in the whole year of 2018 for investigation of pollution in mushroom production. Molecular sequencing and morphological observation were made for identification of 11 isolates of microorganisms, and the influence of the microorganisms on the growth of the hyphae of Hypsizygus marmoreus were analyzed by plate confrontation experiment. The 11 isolates of microorganisms could be assigned to 3 families and 7 genera, of which 10 isolates were bacteria and 1 was a species of fungi. Enterobacteriaceae was the dominant family and Bacillus was the dominant genera. Two isolates of bacteria respectively belonging to Bacillus and Enterobacter were possibly new species. Results of plate confrontation experiment showed that 7 bacteria and 1 fungus significantly inhibit mycelial growth of H. marmoreus. It is proposed that substrate preparation, sterilization and fruiting management should be regarded as 3 critical control points for disease prevention in H. marmoreus cultivation. During low temperature period in autumn and winter, the substrate sterilization and temperature-raising time should be properly prolonged. In summer when the cultivation room needs to cool down using equipment, strict management of cultivation environment during fruiting period can helps reduction of the incidence of disease.
Honghuaerji is known as the home of Pinus sylvestris var. mongolica, one of the main afforestation tree species in northern China with typical mycorrhizal dependence. In this study, rhizosphere soil of natural P. sylvestris var. mongolica in Honghuaerji was sampled for research object of clarifying ectomycorrhizal fungal (ECMF) community structure and protection of local ecosystem. Illumina MiSeq sequencing was used to study the ECMF community structure. P. sylvestris var. mongolica trees were divided into 4 age classes, <10, 11-20, 21-30 and 31-40 years old, respectively graded as Ⅰ, Ⅱ, Ⅲ and Ⅳ. A total of 87 516 ECMF sequences was obtained by sequencing, dividing into 177 OTUs (operational taxonomic units) belonging to 2 phyla, 4 classes, 12 orders, 26 families and 43 genera. It was showed that Ace richness index and Shannon-Wiener diversity index in Ⅳ age class were significantly higher than those in Ⅰ age class (P<0.05). The proportion of dominant genera of ECMF in different age classes was different. Inocybe (42.55%) accounted for the largest proportion in Ⅰ age class. Tricholoma accounted for the largest proportion in Ⅱ and Ⅲ age classes, being 31.64% and 27.69% respectively, while Cortinarius (28.80%) accounted for the largest proportion in Ⅳ age class. Redundancy analysis showed that soil pH had the greatest effect on ECMF, followed by available potassium and available nitrogen. The effects of different physicochemical factors on the proportion of dominant genera were also different.
Based on the property of Concanavalin A (ConA) that specially binds haustoria of rust fungi, ConA-sepharose 4b was used as affinity chromatography medium to isolate haustoria from Gymnosporangium yamadae infected leaves of Malus ‘Radient’ under natural conditions and G. asiaticum infected leaves of Pyrus pyrifolia by artificial inoculation at different time. Intact haustoria of G. yamadae and G. asiaticum were successfully isolated from the infected leaves showing chlorotic flecks, respectively. Vast amount of haustoria showing similar shape were observed under light and transmission electron microscopy. The haustoria are mainly haphal, kidney-shaped or irregular. The ratio of haustoria to chloroplast in both rusts was approximately 1:1. Total RNA of the isolated haustoria of G. yamadae were extracted by traditional TRIzol method. This study provided a basis for further investigating on screening candidate effectors from the haustorial transcriptome and molecular mechanism of pathogen-host interaction.
Under natural conditions, orchid plants depend on mycorrhizal fungi for their nutrition at the germination stage. In this study, four mycorrhizal fungal isolates (WQ17-33, WQ17-43, JST-3 and SL15-7) from roots and protocorms of Bletilla striata were identified based on sequences of internal transcribed spacer (ITS) region, and the effects of the fungi on seed germination under dark and light conditions and seedling rooting were also evaluated. The four isolates were assigned to Coprinus sp., Tulasnella sp., Sebacina sp. and Serendipita sp., respectively. The results of investigation were that JST-13 and SL15-7 treatments obviously promote germination rate of seed and growth of protocorm in the dark condition. Isolate SL15-7 had higher promotive effect on protocorm and seedling development than other isolates. The growth vigor of seedlings treated with JST-13 and SL15-7 was stronger and colonized pelotons were more in comparison with the seedings treated with other isolates. The rooting of seedlings treated with JST-13 and SL15-7 was also better. Facts have proved that different mycorrhizal fungi vary in their ability to facilitate seed germination and root development of B. striata in vitro.
The genetic basis of albino Agrocybe cylindracea was investigated. Monokaryotic strains were obtained by means of protoplast monokaryolization of albino strain Ag.c0002 and brown isolate Ag.c0067. Monokaryotic strains from both parents were paired and four F1 hybrids were obtained. Single spore strains were isolated from fruit-body of two F1 and they were paired with monokaryotic strain “2-2” by back cross. F2 strains were cultivated and their fruit-body color was investigated for inheritance analysis. The results showed that fruit-body with white color had recessive character, and a recessive allele at a single locus encodes the white color. The allele did not link with incompatibility factor either A or B.
Powdery mildews are obligate parasitic Ascomycetes, which cause powdery mildew diseases on a wide range of plants and distribute all over the world. Studies on the mating-type (MAT) genes in powdery mildews would be helpful for understanding life cycle and phylogenetic evolution of the fungi. The amino acid sequences encoded by ten MAT genes and four flanking genes from different ascomycetes were used as seeds to search high similarity sequences from the whole genome sequences of twenty-one powdery mildew isolates. The results showed that ten isolates belonging to MAT1-1 type only include MAT1-1-1 and MAT1-1-3 genes and ten isolates belonging to MAT1-2 type only contain MAT1-2-1 gene. The three mating genes, MAT1-1-1, MAT1-1-3, and MAT1-2-1, were all identified in Erysiphe pulchra. The flanking genes SLA2, APN2, CoxVia, and APC5 were detected in all genomes. The conserved MAT genes and their flanking genes are present in the same scaffold of two species, Blumeria graminis f. sp. hordei RACE1 and Golovinomyces orontii, while they are not present in the same scaffold in the other examined isolates. The structure of MAT loci was also predicted based on the annotation information from nine species. The results showed that MAT gene structure was conserved in the same genus but varied between different genera. The phylogenetic trees were constructed with MAT1-1-1, MAT1-2-1, and MAT1-1-3 gene sequences, and the results showed that Blumeria, Erysiphe, and Golovinomyces were clustered in a distinct clade whereas Erysiphe and Golovinomyces grouped as sister lineages, which were coincided to the phylogenetic relationships of these genera in the previous studies. The results of this study may provide information for mating type genes research and provide a new idea of phylogenetic analyses in powdery mildews.
Fungi are known to secrete multiple effector proteins during their infection of host plants, whereas, the function of the secreted CAP superfamily proteins has been undefined in fungi. Based on CAP homologue analysis, the gene VmPR1c was identified in the genome of Valsa mali, the causal agent of apple valsa canker. Deletion of VmPR1c greatly attenuated V. mali virulence, however, VmPR1c was degraded when transiently expressed in Nicotiana benthamiana. In order to reveal the degradation mechanism of VmPR1c, we determined the functional degradation domain and degradation pathway of VmPR1c. Sequence analysis were performed using BLAST, NCBI CDD web server, SignalP 4.1, and TMHMM 2.0. Sequence truncation and site-directed mutagenesis were used to explore the degradation domain of VmPR1c. To study the degradation pathway of VmPR1c, various protease inhibitors were added during protein expression. Sequence truncation assays showed that, when the α-helix structure or the CBM region of CAP domain was deleted, VmPR1c protein could be easily detected by Western blotting. When the cysteine residues in CAP domain or the CTE region was mutated, VmPR1c protein bands were variously weakened. When the signal peptide of VmPR1c or its cleavage site was substituted, VmPR1c protein bands were differently deepened. When the proline residue in CTE or NTE region was mutated, VmPR1c degraded more evidently. When the proteasome inhibitor MG132 or the lysosome inhibitor chloroquine was added during transient expression of VmPR1c in N. benthamiana, VmPR1c could not be detected by Western blotting. The conclusion is that the CTE region, signal peptide and the cleavage site of VmPR1c, as well as the α-helix structure of the CAP domain, mediate the degradation of VmPR1c. The proline in CTE and NTE could potentially protect VmPR1c from degradation. The degradation of VmPR1c is not dependent on the proteasome and lysosomal pathways.
Trametes gibbosa is a widely distributed medicinal mushroom with reducing blood sugar effect. The optimum culture conditions and domestic cultivation of Trametes gibbosa were studied. The experimental strain ZT-055 was obtained from Red Pine King Park, Lushuihe Town of Baishan City in Jilin Province. Orthogonal experiments revealed that using fructose as carbon source and yeast extract powder as nitrogen source, the mycelia grew well at pH 5.0 and incubation temperature of 30°C. After cultivation for 17 days, the mycelia fully colonized 650g substrate. Primordium began to form in 30 days. In 44 days, the primordium differentiated to form young fruiting body which matured in 57 days. The polysaccharide of T. gibbosa ZT-055 fruiting body was extracted by hot water and ethanol precipitation method, and the quantity was determined using phenol sulfuric acid method. The polysaccharide content was about (80.51±5.1)mg/g. The hydroxyl radical (-OH) scavenging rate reached 45%, and superoxide anion radical (O2 -) scavenging rate reached 48% under polysaccharide concentration of 1.26mg/mL. This indicates that fruiting body of T. gibbosa has high antioxidant activity and is medicinally applicable.
Sparassis latifolia, one of the valuable edible fungi, has been cultivated recently in China. In the present study, the effects of tyrosinase inhibition, antioxidant and moisture-retention of fruiting body water extract of S. latifolia obtained under different temperatures and different drying methods were evaluated. A comparison was made between the effects of tyrosinase inhibition, antioxidant and moisture-retention of the extracts of fresh fruiting body of and those of discarded stipe after being harvested and residual waste during pruning and package. It was found that the water extract of fresh fruiting body of S. latifolia showed strong inhibitory rate against monophenolase and diphenolase [(86.43±3.33)% and (79.03±4.49)%, respectively], slightly lower than the inhibitory rate of the positive control kojic acid. At concentration of 2 000μg/mL all the samples are similar to the positive control BHT in DPPH· scavenging activity, suggesting the antioxidative potential of S. latifolia. The water extract of fresh fruiting body of S. latifolia showed the strongest antioxidant potency with IC50 value of (308.08±6.77)μg/mL. The moisture-retention efficacy of all the samples were slightly higher than that of glycerol and sodium hyaluronate, indicating the potent application in the moisture-retention capability of S. latifolia. The extract of discarded stipes and residual waste had similar DPPH· scavenging and moisture-retention activities to that of fresh fruiting body. The results of this study will be valuable for the application of S. latifolia in cosmetic industry and also provide an idea for the disposal and application of the residual waste after harvest of S. latifolia fruiting bodies.
The effects of Isaria cicadae fruiting bodies (0.75g/kg) on renal tubular epithelial cells was evaluated by measuring the biomarkers of kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-related apolipoprotein (NGAL) in SD male rats after repeated intragastric administration of Isaria cicadae fruiting bodies (0.75g/kg) for 90 days and 28 days after recovery. The effects of different doses of Isaria cicadae fruiting bodies (0.25g/kg, 0.5g/kg and 1.0g/kg) on the proliferation and prolification of renal tubular epithelial cells were also studied. There was no significant difference in serum KIM-1 concentration between the experimental group and the control group in 30, 60, 90 days after administration and 28 days after recovery (P>0.05). In 30 and 60 days after administration, there was no significant difference in serum NGAL concentration between the experimental groups and the control group (P>0.05). In 90 days after administration and 28 days after recovery, the serum NGAL concentration of the experimental groups was lower than that of control group (P<0.05). Serum NGAL concentration in the experimental groups was lower than that in the control group in 90 days after administration and 28 days after recovery (P<0.05), and there was significant difference between the experimental group in 90 days after administration and the control group. Immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and tetramethylazozolium salt microenzyme reaction colorimetry (MTT) showed that Isaria cicadae fruiting bodies could enhance the proliferation of renal tubular epithelial cells, but did not cause apoptosis of renal tubular epithelial cells.
The protective effect of cordycepin on acute liver injury induced by carbon tetrachloride (CCl4) and its molecular mechanism were investigated. An animal model of acute liver injury induced by carbon tetrachloride in mice was established. The hepatoprotective effect of cordycepin was evaluated by detecting the changes of serum biochemical parameters and liver function indexes and observing the pathological changes of tissue sections via HE staining. Western blot analysis was used to study whether cordycepin could increase the body’s ability to resist oxidative damage and inhibit inflammatory factors (TNF-α, TNF-β, IL-6, IL-10) by activating the Nrf-2/Keap1 signaling pathway and expression of its downstream antioxidant factors (HO-1, NQO-1). In comparison with model group, cordycepin can significantly decrease the level (P<0.01) of serum ALT, AST and MDA and significantly increase SOD levels (P<0.01) in the liver. HE staining showed cordycepin could effectively reduce inflammatory cell infiltration and fibrous tissue proliferation in damaged liver tissue. Western blot results indicated that cordycepin could promote the expression of downstream antioxidant and anti-inflammatory factors and thereby reduce inflammation by activating Nrf-2 signaling pathway. Facts have proved that cordycepin has a protective effect on acute liver injury induced by CCl4 in mice, and its mechanism is related to Nrf-2 signaling pathway.
Oxidative stress is thought to be a key risk factor in the development of diabetes mellitus, tumor and hepatic diseases. Blocking or retarding the reactions of oxidation and the inflammatory process by antioxidants could be a promising therapeutic intervention for prevention or treatment of diabetes mellitus. Floccularia luteovirens, previously interpreted as Armillaria luteovirens, as a precious edible-medicinal fungus, is widely distributed in the Qinghai-Tibet Plateau. Its fruiting body has many pharmacological effects such as anti-tumor, anti-inflammation and antioxidant. In this study, we investigated the anti-oxidant and anti-inflammatory effects of the water extract of Floccularia luteovirens (FLW) on STZ-induced type II diabetes mellitus in rats. After treatment with metformin and FLW for 8 weeks, the results showed that FLW improved glucose tolerance and promoted exogenous glucose consumption. MDA and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) levels were reduced and SOD level was advanced by FLW in serum. Taken together, FLW showed antioxidative and anti-inflammatory effects in diabetic rats by inhibiting oxidative stress and the inflammation.
A white rot fungus strain G11 was isolated and purified from the epidermis of immature organic blueberry. According to its morphological characteristics, ITS sequence homology and phylogenetic analysis, the strain was identified as Bjerkandera adusta. The fungus can produce lignin-degrading enzymes such as lignin peroxidase, laccase and manganese peroxidase. The decolorization assays to eight different dyes showed that it was the best of decolorization effect on reactive dyes, and it needed only a short duration to decolorize 90% of reactive dyes. Further assay showed that the strain G11 had remarkable capability to decolorize reactive black and red, with decolorization rate of 99%, 98%, 95%, 94%, and 92% respecfively for 10, 50, 100, 250 and 500mg/L reactive red, and 98%, 97%, 95%, 93%, and 90% respectively for reactive black in decolorization duration of 15d.
Paecilomyces hepiali isolated from fresh Cordyceps sinensis stroma has active ingredients similar to those of Cordyceps sinensis, with anti-tumor, anti-virus and other effects. In this study, the main components of the dried fermentation filtrate of Paecilomyces hepiali were analyzed. The results showed that the dried fermentation filtrate of Paecilomyces hepiali contained 21.33% crude fat, 27.34% total protein, 16.79% hydrolyzed amino acid, 11.16% crude polysaccharide and 71.21% unsaturated fatty acid including oleic acid and linoleic acid. The dried filtrate also contains a variety of nucleoside substances, polysaccharides and other biologically active ingredients, as well as essential trace elements. Preliminary results of acute toxicity experiments indicate that no death and obvious poisoning reaction occur in mice during experimental period, and the morphology of the main organs of mice was normal. There was no significant difference in the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as compared with the experimental control. Fermentation filtrate of Paecilomyces hepiali is worthy to be further researched and developed in the fields of medicine and health care.
Morchella sextelata is a precious edible and medicinal fungus with important economic value. Based on single factor experiment, Box-Behnken design and response surface methodology were used to optimize the extraction process of polysaccharide. The crude polysaccharide was purified by Sevage method and dialysis, and component MSP (Morchella sextelata polysaccharide) was obtained. The structure of MSP was analyzed by gel permeation chromatography-refractive index-multi angle laser light scattering (GPC-RI-MALLS), high performance anion exchange chromatography (HPAEC), fourier transform infrared spectrometer (FTIR) and gas chromatography-mass spectrometry (GS-MS). The free radical scavenging activities of MSP also were determined. The results indicated that the optimum extraction conditions included extraction temperature of 89.94°C, ratio of extracting solution to raw material of 31.07mL/g and extraction time of 162.86min. Under the optimized conditions, the polysaccharide extraction rate was 23.98%. MSP was mainly composed of two components with molecular weight of 4.655×10 6Da and 6.571×10 4Da, and its monosaccharides were mainly glucose, mannose, and galactose with mole percentages of 71.60%, 23.70% and 4.70% respectively. The main glycoside bonds are T-Glc, 1,2-Man, 1,4-Glc, 1,6-Man, 1,3,4-Man, and 1,4,6-Gal. The polysaccharide MSP possessed strong scavenging ability to 2,2‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS) free radicals, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) free radicals and hydroxyl radicals.
Response surface methodology was used to optimize ultrasonic extraction process of N 6-(2-hydroxyethyl) adenosine (HEA) in Isaria tenuipes on the basis of three factors, namely extraction time, pH value and liquid-to-solid ratio. The results showed that liquid-to-solid ratio had priority over pH value in influencing the yield of HEA product, while extraction time was the least influential. The optimum ultrasonic extraction parameters were as follows: ultrasonic duration of 31min, liquid-to-solid ratio of 22:1 (g/mL), pH value of 4.88, and Box-Behnken model predicted value of 0.86mg/g. The actual value of Box-Behnken model is (0.86+0.03)mg/g, with deviation of 0.10%. The method was stable and feasible for the extraction and analysis of adenosine from different Cordyceps s.l. samples, showing its advantages of time saving, high efficiency and energy saving. The method is useful for quality evaluation and product development of Cordyceps and related fungi.
Response surface methodology was used to investigate the effects of corn steep liquor concentration, KH2PO4 concentration and fermentation duration on the yield of exopolysaccharides of Rhizopus nigricans. Commercial potato dextrose broth was used as basic medium. Response surface analysis showed that the factors influencing the response value was arranged in effectivity order of corn steep liquor concentration > fermentation duration > KH2PO4 concentration. The effects of corn steep liquor concentration and fermentation duration were significant on the yield of exopolysaccharides, while that of KH2PO4 concentration were not significant. The optimum fermentation condition was the use of corn steep liquor 3.2mg/mL as culture medium with additional KH2PO4 1.5mg/mL and fermentation for 132h. Under such a condition, the maximum predicted yield of exopolysaccharides was 0.824mg/mL. The technological process was high-yielding, stable, controllable and repeatable for production of Rhizopus nigricans exopolysaccharides.
