Mushrooms are of important source of high-quality proteins. In China, the annual output of edible mushrooms in 2013 is about 32 million tons (data from CEFA, China Association of Edible Fungi), accounting for more than 75% of the world’s total; the direct output value is 170.7 billion RMB, and the indirect output value is 682.8 billion RMB. Over 20 million Chinese people are engaged in edible mushroom production. Mushrooms have become the fifth high-value crop following grain, vegetables, fruits and oil-bearing crops in China at present. Industry of edible mushrooms is of strategic importance for the food safety in China. However, our basic science research on edible mushrooms is seriously deficient, especially on the key mechanism relavant to improvement of yield and quality. This is a hamper for development of mushroom breeding and cultivation techniques, leading to low yield and poor quality of products, hindering extension and upgrade of the industrial chain, and finally resulting in the decline of profits. The shortage of basic science research has become a bottle neck of the development of Chinese mushroom industry at present. “Molecular mechanisms and regulations of yield and quality of edible mushrooms”, a project of National Basic Research Program of China (‘973’ Program) was put into action in 2014. The project is focusing on the scientific issues hampering the development of Chinese mushroom industry, i.e., (1) molecular mechanism of efficient use of lignocellulose by edible mushrooms; (2) regulatory mechanism of fruiting body development of edible mushrooms; (3) molecular mechanism of heat stress response in edible mushrooms; (4) bioactive substances and their biosynthetic pathway in edible mushrooms; (5) genetic basis for the formation of excellent germplasm features of edible mushrooms. The project intends to figure out the following three key scientific issues: (1) nutrient utilization and genetic regulation of mushroom fruiting body formation; (2) molecular mechanisms of temperature response of mushroom; (3) anabolic regulation mechanisms of mushroom bioactive substances. The project aims at establishing a scientific theory system for edible mushrooms, providing scientific basis for technical innovation of Chinese mushroom industry, supporting the production of high yield and quality, and leading the scientific path for increasing benefits of the whole industry.
In this paper, the history of understanding and utilization of mushrooms is reviewed. China’s contribution to mushroom cultivation in early era is known to all and the development of mushroom industry at present is in progress. The development course and current situation of mushroom industry in China is presented. The basic situation of global mushroom industry and developing history, especially button mushroom industry in the West and mushroom industry in Asia is discussed. Changes in the production mode and technology of world mushroom industry are analyzed. The transfer trend of global mushroom industry from developed to developing regions will continue. China will accelerate the change of production mode into systematization, large scale, standardization and specialization.
Pleurotus (Pleurotaceae, Agaricomycetes) is considered as one of the most abundant biological diversity groups in the macrofungi. Pleurotus spp. have abundant bioactive substances, such as polysaccharides, proteins, amino acids and fatty acids, etc. These substances have been extensively used in the fields of food, health care, and pharmaceutical industry. The fungi secrete enzymes such as lignin peroxidases, Mn-peroxidase and laccase, etc., which may degrade PAHs (Polycyclic aromatic hydrocarbons), dyes, agricultural and industrial wastes and thus can be used in treating waste water from industries like textile, paper-making, olive mill and city waste. Due to their strong ability of utilizing diverse substrates, Pleurotus spp. could be cultivated on substrates formulated from agriculture, forestry and light industry wastes. In most cases, their biological efficiency is higher than other edible fungi. Pleurotus spp. have important application value in agricultural production.
The chemistry and the biological activities of the secondary metabolites from Hericium species are reviewed. Up to dates, 83 bioactive compounds belonging to terpeniods, phenolics, fatty acids, steroids, alkaloids have been reported in the mycelia and the fruiting bodies of Hericium species. These compounds show various bioactivities, such as antitumor, antibacterial and hypoglycemic effects, and stimulatory effects to the synthesis of nerve growth factor. The problems existing in researches and the development trends of studies on Hercium medicinal mushrooms are discussed in this review.
The chemistry and biological activities of the secondary metabolites from Pleurotus spp. were reviewed. Natural products, including terpenes, steroids, phenolic derivatives and polyynes, with various biological activities, such as antibacterium, anti-nematode activity, anti-inflammation, and anti-cancer, were reported during the past 60 years. Action mechanism investigation indicated that pleuromutilin and its derivatives interact with the peptidyl transferase center to inhibit the growth of bacteria, and pleuroton B induced apoptosis via the classical Bax/Bak pathway.
Species of the genus Pleurotus are very important edible mushrooms and many of them can be cultivated in commercial scale. Although P. abieticola was originally described from Russian Far East, and then reported from northeastern China and northwestern Russia, its distribution range is still largely unknown. Our morphological and molecular phylogenetic evidence indicated that this species is also distributed in subalpine mountains of southwestern China. This paper documented the taxon based on morphological and ecological features, and DNA sequences generated from materials collected from Sichuan Province and the Tibet Autonomous Region.
Species within the genus Flammulina are among the most important economically cultivated mushrooms in East Asia. Based on examinations of the morphological characters and molecular phylogeny reconstructed from the internal transcribed spacer (ITS) sequences, F. velutipes var. filiformis and F. velutipes var. himalayana are proposed as new varieties, and F. populicola is reported as a new record for China. Detailed descriptions and illustrations of macroscopic and microscopic characters are provided for the taxa known from China, and a key to the taxa is included.
“Heimuer” is one of the most important fungi in China, and it has been cultivated and used as food in the country for more than 1 000 years. Auricularia auricula-judae or Auricularia auricula has been applied for its scientific name for more than 120 years. However, recent molecular studies showed that A. auricula-judae is a species complex, and five species have been found in the complex all over the world. A. auricula-judae has a distribution in Europe only, and grows on angiosperms. Two species are found in North America: A. americana grows on gymnosperms and an undescribed species on angiosperms. Three species are found in China: A. heimuer is a commonly cultivated species growing on angiosperms especially on Quercus, with a wide distribution in most parts of China; A. villosula has been cultivated on a small scale, growing on angiosperms with a wide distribution in northeast China; A. americana has not been cultivated, growing on gymnosperms in northeast and northern China.
The genetic diversity was estimated based on analysis of IGS2-RFLP markers and cultural characteristics for 28 wild ferula mushrooms collected from Xinjiang Autonomous Region, China. It was showed that the percentage of polymorphic bands (PPB) from three restriction endonucleases was 96.4%, Shannon's information index (I) and Nei’s gene diversity (H) were 0.364 and 0.230, respectively. The average similarity coefficient was 0.665, indicating abundant genetic diversity in the wild resources of ferula mushroom. All the samples could be divided into two groups, Pleurotus eryngii var. tuoliensis and P. eryngii var. ferulae, at similarity of 0.36 based on the UPGMA cluster analysis. The genetic diversity of the former was more abundant than that of the latter. The observation of partial cultural characteristics demonstrated the obvious differences among tested samples in optimum temperature, temperature sensitivity and the morphology of colonies. Twenty-eight samples were roughly clustered into two groups, one was featured by fast growing hyphae and stretched colony and another with slow growing hyphae and limited colony. Correlation analysis showed that the cluster based on cultural characteristics was basically consistent with the results of IGS2-RFLP markers. The cultural characteristics might be served as a trait to identify the samples for P. eryngii var. tuoliensis, but the inference remained to be verified by using much more samples.
Based on interferences with Cu2+ (5mg/kg) and guaiacol (1mL/L), the growth characteristics, degradation process, enzyme production and extracellular protein content of the Pleurotus ostreatus CCEF89 were observed and determined. In general terms, its physiological and biochemical characteristics during hyphal growth and fruiting body formation were inferred. 2,2’-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), guaiacol and MnCl2 reduced the hyphal growth. The strain of CCEF89 degraded acid insoluble lignin and hemicellulose during the first ten days, then it digested acid soluble lignin and cellulose in the next ten days. Cu2+ promoted the hyphal growth, fruiting and secreting laccase, while guaiacol had an inhibiting effect. After twelve days, sodium carboxymethyl cellulose enzyme activities (CMCNa activities) had a significant correlation with xylanase activities. In addition, extracellular protein content also had a positive relationship with CMCNa activities.
In order to explore the biochemical pathway of exogenous nitric oxide (NO) improving the heat-tolerance of mycelium of mushroom, the effect of exogenous SNP (sodium nitroprusside, a NO donor) on the response of antioxidant enzymes under heat stress was studied through examining activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) as well as peroxidase (POD) in mycelium of Pleurotus eryngii var. tuoliensis (strain CCMSSC 00489). Results showed that heat stress led to the increase of TBARS content, which revealed enhancement of membrane lipid overoxidation. The application of exogenous SNP had significant effect on overoxidation anesis under high temperature while at the normal temperature SNP showed ineffective. Compared with CK (no exogenous SNP added), the content of TBARS in the mycelium treated with exogenous NO decreased by 31.5%, 25% respectively, at 6h and 8h heat treatment. In addition, the antioxidant enzymes in vivo responded differently to exogenous NO. After addition of SNP under heat stress, the activities of SOD, CAT and GR increased significantly as heating duration went on and reached to the maximum in 72h, being respectively equivalent to 1.73, 7.29 and 4.95 times of those of CK (0h). Among the four antioxidant enzymes, CAT played the most important role in response to high temperature stress and its activity was measured by mmol/L·min-1·mg-1 of protein while others by μmol/L·min-1·mg-1 of protein. Under experimental conditions, the increasement in antioxidant enzymes activities was in accordance of the decrease in TBARS content. In conclusion, the application of SNP could increase the heat-tolerance of the mycelium by improving the activities of SOD, CAT, GR and further mitigating the oxidative damage. The activity of POD decreased under heat stress with exogenous NO application.
To investigate the heat stress response of four cultivated germplasms of Pleurotus eryngii var. tuoliensis (“Zhongnong No.1”, “Huaza 13”, “Zhongnongchibao” and “CCMSSC 00488”), the concentration of thiobarbituric acid-reactive substances (TBARS) and protein carbonyl (PCO) in mycelia was determined, and the colony growth rate, growth vigor and the hyphal morphological characters were detected. Results showed that the responses to heat stress differed in the level of oxidative damage, the colony character, and hyphal morphology, and significant differences of damage existed among the four different cultivated germplasms. The TBARS and PCO content were all significantly increased under heat stress but varied in degree for the four strains in order of CCMSSC 00488 > Huaza 13 > Zhongnongchibao > Zhongnong No.1. The growth recovery duration needed for the four strains after heat stress was ranked as CCMSSC 00488 > Huaza 13 > Zhongnongchibao, Zhongnong No.1, while the colony growth vigor was ranked as Zhongnong No.1 > Zhongnongchibao = Huaza 13 > CCMSSC 00488. The hyphal morphology character, surface growth rate, cell surface area of the distal hyphae, hyphal diameter, and mycelium branching frequency were all significantly reduced, and the strain Zhongnong No.1 suffered the most serious decline. The mycelium branching frequency was relevant to the colony growth vigor.
Laccases are copper-containing polyphenol oxidases, which play an important role in lignin degradation. The production and activity of laccases are regulated by copper ion. The influences of copper on the lignocellulosic degradation and morphological development of Pleurotus ostreatus cultivated in copper-supplemented corn stover substrate were evaluated. The results suggested that copper ion increased laccase activity, and the activity increased by 71.2% over the control under the addition of 3mmol/L Cu2+ on day 7. Copper ion could also induce primordium differentiation and development and of fruiting body.
The mycelial pellets of Pleurotus eryngii were used as receptor for transformation by Agrobacterium-mediated method, and the genetic transformation system of P. eryngii was established. Sensitivity test showed that the tolerance concentration for P. eryngii against hygromycin was 50mg/L. The optimum genetic transformation system for Agrobacterium-mediated P. eryngii mycelia was OD600=0.6-0.7 of the bacterial concentration, 30-35min of infection duration, 2 days of the co-cultivation period and 1mg/mL of acetosyringone (AS) concentration. The results of PCR identification and the histochemical analysis of β-glucuronidase (GUS) activities indicated that the exogenous gene gus was transferred into the mycelia of P. eryngii by hygromycin resistance selection. A stable Agrobacterium-mediated genetic transformation system of P. eryngii was constructed successfully.
Oyster mushroom is widely cultivated in China, and Pleurotus ostreatus and P. cornucopiae are the two representative species. Heat stress affects the quality and productivity of oyster mushroom. Determination of the stress level is the key to the research on the mechanism of heat stress. Heat stress condition includes different elements, such as stress temperature, stress duration and the age of hyphae. Based on TBARS content as the main physiological indexes, combining microbiological parameters such as mycelium morphology and the growth of 11 strains, the level of heat stress conditions for P. ostreatus and P. cornucopiae was studied. Results showed that thiobarbituric acid reactive substances (BARS) content in mycelium under heat stress were positively correlated with temperature (28°C to 40°C) and duration (0h to 48h), but negatively correlated with the recovery rate of mycelium growth. However, TBARS content and recovery rate after heat stress were not related with the natural temperature of mycelium growth and fruiting body formation. TBARS content can be used as a physiological index determining the conditions of heat stress for the mycelium of oyster mushroom. The heat stress conditions are optimized by using 3-day-old mycelium as the material, 12°C higher than the optimal growth temperature, while the heat level for plant is 10°C higher than the optimal growth temperature.
Flammulina velutipes, one of the most popular edible and medicinal mushrooms, has important nutritional and economic values. Many active constituents isolated from F. velutipes fruiting body, such as ergosterol and sesquiterpenoids have powerful antibacterial, anti-cancer and hypolipidemic effects. Previous studies of F. velutipes mainly focused on the nutrition, cultivation and market development. However, the bioactive compound biosynthesis of F. velutipes is still not clear. In the present study, whole-genome of F. velutipes monokaryons L11 was sequenced and analyzed. The reads were assembled and 34.75Mb length genome sequence of L11 was obtained. The structure of 4 key genes involved in terpenoids biosynthesis was identified. Our results indicated that the putative proteins were stable, and had signal peptide function fragments and transmembrane domains. Genomic sequencing of F. velutipes will provide the genetic basis for exploring the medicinal and nutrient properties of the fungus.
Based on the reference genome of monokaryotic strain W23 of Flammulina velutipes obtained previously in our laboratory, comparison of gene expression patterns in the monokaryotic and dikaryotic mycelium revealed that 3 504 genes had differences in gene expression. 2 151 genes were up-regulated and 1 353 genes were down-regulated. These genes contained a number of transcription factors, protein kinases and WD40 repeat-like proteins. According to gene ontology (GO) cluster analyses, all the genes belong to the extracellular region. Genes belonging membrane-enclosed lumen item were up-regulated but those belonging to envelope item were down-regulated. This might be involved in the mechanism of the formation of clamp connection and nuclear migration. Pathway analyses revealed that genes involving with fatty acid, amino acid and most saccharide synthesis were up-regulated. This suggested that dikaryotic mycelium could store nutrition and prepare for the primordium formation at suitable condition in next sexual development stage.
The integration behavior of an RNAi vector was analyzed based on re-sequencing of a mutant and comparison with a previously obtained Flammulina velutipes reference genome. Mutant strain F. velutipes 1382R3 was obtained through Agrobacterium tumefaciens-mediated transformation (ATMT) with RNAi vector fvhmg1-RNAi, and was re-sequenced after PCR-based confirmation of presence of the selection marker. Mapping sequences based on BLAST and new PERL scripts revealed two integration sites where sequence reads deviated from the reference genome sequence and overlapped with the RNAi vector sequence. Integration site 1 contained only a part of the expected transfer DNA (T-DNA) while integration site 2 contained the full T-DNA fragment. Therefore, two integrations could be interfering with endogenous gene expression. This new application of high throughput sequencing for rapid identification of locations and numbers of integration sites in ATMT mutants is an important extension of the available tools for functional genomics and genetic engineering.
A gene (Vv-IF4E) encoding translation initiation factor IF4E from Volvariella volvacea was identified, and its correlating expression with associated transcription factors was analyzed by bioinformatics and RT-qPCR. Eukaryotic initiation factor 4E (IF4E) can combine with the 5′ cap of mRNA, and plays an important role in the translation initiation process of protein synthesis. The results showed that there were many upstream cis-acting elements on Vv-IF4E. There were 4 types of alternative splicing variants, but only one (Vv-IF4E) contained an initiation factor domain. The Vv-IF4E coding sequence (CDS) of 3 083bp encoded a protein with a predicted molecular weight of 87.1kDa and 35 phosphorylation sites. The protein structure of Vv-IF4E differed from Arabidopsis thaliana IF4E, but showed most similarity to that of Auricularia delicata. RT-qPCR results indicated that there was a strong co-expression pattern among Vv-IF4E and its potential target transcription factors YRR1 and ECM22.
Laccases are involved in lignin degradation and mushroom development. In order to further determine the role of laccases in fruiting of Pleurotus ostreatus, expressions of 11 laccase genes and a small subunit of Lacc2 (sspoxa3) in different stages of fungal development were analyzed by real-time PCR. lacc6 and sspoxa3 are highly expressed throughout the growth and development stage. lacc12 is highly expressed during primordium differentiation and fruiting body formation, suggesting that lacc12 functions in fruiting. lacc4, lacc7 and lacc11 are highly expressed in primordium stage, indicating they are related with primordium differentiation. lacc2, lacc3 and lacc8 are relevant to fruiting body differentiation and maturation, with high expression during mature fruiting stage.
Local BLAST analysis of comparing Coprinus cinereus exp1 with the genome of Volvariella volvacea strain PYd21 revealed a vv-exp gene homologue. Comparison between vv-exp gene of PYd21 and that of PYd15 indicated that the sequence was identical in both strains. The 1 937bp sequence contained a coding region of 1 773bp interrupted by three different introns. Analysis of the deduced amino acid sequence demonstrated that the protein encoded by vv-exp contained two HMG-box domains. Expression of vv-exp was higher in the button stage than in vegetative mycelium, suggesting a role in fruiting body formation. Moreover, subsequent analysis showed that the expression of this gene in the pileus of the maturation stage was significantly higher than that in the pileus of the elongation stage, equaling 3 times higher than that in the primordium stage, implying that vv-exp might be involved in expansion of the pileus in V. volvacea in similar manner as in C. cinerea.
Glucose-6-phosphate dehydrogenase as rate-limiting enzyme in pentose phosphate pathway plays an important role in the production of NADPH being necessary to vital activities of cell. In this study, two transcripts of glucose-6-phosphate dehydrogenase encoding gene were cloned, and their expression levels were determined in homokaryon and heterokaryon strains of Volvariella volvacea, respectively. The results showed that gDNA sequence of g6pdh spanning 1 954bp contains seven introns, the fifth of them exists intron retention. Therefore, gene g6pdh can produce two transcripts: one in which all introns were removed (g6pdhID), encoding 515aa glucose-6-phosphate dehydrogenase with integral conserved domain; another is alternative splicing variant with the fifth intron retention (g6pdhIR), encoding 316aa presumptive protein without integral domain. The results of quantitative PCR showed that the expression levels of g6pdhIR were extremely low in both homokaryon and heterokaryon strains, suggesting that g6pdhID was the main transcript of g6pdh gene. The expression level of g6pdh gene in rapid-growing heterokaryon strain was much higher than that in both of two slow-growing homokaryon strains. It is suggested that activity of glycometabolism may be involved in the growth and development of mushroom.
Dye-decolorizing peroxidase (DyP) is a member of a novel heme peroxidase family (DyP-type peroxidase family), which participates in the process of regulating cell oxidative stress response and matrix degradation. A gene coding DyP-type peroxidase from Volvariella volvacea was obtained, and named as VvDyP. The DyP-type peroxidase gene contains 2 333bp, including 8 exons and 7 introns; besides the open reading frame (ORF) is 1 485bp, encoding a polypeptide of 818 amino acids. The analysis of phylogenetic tree showed that the amino acid sequence of DyP-type peroxidase from V. volvacea shared the highest homology with that from Coprinopsis cinerea and Pleuotus ostreaus. The digital gene expression profiling (DGE) about this gene was studied and a RT-qPCR was designed to verify the former result. It’s turned out that the DyP-type peroxidase encoding gene has the highest expression in the primordial stage of V. volvacea fruit body development, inferring that the DyP-type peroxidase gene can clear the excess of reactive oxygen species in order to ensure the normal formation of primordia.
Edible (medicinal) fungi produce a variety of enzymes for the degradation of plant lignocellulose materials to facilitate infection and gain nutrition. Identifying and comparing enzymes from edible (medicinal) fungi with different nutritional modes may provide information for better understanding of their life styles and further improving of their culture conditions. Glycoside hydrolases (GHs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), glycosyltransferases (GTs), carbohydrate-binding modules (CBMs), auxiliary activities (AAs) and cytochromes P450 were systemically identified in the predicted genomes of 46 edible (medicinal) fungi and 3 representative lignocelluloses-degrading fungi. Comparative analysis revealed that edible (medicinal) fungi exhibited tremendous diversity in the number and variety of enzymes that play major roles in lignocellulose degradation. The result indicated the relationship between enzyme diversity and nutrition models. In general, saprophytic fungi have greater number of enzymes than symbiotic fungi, and brown rot fungi have smaller number of enzymes than white rot fungi and straw rot fungi.
Three Flammulina velutipes strains were used to study the effects of corncob and cottonseed hull as well as different carbon and nitrogen sources on cellulose, hemicellulose and lignin decomposition enzyme activities. The activities of CMCase, xylanase and laccase indicated significant differences among Flammulina velutipes strains (P<0.001), and cultural media significantly affected the activities of CMCase, xylanase and laccase in Flammulina velutipes (P<0.001). The CMCase and xylanase activities on medium with simple carbon sources were significantly reduced than those on medium with complicated carbon sources (P<0.05). The CMCase and xylanase activities on complete yeast medium were significantly decreased than those on media without glucose (P<0.05) or simple nitrogen sources (P<0.05). The laccase activities on media without simple nitrogen sources were significantly lower than those on complete yeast media (P<0.05) and media without glucose (P<0.05), indicating the laccase activities on complicated media were lower than those on media with simple carbon and nitrogen sources.
Flammulina rossica has a wide-ranging medical and industrial applications. The purpose of this investigation is to evaluate the anti-tumor and immunomodulatory activities of exopolysaccharide from Flammulina rossica. After 10 days’ oral administration to Hepatoma 22 (H22) tumor-bearing mice, exopolysaccharide from Flammulina rossica could significantly inhibit the growth of H22 and alleviate the phenomenon of the splenomegaly and thymus atrophy, especially at the high-dose of exopolysaccharide. In the meantime, exopolysccharide could promote the secretion of the cytokines. These data demonstrated that expolysaccharide from Flammulina rossica had potential immunomodulatory activity and might be employed as effective therapeutic agents for the prevention of cancer.
The content of exopolysaccharide and protein were determined from Lentinus lepideus. The polysaccharide content was 21.87%, and protein content was 7.14%. The exopolysaccharide of L. Lepideus was orally administered to S180 tumor bearing mice. The results demonstrated that the exopolysaccharide could significantly inhibit the growth of S180 sarcoma and alleviate the phenomenon of splenomegaly and thymus atrophy, especially at the high-dose(500mg/kg) treatment, and the rate of anti-tumor was 39.44%. The treated groups administrated with low and moderate doses(250mg/kg, 125mg/kg) can also inhibit the growth of tumor, and the tumor inhibition rates were respectively 30.43% and 23.40%. The ELISA method was used to determine various cytokines in serum, and the results showed that the high dose treatment group could promote the secretion of cytokines TNF-α and IL-12. The low dose treatment could induce the increase of IFN-γ and IL-10. These data demonstrate that expolysaccharide from L. lepideus possesses potential immunomodulatory activity. The anti-tumour activity of the exopolysaccharide may be related to its immunomodulatory effects that restore immune organ function, and increase the content of TNF α, IL - 12, IFN - γ and IL - 10.
The culture medium for Pleurotus pulmonarius fermentation was optimized by the single factor experiment of carbon source and nitrogen source and the orthogonal experiment. P. pulmonarius intracellular polysaccharide extraction technique was optimized by using extraction rate as index in the orthogonal experiment with three factors, extraction duration, temperature and liquid-solid ratio. The results showed that the appropriate submerged fermentation culture medium of P. pulmonarius included saccharose 1.5%, bran 5%, peptone 0.6%, KH2PO4 0.15%, MgSO4 0.75% and VB1 0.01%. The highest extraction rate was obtained on condition that liquid and solid ratio was at 50:1 and extracting duration 2h at 90°C. Under such a condition, the polysaccharide extraction rate was 34.35%.
