Ganoderma tsugae, a significant medicinal fungus, has abundant germplasm resources and various bioactive substances, having a long collection and utilization history in China. This paper is the research progress of the germplasm resources and bioactive substances of G. tsugae. G. tsugae is mainly distributed in the northeast region of China. Its artificial cultivation mainly adopts cut-log cultivation mode, using the wood of larch and Mongolian oak as culture medium. Significant achievements have been made in biomimetic cultivation under forest, wild cultivation under forest, greenhouse cultivation and other modes. G. tsugae is rich in active ingredients such as polysaccharides, terpenoids, and sterols, among them polysaccharides and triterpenes are important. Previous studies have shown that they can enhance the body’s immune function, inhibit tumor cell growth and metastasis, and have protective effects on the cardiovascular and cerebrovascular systems. Further research should focus on the extraction and purification of active ingredients, exploration of pharmacological mechanisms, and improvement of artificial cultivation techniques in order to achieve their more wide-ranging development and utilization.
Based on 27 strains of the Auricularia delicata complex collected from China, molecular identification was carried out by using ITS sequences, and 16 phenotypic characteristics were evaluated by means of analyses of diversity, correlation, cluster, and principal component. The results showed that the tested strains were identified as Auricularia sinodelicata and A. delicata of the Auricularia delicata complex. The Shannon-Wiener’s diversity index of 16 phenotypic traits ranged from 0.42 to 1.60, with an average of 0.97; the variation coefficients of 4 quantitative characteristic scope were 4.35% to 26.49%; there is a correlation between some phenotypic characteristics. Cluster analysis indicated that 3 groups were divided at Euclidean distance of 1.54 in accordance with 5 phenotypic characteristics, namely, mycelium colloid, color change of mycelia (cultivated species), main color of ventral side of fresh basidiocarp, texture of basidiocarp and mature period of fruiting body. Principle component analysis changed 16 phenotypic characteristics into 5 principal components, and the cumulative contribution rate was 81.55%; the variance contribution rate of the first principal component was 26.67%, named as the fruit body color characteristic factor, and that of the second principal component was 23.05%, named as mycelium characteristic factor; the variance contribution rate of the third principal component was only 12.89%, defined as growth period-commodity characteristic factor. Through comprehensive analysis, three phenotypic characteristics, mycelium colloid, main ventral color of fresh basidiocarps and fruiting body mature period, were selected as the key indexes for germplasm evaluation.
A wild mycelium sample growing on rotten stump of Pinus yunnanensis collected from the Kunming Botanical Garden in Yunnan Province was identified as Pleurotus citrinopileatus by using molecular phylogenetic method. Morphological characteristics of asexual mycelium and spores were observed under stereomicroscope and light microscope. It was observed that asexual mycelium of P. citrinopileatus were dominated by dikaryotic hyphae with clamp connections and produced asexual spores in three ways: (1) spherical binucleate conidia with size ranging from 1.50 to 4.57 μm formed from short and unbranched conidiophores laterally differentiated from dikaryotic hyphae; (2) elliptical binucleate oidia with size ranging from 1.17 to 3.99 μm and cylindrical binucleate oidia with size being 2.78-5.40×1.57-3.03 μm formed from lateral branches of dikaryotic hyphae; (3) cylindrical uninucleate oidia with size being 3.71-6.70× 3.60-4.02 μm formed in chains from lateral monokaryotic branches of hyphae. The microscopic characters of asexual mycelium and spores were described and discussed in detail for the first time, with attached figures or drawing.
Molecular mechanism of Flammulina filiformis fruiting body development and differentiation from primordium formation to stipe elongation stages remains unclear. In this study, combined analyses of transcriptome and proteome were carried out to reveal the expression patterns of genes and proteins during the development of Flammulina filiformis fruiting bodies from primordium formation to stipe elongation stages. Comparative analysis of the transcriptome and proteome in primordium and stipe elongation stages revealed that there were 1 220 differentially expressed genes (DEGs) and 97 differentially expressed proteins (DEPs) in the stipes, and 2 329 DEGs and 387 DEPs in the pileus. In the functional clustering of GO (gene ontology), these DEGs and DEPs mainly enriched in catalytic activity and cell components. Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis showed that in primordium and elongated stipe 105 DEGs were annotated to 20 pathways in which 19 pathways were related to metabolism; in primordium and pileus of stipe elongation stage 163 DEGs were annotated to 20 pathways in which 18 pathways were related to metabolism. Additionally, the differential protein interaction network was analysed and eight differentially expressed genes were randomly selected for real-time quantitative PCR (RT-qPCR). In this study a data reference for further understanding the growth and development mechanism of Flammulina filiformis fruiting bodies was provided, and candidate functional genes were screened for further study of molecular regulatory mechanisms of fruiting body differentiation and development of Flammulina filiformis and other edible fungi.
The stipe is the main edible part of Flammulina filiformis. The elongation position of the stipe is mainly located at the upper part, however, the regulation mechanism of stipe elongation is not well understood so far, and the related genes are rarely reported. In this study, the elongation characteristics of the upper, middle and lower parts of the stipe were analyzed, and the transcriptomic analysis and qRT-PCR verification were performed. The results showed that there were 532 DEGs in the middle part vs. lower part and 3 522 DEGs in the upper part vs. middle part of the stipe. GO function annotation of DEGs of upper part vs. middle part mainly included ribosome structure composition, structural molecular activity, transport activity and transmembrane transporter activity. KEGG analysis showed that up-regulated genes were significantly enriched in ribosome, steroid biosynthesis, DNA replication and other pathways, while down-regulated genes were significantly enriched in secondary metabolites and amino acid biosynthesis, glycolysis and other metabolic pathways. The DEGs with the highest difference multiples included chitinase, glycosyl hydrolase and transcription factors, etc. qRT-PCR results proved that the transcriptome data were accurate and reliable. This work comprehensively demonstrated the pathways and candidate genes that may be involved in the regulation of stipe elongation of F. filiformis, providing gene resources for subsequent gene function studies and molecular breeding of F. filiformis.
Auricularia heimuer is an important edible and medicinal fungus, which contains rich nutrients and bioactive components, and is widely cultivated in China. Targeted metabolomics was used to examine the variations in sugar, flavonoid, polyphenol, and essential amino acid content in A. heimuer grown on sawdust (SW), corncob (CO), and cottonseed shell (CH). It was found that the lowest total sugar content was demonstrated in the fruiting bodies grown on SW substrate. There was no significant difference in total flavonoid content in the fruiting bodies grown on the different substrate. Vitexin content was the highest in the fruiting bodies grown on CO substrate, but the lowest in those grown on CH substrate. Polyphenol content was usually higher in the fruiting bodies grown on SW substrate. The total content of amino acids and their derivatives was the highest in the fruiting bodies on CH substrate, and the levels of nine essential amino acids, especially lysine and histidine having substantial biological activities, were much higher in fruiting bodies on CH substrate than in those on the SW and CO substrates. The comprehensive determination of the content of sugars, flavonoids, polyphenols and essential amino acids in the fruit bodies of A. heimuer cultivated in different substrates can provide clues and basis for identification of nutritional quality of A. heimuer and development of functional products with different functions.
Biotic perception and response to abiotic stress can improve the environmental adaptability of organisms. Cdc24 gene is usually called guanylate exchange factor of small molecule G protein Rho family, involving in biological processes such as cell division, proliferation and differentiation. The function of Cdc24 gene in edible fungus Flammulina filiformis has not been studied. In this study, RNAi transformants of Cdc24 gene in F. filiformis were constructed by Agrobacterium-mediated genetic transformation. The results showed that the interference of Cdc24 gene significantly reduced the tolerance of mycelium to abiotic stresses such as salt, high temperature, low temperature, oxidation and acid-base. The polysaccharide and polyphenol content of RNAi transformant strains were lower than that of wild-type strains. The number of fruiting bodies and biological efficiency of RNAi transformant strains were also significantly lower than those of wild-type strains. This study shows that Cdc24 gene plays an important role in abiotic stress tolerance and fruiting body development of F. filiformis, and provides some data reference for variety improvement and efficient cultivation and production of F. filiformis.
Dikaryon-monokaryon (di-mon) mating is an important breeding method of edible basidiomycete mushrooms. Currently, research on the leading nucleus in di-mon mating is not sufficient. In this study, the genetic patterns of the leading nuclei in di-mon mating were explored. A complete compatibility di-mon mating test was conducted by using three Hypsizygus marmoreus dikaryon strains, B3, X3, and NN12, and nine monokaryon strains, H3-8, H4-4, H4-10, H13-3, H13-6, H14-10, NN12-1, X3-1A, and X3-13, as tested strains. Molecular markers based on mating type genes were developed to identify the leading nuclei. The results showed that hybridization pairs using B3, X3, and NN12 as the nuclear donors obtained 89, 87, and 75 di-mon mating hybrid strains, respectively. The results of mating type molecular marker identification showed that the ratio of the two nuclei becoming leading nuclei in the nuclear donors B3, X3, and NN12 were 41:48, 44:43 and 36:39, respectively, all conforming to the statistical ratio of 1:1, indicating that the probability of the two nuclei in the tested dikaryotic strains of H. marmoreus serving as leading nucleus is the same.
Vegetative incompatibility (Ⅵ) is ubiquitous between single-ascospore isolates originated from the same ascocarp of Morchella importuna. In this study, ascocarps of M. importuna with different genetic background were used as materials for obtaining single-ascospore isolates from the same asci, and the vegetative compatibility relationship between the isolates originated from the same asci and different asci were analyzed. Mating type and the genotype of mimpvic32 and mimpvic33 of the isolates were identified. The results showed that no more than four vegetative compatibility groups (VCGs) were divided among the offsprings of YPL6 populations. Parent Y282 and Y346 were vegetatively compatible, and eight isolates originated from the spores of same asci of the progeny ascocarps were vegetatively compatible, and all the progeny single-ascospore isolates were compatible and formed a VCG with the parent. Hybridization were performed between Y282 and Y134 showing typeⅠbarrage, and between Y282 and Y88 showing typeⅡbarrage, and eight isolates originated from spores of the same asci of the progeny ascocarps formed 2-3 VCGs. When Y282 crossed with the wild isolates ZQW10 and ZQW31 respectively, eight isolates originated from spores of the same asci of the progeny ascocarps were divided into 4 VCGs, and the single-ascospore isolates originated from different asci of the same ascocarp were generally incompatible and produced more VCGs. It is deduced that the Ⅵ is controlled by multiple loci in M. importuna, and all the progeny isolates become a VCG when there are no genetic differences in the Ⅵ regulatory loci between parents. The more the numbers of regulatory loci with genetic difference exist between parents, the more the numbers of VCG are formed by the progeny single-ascospore isolates. During spore development in an ascus, the ways of chromosome recombination, exchange and segregation are different at the stage of meiosis, and genetic and phenotypic differences produce between spores.
A wild Morchella strain MS-1 was isolated from an ascocarp collected from high-altitude areas in Zhouqu County, Gansu Province. Identification based on multi-gene analysis of ITS, RPB1, RPB2 and EF1-α proved that this wild strain was M. sextelata. The optimal temperature for the growth of wild strain MS-1 during the stock culture was 18-20 ℃; the minimum growth temperature was 1 ℃, and the maximum 33 ℃. The optimal pH value is 6-7. The optimal carbon source is lactose and soluble starch. The optimal nitrogen source is yeast extract and beef dipping powder. The optimal C/N ratio is 40:1-45:1. The lethal temperature under 2 h high-temperature treatment during stock culture is 44 ℃, while that during expanded cultivation is 48 ℃; the yield per unit area is 0.6 kg/m2, significantly higher than that of commercial production strains using for comparison. Its agronomic traits are excellent, with a medium cultivation period of 140 d. Comprehensive analysis shows that the wild M. sextelata MS-1 has potential to be further developed as a new commercial production strain.
A wild Panus conchatus strain collected from Milin County, Nyingchi, Xizang Autonomous Region was identified by morphological observation and ITS sequencing analysis. The biological characteristic research and cultivation experiments were carried out. The results showed that the most suitable carbon source for domestication was fructose, and the most suitable nitrogen source was yeast powder. The fungus grew well at the optimal pH of 6.0 and the optimal temperature of 30 ℃. Bag-cultivation showed that the mycelium bagful time was 22 days; the primordium appeared in 20-30 days and the harvest time was 10 days. The average fresh weight of a single fruiting body was 24.12 g. This study provides a scientific reference for further industrial production of Panus conchatus.
The fruiting bodies of a wild Sparassis species collected from Milin, Xizang, were identified on the basis of morphology and molecular analysis as Sparassis subalpina. The pure mycelium (XQ-1) was isolated from the fruiting body by tissue isolation method. The optimal stock culture medium was screened, and biological characteristics and domestication of the strain were studied. The most suitable stock culture medium for XQ-1 strain was carrot pine sawdust dextrose agar (CSDA) medium. Single factor experiments showed that the optimal carbon and nitrogen sources for mycelium growth were glucose and malt powder, respectively, and the optimal carbon-nitrogen ratio was 5:1 and 10:1. The optimal pH was 6.0-6.5; the optimal inorganic salt was magnesium sulfate; adding vitamin C and vitamin B1 (respectively 20 mg/L) to the medium could stimulate the mycelium growth; the optimal growth temperature was 21 ℃. Orthogonal experiments showed that fructose 20.0 g/L, malt extract 2.0 g/L, pH 6.5 and magnesium sulfate 0.1 g/L were the best medium combinations. The highest enzyme activity of hydroxymethyl cellulase was 191.62 U/mL. Adding wheat flour, barley flour and corn flour to the cultivation substrate could successfully speed up fruiting body production, and peanut flour, barley flour and glutinous rice flour could play a positive role in the growth of mycelium.
A wild polypore strain XHMK collected from chestnut cultivation area in Qianxi County, northern China, was identified as Trametes sanguinea based on morphological features and molecular data. The effects of different factors on the mycelial growth of this strain were explored through single factor experiments. The optimal carbon source, nitrogen source, and inorganic salt for the mycelial growth were fructose, yeast extract powder, and potassium dihydrogen phosphate, respectively. The optimal pH and temperature for the mycelial growth were 7 and 35 ℃, respectively. The orthogonal experiment showed that the optimal combined conditions of mycelium growth for this strain were fructose 25 g/L, yeast extract 2 g/L, potassium dihydrogen phosphate 1 g/L, and pH 6.5. The optimized cultivation substrates for domestication were 70% chestnut sawdust, 10% corn cob, 19% bran, and 1% gypsum. It took 36 days to fulfill the cultivation period, and the biological efficiency was 9.49%.
Domestication and cultivation studies of wild Pholiota limonella strains collected from Shandong Province were carried out. Formulation consisting of 40% of cottonseed shell, 40% of wood chips, 10% of bran, 8% of corn flour and 2% of gypsum powder was suitable for artificial cultivation. By using this formula, the biological efficiency of the first two crops was 54%, and the biological efficiency increased by 7.2%, reaching 57.90%, after three consecutive generations of domestication. The number of fruiting bodies per cluster increased by 1.66 times as against the original wild strain. The commodity character is good, and the domestication cultivation effect is remarkable. The biological efficiency of the top fruiting mode of bag-cultivation was 56.30%, significantly higher than that of the porous fruiting mode in perforated bag. The results of orthogonal experiment showed that the four factors tested had significant effects on the biological efficiency of P. limonella, among which the combination of 50% of Lentinula edodes residue, 20% of wheat bran, 2% of shell powder, 1% of gypsum powder and 27% of wood chips was the optimized alternative formula. Nutrient analysis of fruit body protein and amino acid showed that the protein content was 35.17% and the amino acid scored 64.7 points, and umami amino acid content accounted for 44.52% of the total amino acid, indicating high nutritional value. It is considered that P. limonella is a rare edible fungus with great development potential, and this study provide reference for artificial cultivation and application of this mushroom.
Diabetes is one of the most common chronic diseases, capable of generating various complications. α-Glucosidase serves as a crucial therapeutic target for diabetes, and the exploration of novel α-glucosidase inhibitors holds significant importance for diabetes treatment. The chemical constituents from fruiting body of Phellodon cinereofuscus and their inhibitory activities against α-glucosidase were studied. Sixteen compounds from 70% ethanol extract of P. cinereofuscus were isolated and purified by silica gel column, HP20 column chromatography, ODS column chromatography and high performance liquid chromatography (HPLC). The structures of them were identified by nuclear magnetic resonance (NMR) as (22E,24R)-3β- hydroxy-24-methylcholesta-5,22-dien-7-one (1), (22E,24R)-ergosta-4,6,8(14),22-tetraen-3,15-dion (2), 5α,8α-epidioxyergosta-6,9,22-trien-3β-ol (3), (22E,24S)-cerevisterol (4), 20R,24R,9α-hydroxy- Δ4,6,8(14),22-tetraene-3,15-dione-ergosterol (5), glaucoposterol B (6), (22E,24R)-5α,6α-epoxyergosta- 8(14),22-diene-3β,7α,15α-triol (7), 3β-Hydroxyl-(22E,24R)-ergosta-5,8,22-trien-7,15-dione (8), 5α,6α-epoxy-(22E,24R)-ergosta-8(14),22-diene-3β,7α-diol (9), (22E,24R)-5α,6α-epoxy-ergosta- 8,22-dien-3β,7α-diol (10), demethylincisterol A3 (11), betulinic acid (12), lup-20(29)-en-3α,28- diol (13), (Z)-6-nonadecenoic acid (14), tetradecanoic acid (15), hexacosane (16). All the compounds were obtained from the genus of Phellodon for the first time. The inhibitory activities of compounds 1−16 against α-glucosidase were evaluated. Compounds 3, 6, 11, 13 and 14 showed significant inhibitory activity against α-glucosidase with IC50 values of (19.51±5.68), (6.29±1.16), (12.15±2.21), (6.81±0.26), (10.26±1.84) μmol/L respectively. The results provide theoretical support for further research of the medicinal function of P. cinereofuscus.
Single factor and orthogonal experiments were conducted on the biological characteristics of the wild strain of Fulvifomes nonggangensis collected from Nonggang Nature Reserve in Guangxi, and domestication cultivation of the fungus was attempted. Preliminary research was also conducted on the antioxidant activity of water extract of the cultivated fruiting bodies. The results showed that the optimal combination of culture condition was fructose as the carbon source, yeast extract as the nitrogen source, culture temperature of 28 ℃, and pH value of 6.5; temperature had the greatest influence on mycelial growth. Domestication cultivation experiment indicated that formulation of cottonseed hulls 40%, sawdust 38%, bran 20%, lime 1%, and gypsum 1% was the best combined substrate, displaying colonial growth rate of 0.33 cm/d, average yield of 27.26 g/bag, and biological efficiency of 12.98%. Water extract of fruiting body was obtained by using ultrasonic method. When the extract concentration was up to 5 mg/mL, the clearance rate of DPPH reached 84.45%, when it was only 2.5 mg/mL, the clearance rate of ABTS still reached 93.82%, indicating fairly good antioxidant activity of the fungus. This data hopefully gives support to the large-scale cultivation and industrial utilization of F. nonggangensis in future.
At present, there is limited research on the nutritional physiology of Phlebopus portentosus, and there are no reports on the changes of micromolecule carbohydrates in the fruiting body of P. portentosus at different growth stages. In this study, the content of micromolecule carbohydrates in fruiting bodies of P. portentosus was determined and analyzed at different stages of fruiting body growth. The results showed that the content of trehalose and mannitol was relatively high in the fruiting bodies of P. portentosus, and the range of change was great during the growth process. The trehalose content at harvest stage was 13.72 times that at initial stage. Trehalose and mannitol played an important role in the transportation of carbon source in P. portentosus. Adding trehalose and mannitol to the culture medium increased the yield of fruiting bodies, and the yield increased by 4.15% when solely adding trehalose. Further research on revealing the nutritional and physiological rules of P. portentosus and improving the formula of culture medium will be continued.
For increasing the melanin yield in Auricularia heimuer fermentation broth, fermentation medium formula of the experimental strain A. heimuer 1703 was optimized by single factor experiments and response surface methodology. The stability and antioxidant activities of melanin were determined. The results showed that sucrose, peptone, and MgSO4 had a significant impact on the melanin yield of A. heimuer fermentation broth. The optimal fermentation medium for increasing A. heimuer melanin yield was potato of 200 g/L, sucrose of 21.40 g/L, peptone of 5.34 g/L, and MgSO4 of 0.56 g/L. Under these conditions, the melanin yield was 0.050 g/100 mL. A. heimuer melanin has good stabilities under different illumination condition and temperature, and in solution containing different metal ions, showing pigment retention rate of over 90%. A. heimuer melanin has strong DPPH free radical, superoxide anion free radical scavenging abilities, and iron reducing ability. The IC50 values for DPPH free radical and superoxide anion free radical were 0.021 mg/mL and 0.90 mg/mL, respectively. This study provides theoretical reference for the liquid fermentation production and application of A. heimuer melanin.
Three new varieties of ‘Sichuan Morel Series’ were bred by systematic breeding method. ‘Sichuan Morel No. 7’ is Morchella eximia, with typical characteristics of having grayish-brown and almost conical pileus with round and blunt-top and without obvious ridge. ‘Sichuan Morel No. 8’ is M. sextelata, characterized by reddish-brown to dark reddish-brown pileus with moderately dense ridge; longitudinal ridge is extremely obvious, and the colour of hymenia is fleshy-pink. ‘Sichuan Morel No. 10’ is M. importuna, characterized by grayish-brown, nearly triangular, blunt-topped and moderately ridgy pileus, with light grayish-brown hymenia.
Flammulina filiformis ‘Nongwanjin 8’ is a new factory-cultivated white variety bred and screened through monospore hybridization of ‘F0321’ and ‘F0323’. The fruiting bodies are clustered and white, with less villi at the base; the cap is slightly larger, and the stipe is slightly thicker, and the fruiting body is firm and storable. Its average yield is 365 g/bottle, and the cultivation cycle is 45 days. F. filiformis ‘Nongwanjin 9’ is also a new factory-cultivated white variety bred and screened through double-mono hybridization of ‘F0321’ and ‘F0323’. The fruiting bodies are white, with small and neat caps, hard stipes and tight roots; the longitudinal section of pores are even; the moisture content is low; the properties are good, and the average yield is 436 g/bottle, and the cultivation cycle is 45 days. Hypsizygus marmoreus ‘Nongwanzhen 1’ is a new factory-cultivated white variety bred and screened through monospore hybridization of ‘B01’ and ‘B02’ with more fruiting, thin stipes and appropriate hardness. The fruiting body is slightly whiter than that of the parent ‘B01’ and plump. The average yield is 284 g/bottle and the cultivation cycle is 113 days.
