The scientific name of the widely cultivated Ganoderma species in China is discussed. The historical changes and disputes of the different names used by different authors are reviewed. It is confirmed that the scientific name of the widely cultivated Ganoderma species in China should be G. lingzhi. Ganoderma sichuanense is an independent species different from G. lingzhi in morphology and molecular sequence. The location of the epitype of G. sichuanense is far away from that of the holotype, and the altitude and ecological habits of the epitype are also different from those of the holotype. The epitype of G. sichuanense represents a different species from G. sichuanense and thereby should be abolished.
Dielectric barrier discharge (DBD) plasma was applied to treat the protoplast of Ganoderma lingzhi for obtaining high-nano-selenium mutant. Under the condition of experimental parameters of voltage 15.6kV, current 1.8mA, discharge frequency 1.8kHz, and the treatment duration of 2.5min, the optimal effect of DBDP mutagenesis was obtained, and six mutants were screened by random amplified polymorphic DNA (RAPD) labeling approach. Multiple-generation culture showed that mutant H10 produced the highest yield of nano-selenium. Compared with the original strain, the selenium content in the mycelium of H10 increased by about 30%. This study proves that plasma mutagenesis seems feasible for obtaining excellent Ganoderma lingzhi mutant strains with the high-yield of nano-selenium.
Mitochondria are “power plants” of the cell that are capable of oxidative metabolism for energy conversion, participating in the tricarboxylic acid cycle to form ATP. With the development of molecular biology and bioinformatics technology, several mitochondrial genomes of Ganoderma species have been reported recently. However, there has been no report on the mitochondrial genome of G. multipileum. In this study, the mitochondrial genome of G. multipileum YX was assembled and annotated, and the differences between the mitogenome and that of other Ganoderma species were compared and analyzed. According to differential sequences, the molecular markers were constructed. It is indicated that the mitogenome of G. multipileum has a closed loop structure of 67 340bp, containing 15 common protein coding genes, 28 tRNA genes, and subunit gene of rRNA. There are five genes in total, containing 12 introns which are of mainly IB type and partly ID type and including LAGLIDADG_1 superfamily, and GIY-YIG_Cterm superfamily conserved domain. According to the specific primers designed by the mitochondrial cox2 gene of G. multipileum, the cox2 gene fragments of four G. multipileum strains can be accurately amplified while the cox2 gene fragments of other Ganoderma species cannot be amplified. Based on the systematic evolutionary tree constructed by mitochondrial cox2 gene sequences of G. multipileum strains, four strains of G. multipileum can be classified into the same branch, with homology of 98%, being consistent with the ITS identification results. The result proves that G. multipileum can be quickly identified by mitochondrial gene-based cox2-specific primer, and we only need to observe the presence or absence of the amplified band without sequencing.
The agronomic characteristics, microstructures and phytochemical content of four cultivated varieties (G-HN01, G-LQ01, G-XZ01 and G-LQ02) of Ganoderma lingzhi were compared. The four strains were cultivated in cultivation sacks. The result was that the four varieties were significantly different in mycelial growth rate and cultivation characteristics. Observation under the scanning electron microscope (SEM) showed that no-sporulation variety exhibited distinct feature in microstructure of fruit body as compared with other three sporulation ones. Comparsion of phytochemical content (polysaccharides, total triterpenoids and total phenol) of fruitbody and antioxidant capacity showed significant difference between three sporulation varieties, but no difference was found between sporulation varieties and no-sporulation variety.
Mitochondrion is an important organelle carrying its own DNA, being essential for conversion of ATP into energy in eukaryotic cells. Many mitochondrial genomes of the genus Ganoderma have been reported so far. However, the intraspecific differences in nuclear and mitochondrial genomes of Ganoderma species are rarely reported. In this study, mitochondrial genomes of two Ganoderma lingzhi strains were assembled and annotated, and molecular markers were developed based on differential mitogenome fragments for intraspecific classification. The results showed that the mitogenome of G. lingzhi is a closed ring structure ranged from 49 233bp to 61 563bp in size, containing 15 common protein coding genes, small and large subunit genes of rRNA and 26 amino acids carrying tRNA genes. The main differential fragments located in introns, large subunit and intergenic regions. The phylogenetic tree was constructed based on different fragment sequences, and the results indicated that the sequences of cob gene and cox2 gene could be used in interspecies identification of Ganoderma. Monokaryotic commercial strain 119 (Fujian, China) and sporeless strains W (Fujian, China) of G. lingzhi were used in construction of the isonuclear alloplasmic TY-W and TY-119 and the effects of mitochondria on the morphology, mycelial growth rate and triterpenoid composition of their fruiting bodies were evaluated. It was found that some differences appeared in the colony morphology of isonuclear alloplasmic TY-W and TY-119. The mycelial growth rate of TY-W was 4.77mm/d, 1.06 times faster than the 4.5mm/d growth rate of homonuclear heterogeneous TY-119. The content of polysaccharide in mycelial stage and fruiting stage of TY-W was 4.45mg/g and 12.14mg/g respectively, being 1.38 times and 1.19 times the content in mycelial stage (3.23mg/g) and fruiting stage (10.24mg/g) of TY-119. Triterpenoids in mycelial stage and fruiting stage of TY-W was 6.82mg/g and 11.45mg/g, respectively, being 0.74 times and 1.26 times the content in mycelial stage (9.26mg/g) and fruiting stage (9.10mg/g) of TY-119. Based on high performance liquid chromatography (HPLC) analysis, the content of ganoderic acids A (3.77μg/mL), E (14.92μg/mL) and F (12.91μg/mL) of TY-W in fruiting stage is respectively 1.46 times, 1.17 times and 1.01 times that of TY-119. The key genes’ (pgm, ugp, gls, hmgs, hmgr, mvd, ls, fps and sqs) expression levels of polysaccharide and triterpenoid synthesis pathways in isonuclear alloplasmic fruiting bodies showed significantly different between TY-W and TY-119. In conclusion, the differences of mitochondria influenced the colony morphology, mycelial growth rate and the polysaccharide and triterpenoid content as well as key genes expression in G. lingzhi strains. Further studies are needed to improve our understanding of fungal mitochondrial genome.
As a key transcription factor in fungal nitrogen metabolism, AreA functions in growth, secondary metabolism and stress resistance. In this study, the full-length sequence of AreA was obtained by bioinformatics comparison analysis, and the influence of AreA on growth, resistance to cell wall stress and secondary metabolism were studied. The C terminal domain of AreA contained a conserved zinc finger domain. qRT-PCR analysis indicated that AreA could response to different nitrogen sources. AreA transcription level increased under low nitrogen concentration condition as compared with that under high nitrogen concentration condition. The growth rate of AreA silenced strains was significantly lower (decreased by 70%) than that of the control strains. It was found that the hyphae of AreA silenced strains were fluffier than those of the control strains. Detection of the composition of cell wall indicated that the content of β-glucan and chitin decreased by 65% and 40%-60%, respectively as compared with wild type (WT) and CK strains. The tolerance to cell wall stress was examined and the results showed that AreA silenced strains were sensitive to Congo red and SDS, and the growth stagnated on the plates. The content of ganoderic acid in AreA silenced strains decreased by 23% as compared with that in the control strains. Our study indicated that AreA as a transcription factor could response to the nitrogen source and play important role in growth, secondary metabolism and resistance to the cell wall stress in G. lingzhi.
Ganoderic acid (GA) is one of the major secondary metabolites in Ganoderma lingzhi, a basidiomycete with high commercial value. Calmodulin (CaM) is a Ca 2+ sensor in eukaryotes and participates in signaling pathways that regulate many crucial processes such as growth, development and secondary metabolism. Sequence analysis revealed that CaM of G. lingzhi (GlCaM) encoded 149 amino acids with a high evolutionary conservation and comprised four E-F hand domains, each containing one conserved D-x-D motif. CaM gene-silencing strains were generated by RNA interference (RNAi) to examine the effect of CaM on the growth, development and secondary metabolism in G. lingzhi. The result showed that GA level in wild-type strain (WT) was 34% higher than that in GlCaM-silenced strain. The growth rate of GlCaM-silenced strain was significantly lower than that of WT. The results obtained in this study may facilitate further investigation of the mechanism underlying CaM function and pathway in fungi.
The dynamic characteristics of triterpene acid produced by submerged fermentation of Ganoderma lingzhi before and after adding 9,10-methylene hexadecanoic acid (9,10-CMA) were studied by using Sigmoid model. The results showed that triterpene acids were mainly synthesized at days 4-9 and reached the maximum at day 9 (268.62mg/L) in experimental control, while the synthesis rate was relatively fast, reached the maximum at day 8 (343.52mg/L) under the treatment of adding 9,10-CMA. After adding 9,10-CMA, the utilization rate of substrate glucose in G. lingzhi cells increased, and the cell specific growth rate (μ) reached the maximum of 0.94d -1at day 3.2 (μmax), being higher than that of the experimental control (0.88d -1 obtained at day 3.4); the specific consumption rate of glucose reached the maximum of 8.34d -1 at day 1.7 (qS, max), significantly higher than that of the control (6.80d -1 acquired on day 2.1). Intracellular triterpene acids were synthesized at a rate of 13.76d -1 (qITA, max) at days 6.2, being 1.42 times higher than that of the control (9.66d -1). The triterpene acid synthesis of G. lingzhi in both experimental groups showed a partial coupling relationship with cell growth. After adding 9,10-CMA, there was no change in the relationship between cell growth and triterpene acid synthesis in the fermentation process.
Ganoderma boninense is one of the most important wood-inhabiting fungi mainly distributed in tropical areas and is a serious pathogen of oil palms in Malaysia and Indonesia. In this study, three strains of the fungus isolated from basidiocarps growing on the base of living oil palms in the plantation land of Jeram, Selangor, Malaysia, were evaluated for their antioxidant activity. The mycelia and fermentation broth were sampled on the second, fourth, sixth, eighth, tenth, twelfth and fourteenth days during liquid cultivation. Mycelial biomass and the content of crude polysaccharides, polyphenols, flavonoids and ascorbic acids, as well as eight antioxidant indicators of fermentation broth were determined. The supernatant of the fermentation broth of the three strains all showed significant antioxidant activities, although different strains showed variation in level and appearance time of the content of certain secondary metabolites and antioxidant activities. Our results indicated that G. boninense had higher content of polysaccharides and polyphenols and stronger ferric ion reducing antioxidant power than previously well-studied medicinal Ganoderma lingzhi and Sanghuangporus quercicola. Facts have proved that G. boninense showed valuable for artificial cultivation and further intensive medicinal studies.
The respiratory rate, ganoderic acid (GA) content, total phenolic content, reactive oxygen species (ROS) content, malondialdehyde (MDA) content, antioxidant enzyme activities, xanthineoxidase (XOD) activity, succinic dehydrogenase (SDH) activity, H +-ATPase and Ca 2+-ATPase in Ganoderma lingzhi fruiting body were investigated under the treatment of 60% oxygen. The results showed that high oxygen inhibited the respiratory rate of G. lingzhi fruiting body, and the content of H2O2 and O2 -? were higher than that in experimental control in the first day. However, the production of ROS retarded with the development of high oxygen treatment, and the MDA content reduced. The activities of SOD, CAT and SDH were enhanced and the content of GA and total phenol increased. It is suggested that suitable environmental stress can stimulate the fungus to activate its antioxidant system to protect itself from oxidative damage, and enhance the biosynthesis of related secondary metabolites.
Sugarcane fiber was used as the immobilized carrier of Ganoderma lingzhi mycelia. The mycelial immobilized time of the carrier was determined by scanning electron microscope observation, and the carrier shape, size and quantity were determined by mycelial pellet size, biomass, extracellular polysaccharide and extracellular triterpene content of liquid-fermented G. lingzhi. The results showed that inoculation of six pieces of cylindrical-shaped and 1.5cm×1.5cm sized (diameter×height) (Y1.5) mycelial carriers undergoing immobilized period of seven days could successively and stably maintain fermentation for seven generations. The liquid fermentation seeds were prepared by fixed fermentation of sugarcane fiber carrier. The size of mycelial pellets was uniform after fermentation, and the biomass, extracellular polysaccharide content and triterpenoid content increased by 78%, 84% and 60%, respectively.
A crude polysaccharide was isolated from basidiospores of Ganoderma lingzhi by hot water extraction and ethanol precipitation, and two main constituents, LBPI and LBPII, were collected from Sepharose-CL 6B gel chromatography assay. The molecular weight of LBPI and LBPII was determined to be 9.17×10 4 and 1.86×10 4 respectively by HPLC. GC analysis, periodate oxidation, methylation and GC-MS analysis were used to elucidate the monosaccharide composition and structure of LBPI and LBPII. The results indicated that LBPI was consisted of 1→ linked, 1→4,6 linked and 1→3,6 linked glucose residues, 1→6 linked galactose residue and 1→3,6 linked mannose residue; LBPII was consisted of 1→ linked rhamnose residue, 1→ linked, 1→4 linked, 1→6 linked, 1→4,6 linked and 1→3,6 linked glucose residues, 1→6 linked galactose residue and 1→2,3,6 linked mannose residue. Both LBPI and LBPII are multi-branched heteropolysaccharides, however, the monosaccharide composition and linkage of the two are different. Both polysaccharides are reported for the first time.
Eleven compounds were isolated from mycelial solid culture of sporeless Ganoderma lingzhi Longzhi No. 2 by preparative high-performance liquid chromatography (HPLC), gel chromatography and preparative thin layer chromatography (PTLC), etc. On the basis of their spectral data, they were identified as ganoderic acid P (1), ganoderic acid T1 (2), ganoderic acid Mk (3), ganoderic acid S (4), ganoderic acid T (5), ganodermanondiol (6), ganoderic acid Me (7), 5α, 8α-epidioxyergosta-6, 22-dien-3β-ol (8), ganoderic acid R (9), lanosta-7, 9(11), 24-trien-3α-hydroxy-26-oic acid (10), and ganodermenonol (11). Ganoderic acid T1 was first discovered as natural product. All the compounds showed strong proliferation inhibition to tumor cell L1210 with IC50 values of <39.69μmol/L.
The content of four heavy metals (lead, cadmium, mercury and arsenic) in culture substrate and different growth stages of fruiting bodies of different varieties of Ganoderma lingzhi cultivated in different substrates by use of different cultivation methods was tested. The results showed that the content of heavy metals in the fruiting bodies of G. lingzhi cultured in “juncao” and that cultured in sawdust not exceeded the provided national standards. Although the heavy metal content in “juncao” substrate was higher than that in sawdust substrate, the heavy metal enrichment rate of G. lingzhi cultured in “juncao” was much lower than that cultured in sawdust. Therefore, “juncao” is an ideal substrate for replacing sawdust for cultivation of G. lingzhi.
Fruiting bodies of two cultivated strains of Ganoderma lingzhi were cultivated in two different substrates using two different management methods and then collected at different growth stages. The triterpenoids of fruiting bodies were extracted with 95% ethanol by ultrasound. The content of triterpenoids and sterols was determined by chemical spectrophotometry, and the composition of triterpenoids was analyzed by high performance liquid chromatography (HPLC) and the antitumor activity was tested in vitro. The results indicated that the content of triterpenoids and sterols was 4.60-6.20mg/g. Analysis of HPLC showed that there were significant differences in species and content among 10 triterpenoids obtained. All the extracts showed inhibiting effect on proliferation of tumor cell L1210 in vitro to a certain extent, and the inhibiting activity of the extracts obtained in cap formation stage was stronger than that obtained in other growth stages.
Effects of ethanol concentration on the extraction of triterpenoids in Ganoderma lingzhi and the activity of the extract against prostate cancer cell LNCaP are evaluated. Nine representative triterpenoids in Ganoderma lingzhi were determined with the help of HPLC. The activities of extracts obtained under different ethanol concentration inhibiting LNCaP from proliferation and migration were determined. HPLC results showed that under ethanol concentration of 50%-80%, the content of nine triterpenoids increased with the increase of ethanol concentration. The content of triterpenoids decreased when ethanol concentration attained to 95%. Cell experiments showed that all ethanol extracts obtained under different ethanol concentration had a good effect in inhibiting the proliferation and retarding the migration of LNCaP, and concentration of 80% ethanol is more appropriate for the production of Ganoderma lingzhi products with anti-prostate cancer activity.
The effects of 14 additives or compounds on the growth and synthesis of polysaccharides and beta-glucan of fermentative Ganoderma lingzhi were studied. The results showed that the aqueous extract of Forsythia suspensa fruits (3g/L) had significant promoting effect on the growth of G. lingzhi, and coixenolide (3g/L) had stimulatory effect on the synthesis of intracellular polysaccharides and beta-glucan. However, the water extract of Platycodon grandiflorum roots, cerium ammonium nitrate, praseodymium nitrate, methyl jasmonate and sodium nitroprusside inhibited the growth and polysaccharide synthesis of G. lingzhi. A quadratic polynomial model for the production of beta-glucan using coixenolide as additive was established by Box-Behnken design and response surface methodology. The optimum fermentative conditions for obtaining high yield of β-glucan included adding coixenolide 10.5g/L in culture medium containing inoculum 16% at 88th hour in the process of fermentation under initial pH 7.00. Under such a condition, the yield of beta-glucan reached (40.67±8.43)mg/L, 41.86% higher than the yield obtained in experimental control without adding coixenolide, and the yield of polysaccharide was (1.13±0.21)g/L, 50.66% higher than that in the control. The results indicated that by adding coixenolide the optimized submerged fermentation of G. lingzhi could induce the synthesis capability in a directional way and thus increased the production of polysaccharide and beta-glucan.
Ganoderic acid (GA) possesses significant pharmacological activities, however, low production of GA has become a bottleneck for its widespread application, therefore, it is necessary to improve GA production in fermentation of Ganoderma lingzhi. The effect of microparticle Talc on GA production in submerged culture of G. lingzhi was studied. Our results showed that addition of Talc significantly reduced the diameter of G. lingzhi pellet. The equivalent diameters were (3.33±0.16)mm for experimental control and (2.04±0.12)mm for the experimental groups of adding 0 and 15g/L Talc to liquid fermentation of G. lingzhi. Addition of Talc significantly increased the levels of individual and total GAs as compared to those in the control (without addition of Talc). The obtained maximum content of GA-Mk, GA-T, and GA-Me reached (6.02±0.29), (5.08±0.14), and (1.71±0.09)μg/100mg dry cell weight (DCW), respectively, and the total content of GAs reached (1.51±0.02)mg/100mg DCW under the condition of additional 15g/L Talc in submerged culture, 4, 1.9, 1.4 and 1.6-folds higher than the content in the control, respectively. The maximum accumulated quantities of lanosterol and squalene were (3.69±0.23) and (34.86±6.41)μg/100mg DCW respectively under the condition of additional 15g/L Talc in submerged culture, 2.6 and 4.2 times higher than those in control. The transcription levels of farnesyl diphosphate synthase gene and a CYP450 gene cyp-5150l8 in the GA biosynthetic pathway were up-regulated by 2.35 and 1.53-folds under the condition of additional 15g/L Talc as compared with those in the control.
High-performance liquid chromatography method was established to determine the content of triterpenoids in Ganoderma lingzhi spore powder. 40% ethanol was proved to be the best solvent for extracting the medium-grade triterpenic acid in spore powder, and the extract was concentrated 50 times. The determination method of 13 standard products including ganoderic acid I, ganoderenic acid C, and ganoderic acid C2 were established by optimizing the column and elution conditions. The methodological investigation showed that RSD values of method precision, repeatability and stability were less than 5%, indicating the method was appropriate for the quantitative detection of medium-polar triterpenoids in Ganoderma lingzhi spore powder. The analysis of multiple samples showed that ganoderic acid C6, ganoderic acid G, ganoderic acid A, ganoderic acid D and ganoderic acid F were the main triterpenoids in Ganoderma lingzhi spore powder, and the content of ganoderic acid A was the highest, whose average proportion in the total quantity of triterpenoids was 19.71%. There was no correlation between triterpene content and wall breaking treatment. The content of triterpenoids in spore powder was 14.24-99.70μg/g, only accounting for one percent of triterpenoid content of the fruiting body. The content of triterpenoids in the Ganoderma lingzhi spore oil was less than 50μg/g. The results suggested that the content of triterpenoids in Ganoderma lingzhi spore powder and oil was too low to be regarded as the quantitative detection index of Ganoderma lingzhi spore powder products.
A rapid method for isolation of ganoderenic acid B from fruiting bodies of Ganoderma lingzhi is described. The extract of ganoderma triterpenoid acids (GTA) was obtained from G. lingzhi Hunong No. 1 by using D101 macroporous resin as filler and the extract was eluted by 60% ethanol. The separation of obtained GTA sample was performed by high-speed countercurrent chromatography (HSCCC). The optimal solvent system was determined to be hexane-ethyl acetate-methanol-water (V/V/V/V, 5:5:2:9) with the upper-layer as stationary phase and the sub-layer as mobile phase. The single-factor experiments and orthogonal experiments were carried out to determine the optimal processing for the preparation of ganoderenic acid B. Flow rate of 2.0mL/min, rotational speed of 900r/min, and loading quantity of 500mg were proved to be optimal for the preparation process, and the yield of ganoderenic acid B reached (9.07±0.16)% and the purity reached (85.04±3.45)%. The structure of ganoderenic acid B was identified by nuclear magnetic resonance (NMR) and mass spectroscopy. This method is simple and efficient, and suitable to large scale preparation of ganoderenic acid B.
