Intraspecific Identification of Lingzhi Medicinal Mushroom, Ganoderma lingzhi (Agaricomycetes), Using ISSR Markers.

This study aimed to obtain a set of specific inter simple sequence repeat (ISSR) primers and establish a stable and accurate intraspecific identification method for Ganoderma lingzhi. A total of 117 G. lingzhi strains were identified using internal transcribed spacer sequences from 147 strains determined as G. lingzhi via simple morphological identification. Based on the sequences obtained, specific ISSR primers for G. lingzhi were screened and validated, and 15 specific ISSR primers showed polymorphic banding pattern with clear band resolution. Subsequently, ISSR PCRs of the 15 specific primers were performed for the 117 G. lingzhi strains. As expected, DNA analysis of the ISSR markers could distinguish G. lingzhi strains, with similarity coefficients ranging from 0.11 to 0.89. Thus, the 15 specific ISSR primers can be used for intraspecific identification and polymorphism analysis of G. lingzhi.

Identification of non-ribosomal peptide synthetase in Ganoderma boninense Pat. that was expressed during the interaction with oil palm.

Basal stem rot (BSR) of oil palm is a disastrous disease caused by a white-rot fungus Ganoderma boninense Pat. Non-ribosomal peptides (NRPs) synthesized by non-ribosomal peptide synthetases (NRPSs) are a group of secondary metabolites that act as fungal virulent factors during pathogenesis in the host. In this study, we aimed to isolate NRPS gene of G. boninense strain UPMGB001 and investigate the role of this gene during G. boninense-oil palm interaction. The isolated NRPS DNA fragment of 8322 bp was used to predict the putative peptide sequence of different domains and showed similarity with G. sinense (85%) at conserved motifs of three main NRPS domains. Phylogenetic analysis of NRPS peptide sequences demonstrated that NRPS of G. boninense belongs to the type VI siderophore family. The roots of 6-month-old oil palm seedlings were artificially inoculated for studying NRPS gene expression and disease severity in the greenhouse. The correlation between high disease severity (50%) and high expression (67-fold) of G. boninense NRPS gene at 4 months after inoculation and above indicated that this gene played a significant role in the advancement of BSR disease. Overall, these findings increase our knowledge on the gene structure of NRPS in G. boninense and its involvement in BSR pathogenesis as an effector gene.

Transcriptional profile of oil palm pathogen, Ganoderma boninense, reveals activation of lignin degradation machinery and possible evasion of host immune response.

BACKGROUND: The white-rot fungi in the genus Ganoderma interact with both living and dead angiosperm tree hosts. Two Ganoderma species, a North American taxon, G. zonatum and an Asian taxon, G. boninense, have primarily been found associated with live palm hosts. During the host plant colonization process, a massive transcriptional reorganization helps the fungus evade the host immune response and utilize plant cell wall polysaccharides. RESULTS: A publicly available transcriptome of G. boninense - oil palm interaction was surveyed to profile transcripts that were differentially expressed in planta. Ten percent of the G. boninense transcript loci had altered expression as it colonized oil palm plants one-month post inoculation. Carbohydrate active enzymes (CAZymes), particularly those with a role in lignin degradation, and auxiliary enzymes that facilitate lignin modification, like cytochrome P450s and haloacid dehalogenases, were up-regulated in planta. Several lineage specific proteins and secreted proteins that lack known functional domains were also up-regulated in planta, but their role in the interaction could not be established. A slowdown in G. boninense respiration during the interaction can be inferred from the down-regulation of proteins involved in electron transport chain and mitochondrial biogenesis. Additionally, pathogenicity related genes and chitin degradation machinery were down-regulated during the interaction indicating G. boninense may be evading detection by the host immune system. CONCLUSIONS: This analysis offers an overview of the dynamic processes at play in G. boninense - oil palm interaction and provides a framework to investigate biology of Ganoderma fungi across plantations and landscape.

On the Typification of Ganoderma sichuanense (Agaricomycetes)-the Widely Cultivated Lingzhi Medicinal Mushroom.

The well-known and widely cultivated lingzhi has had a significant impact on Chinese culture and is now an important fungal crop providing medicinal benefits to human health and economic value to social development within China and around the world. The European mushroom name, Ganoderma lucidum, has been misapplied to this species for over 100 years until recently reidentified as G. sichuanense. Soon after this, a new species name, G. lingzhi, was also proposed for the fungus because of an unusual internal transcribed spacer (ITS) sequence purportedly of the holotype of G. sichuanense. This extraordinary ITS sequence, which apparently belongs to another species, created an inconsistency between morphological characteristics and molecular data of the holotype making it "demonstrably ambiguous"; this led to an epitypification to support the holotype for the precise application of the name, according to the International Code of Nomenclature for algae, fungi, and plants. However, arguments concerning the names G. sichuanense and G. lingzhi are still heating up, including attempts to reject the epitype of G. sichuanense. To clarify the confusion, the typification of G. sichuanense is reviewed here to demonstrate that the epitype of G. sichuanense was appropriately designated for the purpose to support the holotype of the name, the fact that both G. sichuanense and G. lingzhi are conspecific, and that the name G. lingzhi was based on the unwarranted ITS sequence claimed to be of the holotype of G. sichuanense. Suggestions are made for this case to make a way forward, especially re-examination of relevant fungarium collections to reach a consensus to stabilize the use of the name.

Improved production of polysaccharides in Ganoderma lingzhi mycelia by plasma mutagenesis and rapid screening of mutated strains through infrared spectroscopy.

As a traditional Chinese medicine, Ganoderma lingzhi has attracted increasing attention for both scientific research and medical application. In this work, in order to improve the production of polysaccharides from an original wide-type (WT) strain (named "RWY-0") of Ganoderma lingzhi, we applied atmospheric-pressure dielectric barrier discharge (DBD) nonthermal plasma to the protoplasts of RWY-0 for mutagenesis treatment. Through a randomly amplified polymorphic DNA (RAPD) assay, at least 10 mutagenic strains were confirmed. They also showed different mycelium characteristics in terms of shape, color, size and biomass in liquid fermentation. The mutant strains were examined by infrared spectroscopy, and based on the established near-infrared (NIR) quantification model, the polysaccharide contents in these mutants were quantitatively evaluated. As a result, we found that the Ganoderma polysaccharide contents in some of the mutant strains were significantly changed compared with that of the original WT strain. The polysaccharide content of RWY-1 G. lingzhi was considerably higher than that of the WT strain, with an increase of 25.6%. Thus, this preliminary work demonstrates the extension of the plasma mutagenesis application in acquiring polysaccharide-enhanced Ganoderma lingzhi mutants and shows the usefulness of NIR spectroscopy in the rapid screening of mutagenic strains for other important ingredients.

Cloning and Characterization of Promoters of the Fungal Immunomodulatory Protein Genes from Ganoderma spp. (Agaricomycetes) and Their Response to Methyl Jasmonate and Salicylic Acid.

Ganoderma mushrooms for medicinal use contain various bioactive compounds, but the genetic elements available for these medicinal mushrooms are still limited. In this study we cloned and analyzed the promoters of fungal immunomodulatory protein (FIP) genes from G. lucidum and G. atrum. FIP gene expression was induced by different concentrations of methyl jasmonate (MeJA) and salicylic acid (SA), and messenger RNA expression was detected by quantitative reverse-transcription polymerase chain reaction. The results provided 5' upstream sequences of FIP genes from G. lucidum and G. atrum. Sequence analysis showed that the FIP-glu promoter sequence contained 11 CAAT boxes, 3 TATA boxes, 3 MeJA-responsive elements, 3 MYB binding site (MBS) motifs, 1 abscisic acid responsive element, 1 TGA, 1 anaerobic inducible element, 2 circadian elements, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. The 5' flanking region of FIP-gat included 9 CAAT boxes, 4 TATA boxes, 3 MeJA-responsive elements, 1 AuxRR core, 1 GC motif, 1 MBS, 1 fungal elicitor, 1 meristem-specific activation element, 3 Skn-1 motifs, and several light-responsive elements. On the transcriptional level, both FIP-glu and FIP-gat reached their highest expression after treatment with MeJA at 500 µmol/L. FIP-glu expression depended on the concentration of SA (0-1000 mg/L); the expression of the FIP-gat gene was highest at a concentration of 100 mg MeJA/L. This research lays the foundation to use Ganoderma mycelia as bioreactors for producing FIPs.

Transcriptional profiling provides new insights into the role of nitric oxide in enhancing Ganoderma oregonense resistance to heat stress.

Ganoderma is well known for its use in traditional Chinese medicine and is widely cultivated in China, Korea, and Japan. Increased temperatures associated with global warming are negatively influencing the growth and development of Ganoderma. Nitric oxide is reported to play an important role in alleviating fungal heat stress (HS). However, the transcriptional profiling of Ganoderma oregonense in response to HS, as well as the transcriptional response regulated by NO to cope with HS has not been reported. We used RNA-Seq technology to generate large-scale transcriptome data from G. oregonense mycelia subjected to HS (32 °C) and exposed to concentrations of exogenous NO. The results showed that heat shock proteins (HSPs), "probable stress-induced proteins", and unigenes involved in "D-amino-acid oxidase activity" and "oxidoreductase activity" were significantly up-regulated in G. oregonense subjected to HS (P < 0.05). The significantly up-regulated HSPs, "monooxygenases", "alcohol dehydrogenase", and "FAD/NAD(P)-binding domain-containing proteins" (P < 0.05) regulated by exogenous NO may play important roles in the enhanced HS tolerance of G. oregonense. These results provide insights into the transcriptional response of G. oregonense to HS and the mechanism by which NO enhances the HS tolerance of fungi at the gene expression level.

Screening and analysis of potential anti-tumor components from the stipe of Ganoderma sinense using high-performance liquid chromatography/time-of-flight mass spectrometry with multivariate statistical tool.

According to Chinese Pharmacopoeia 2015 edition, Ganoderma (Lingzhi) is a species complex that comprise of Ganoderma lucidum and Ganoderma sinense. The bioactivity and chemical composition of G. lucidium had been studied extensively, and it was shown to possess antitumor activities in pharmacological studies. In contrast, G. sinense has not been studied in great detail. Our previous studies found that the stipe of G. sinense exhibited more potent antitumor activity than the pileus. To identify the antitumor compounds in the stipe of G. sinense, we studied its chemical components by merging the bioactivity results with liquid chromatography-mass spectrometry-based chemometrics. The stipe of G. sinense was extracted with water, followed by ethanol precipitation and liquid-liquid partition. The resulting residue was fractionated using column chromatography. The antitumor activity of these fractions were analysed using MTT assay in murine breast tumor 4T1 cells, and their chemical components were studied using the LC-QTOF-MS with multivariate statistical tools. The chemometric and MS/MS analysis correlated bioactivity with five known cytotoxic compounds, 4-hyroxyphenylacetate, 9-oxo-(10E,12E)-octadecadienoic acid, 3-phenyl-2-propenoic acid, 13-oxo-(9E,11E)-octadecadienoic acid and lingzhine C, from the stipe of G. sinense. To the best of our knowledge, 4-hyroxyphenylacetate, 3-phenyl-2-propenoic acid and lingzhine C are firstly reported to be found in G. sinense. These five compounds will be investigated for their antitumor activities in the future.

Discovery of novel xylosides in co-culture of basidiomycetes Trametes versicolor and Ganoderma applanatum by integrated metabolomics and bioinformatics.

Transcriptomic analysis of cultured fungi suggests that many genes for secondary metabolite synthesis are presumably silent under standard laboratory condition. In order to investigate the expression of silent genes in symbiotic systems, 136 fungi-fungi symbiotic systems were built up by co-culturing seventeen basidiomycetes, among which the co-culture of Trametes versicolor and Ganoderma applanatum demonstrated the strongest coloration of confrontation zones. Metabolomics study of this co-culture discovered that sixty-two features were either newly synthesized or highly produced in the co-culture compared with individual cultures. Molecular network analysis highlighted a subnetwork including two novel xylosides (compounds 2 and 3). Compound 2 was further identified as N-(4-methoxyphenyl)formamide 2-O-ß-D-xyloside and was revealed to have the potential to enhance the cell viability of human immortalized bronchial epithelial cell line of Beas-2B. Moreover, bioinformatics and transcriptional analysis of T. versicolor revealed a potential candidate gene (GI: 636605689) encoding xylosyltransferases for xylosylation. Additionally, 3-phenyllactic acid and orsellinic acid were detected for the first time in G. applanatum, which may be ascribed to response against T.versicolor stress. In general, the described co-culture platform provides a powerful tool to discover novel metabolites and help gain insights into the mechanism of silent gene activation in fungal defense.

Diversity of Polypore Bracket Mushrooms, Polyporales (Agaricomycetes), Recorded in Armenia and Their Medicinal Properties.

The list of polypore bracket mushrooms (Polyporales) recorded in Armenia is presented. The order Polyporales in Armenia is currently represented by 87 species (4 varieties) belonging to 47 genera. Information regarding the study of the medicinal properties (e.g., antifungal, antibacterial, mitogenic, regenerative, antioxidant, proteolytic) of genetically identified mycelial collections of several polypore species-mainly from the genera Daedalea, Fomes, Fomitopsis, Ganoderma, Laetiporus, Piptoporus, Polyporus, and Trametes-is reported, as well.

Chromosome-level genome map provides insights into diverse defense mechanisms in the medicinal fungus Ganoderma sinense.

Fungi have evolved powerful genomic and chemical defense systems to protect themselves against genetic destabilization and other organisms. However, the precise molecular basis involved in fungal defense remain largely unknown in Basidiomycetes. Here the complete genome sequence, as well as DNA methylation patterns and small RNA transcriptomes, was analyzed to provide a holistic overview of secondary metabolism and defense processes in the model medicinal fungus, Ganoderma sinense. We reported the 48.96 Mb genome sequence of G. sinense, consisting of 12 chromosomes and encoding 15,688 genes. More than thirty gene clusters involved in the biosynthesis of secondary metabolites, as well as a large array of genes responsible for their transport and regulation were highlighted. In addition, components of genome defense mechanisms, namely repeat-induced point mutation (RIP), DNA methylation and small RNA-mediated gene silencing, were revealed in G. sinense. Systematic bioinformatic investigation of the genome and methylome suggested that RIP and DNA methylation combinatorially maintain G. sinense genome stability by inactivating invasive genetic material and transposable elements. The elucidation of the G. sinense genome and epigenome provides an unparalleled opportunity to advance our understanding of secondary metabolism and fungal defense mechanisms.

Phylogenetic analysis of widely cultivated Ganoderma in China based on the mitochondrial V4-V6 region of SSU rDNA.

Ganoderma mushroom is one of the most prescribed traditional medicines and has been used for centuries, particularly in China, Japan, Korea, and other Asian countries. In this study, different strains of Ganoderma spp and the genetic relationships of the closely related strains were identified and investigated based on the V4-V6 region of mitochondrial small subunit ribosomal DNA of the Ganoderma species. The sizes of the mitochondrial ribosomal DNA regions from different Ganoderma species showed 2 types of sequences, 2.0 or 0.5 kb. A phylogenetic tree was constructed, which revealed a high level of genetic diversity in Ganoderma species. Ganoderma lucidum G05 and G. eupense G09 strains were clustered into a G. resinaceum group. Ganoderma spp G29 and G22 strains were clustered into a G. lucidum group. However, Ganoderma spp G19, G20, and G21 strains were clustered into a single group, the G. lucidum AF214475, G. sinense, G. strum G17, G. strum G36, and G. sinense G10 strains contained an intron and were clustered into other groups.

Molecular defense response of oil palm to Ganoderma infection.

Basal stem rot (BSR) of oil palm roots is due to the invasion of fungal mycelia of Ganoderma species which spreads to the bole of the stem. In addition to root contact, BSR can also spread by airborne basidiospores. These fungi are able to break down cell wall components including lignin. BSR not only decreases oil yield, it also causes the stands to collapse thus causing severe economic loss to the oil palm industry. The transmission and mode of action of Ganoderma, its interactions with oil palm as a hemibiotroph, and the molecular defence responses of oil palm to the infection of Ganoderma boninense in BSR are reviewed, based on the transcript profiles of infected oil palms. The knowledge gaps that need to be filled in oil palm-Ganoderma molecular interactions i.e. the associations of hypersensitive reaction (HR)-induced cell death and reactive oxygen species (ROS) kinetics to the susceptibility of oil palm to Ganoderma spp., the interactions of phytohormones (salicylate, jasmonate and ethylene) at early and late stages of BSR, and cell wall strengthening through increased production of guaiacyl (G)-type lignin, are also discussed.

Global diversity of the Ganoderma lucidum complex (Ganodermataceae, Polyporales) inferred from morphology and multilocus phylogeny.

Species of the Ganoderma lucidum complex are used in many types of health products. However, the taxonomy of this complex has long been chaotic, thus limiting its uses. In the present study, 32 collections of the complex from Asia, Europe and North America were analyzed from both morphological and molecular phylogenetic perspectives. The combined dataset, including an outgroup, comprised 33 ITS, 24 tef1α, 24 rpb1 and 21 rpb2 sequences, of which 19 ITS, 20 tef1α, 20 rpb1 and 17 rpb2 sequences were newly generated. A total of 13 species of the complex were recovered in the multilocus phylogeny. These 13 species were not strongly supported as a single monophyletic lineage, and were further grouped into three lineages that cannot be defined by their geographic distributions. Clade A comprised Ganoderma curtisii, Ganoderma flexipes, Ganoderma lingzhi, Ganoderma multipileum, Ganoderma resinaceum, Ganoderma sessile, Ganoderma sichuanense and Ganoderma tropicum, Clade B comprised G. lucidum, Ganoderma oregonense and Ganoderma tsugae, and Clade C comprised Ganoderma boninense and Ganoderma zonatum. A dichotomous key to the 13 species is provided, and their key morphological characters from context, pores, cuticle cells and basidiospores are presented in a table. The taxonomic positions of these species are briefly discussed. Noteworthy, the epitypification of G. sichuanense is rejected.

The cloning, characterization, and functional analysis of a gene encoding an isopentenyl diphosphate isomerase involved in triterpene biosynthesis in the Lingzhi or reishi medicinal mushroom Ganoderma lucidum (higher Basidiomycetes).

An isopentenyl diphosphate isomerase (IDI) gene, GlIDI, was isolated from Ganoderma lucidum, which produces triterpenes through the mevalonate pathway. The open reading frame of GlIDI encodes a 252 amino acid polypeptide with a theoretical molecular mass of 28.71 kDa and a theoretical isoelectric point of 5.36. GlIDI is highly homologous to other fungal IDIs and contains conserved active residues and nudix motifs shared by the IDI protein family. The color complementation assay indicated that GlIDI can accelerate the accumulation of ß-carotene and confirmed that the cloned complementary DNA encoded a functional GlIDI protein. Gene expression analysis showed that the GlIDI transcription level was relatively low in the mycelia and reached a relatively high level in the mushroom primordia. In addition, its expression level could be up-regulated by 254 µM methyl jasmonate. Our results suggest that this enzyme may play an important role in triterpene biosynthesis.

Determination of the antioxidant activity and polyphenol contents of wild Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt. Fr.) P. Karst. (higher Basidiomycetes) from central Himalayan hills of India.

The antioxidant potential of wild strain of Lingzhi or Reishi medicinal mushroom Ganoderma lucidum from Central Himalayan Hills (2000 m MSL) was evaluated, and compared with its in vitro cultured mycelia grown on malt extract broth in the laboratory. Antioxidant activities of both wild and cultivated G. lucidum in terms of IC50 (mg/ mL) were determined against different in vitro radical systems such as DPPH (1, 1-diphenyl-2-picrylhydrazyl), ABTS [2,2'-azinobis (3-ethylenebenzothiazoline-6-sulphonic acid)] and hydroxyl radicals, in addition to ferric reducing antioxidant power assay. Polyphenol contents were also determined, in order to assess their effects on the antioxidant activity of extracts. All the extracts showed significant antioxidant activity, and maximum scavenging was observed in the case of methanolic extracts of wild G. lucidum with minimum IC50 values 0.953 ± 0.040, 0.690 ± 0.014 and 3.295 ± 0.027 mg/mL, respectively, for DPPH, ABTS, and hydroxyl radicals. The efficacy of wild G. lucidum as a rich source of natural antioxidant was established for nutraceutical development.

Ganoderma oerstedii (Fr.) murrill (higher Basidiomycetes), a tree parasite species in Mexico: taxonomic description, rDNA study, and review of its medical applications.

We present a description of macro- and microscopic taxonomical features of a medicinal mushroom, Ganoderma oerstedii, based on Mexican specimens from the states of Chiapas, Morelos, Sinaloa, and Veracruz, and discuss its relationships with species of the G. lucidum complex. A phylogenetic study based in rDNA sequences of a specimen of G. oerstedii from Veracruz is presented, as well as a review of traditional and modern uses in medicine.

[AFLP analysis for genetic diversity of Ganoderma].

OBJECTIVE: To investigate the genetic diversity of Ganoderma cultivars provided for the genuineness study, germ-plasm resource identification, genetic relationship study, breeding, introduction and cultivante of Ganoderma strains. METHOD: With the software of NTSYSpc 2. 1, 24 materials, of G. lucidum and G. sinense, were studied using AFLP to construct the dendrogram. RESULT: There were 177 polymorphic bands with 14 primer combinations. And all materials could be identified with AFLP. CONCLUSION: There actually existed much genetic diversity at the molecular level among the germplasm resources of Ganoderma strains, and all the strains were clustered into G. lucidum group and G. sinense group at the similarity coefficient 0. 676.

Comparison of rapid DNA extraction methods applied to PCR identification of medicinal mushroom Ganoderma spp.

Four different DNA extraction methods were used to extract genomic DNA of the medicinal mushroom Lingzhi from its developing stage materials, such as mycelium, dry fruiting body, or sliced and spore powder or sporoderm-broken spore powder. The DNA samples were analyzed using agarose gel electrophoresis, UV spectrophotometer, and PCR amplification. According to the average yields and purity of DNA, high salt concentrations and low pH methods were the best for DNA extraction. The mycelia and sporoderm-broken spore powder yielded higher and purer DNA. The method developed could effectively eliminate the influence of the secondary metabolites to DNA extraction. The DNA samples extracted from the developed method could be successfully used for PCR applications.

Identification of medicinal mushroom species based on nuclear large subunit rDNA sequences.

The purpose of this study was to develop molecular identification method for medical mushrooms and their preparations based on the nucleotide sequences of nuclear large subunit (LSU) rDNA. Four specimens were collected of each of the three representative medicinal mushrooms used in Korea: Ganoderma lucidum, Coriolus versicolor, and Fomes fomentarius. Fungal material used in these experiments included two different mycelial cultures and two different fruiting bodies from wild or cultivated mushrooms. The genomic DNA of mushrooms were extracted and 3 nuclear LSU rDNA fragments were amplified: set 1 for the 1.1-kb DNA fragment in the upstream region, set 2 for the 1.2-kb fragment in the middle, and set 3 for the 1.3-kb fragment downstream. The amplified gene products of nuclear large subunit rDNA from 3 different mushrooms were cloned into E. coli vector and subjected to nucleotide sequence determination. The sequence thus determined revealed that the gene sequences of the same medicinal mushroom species were more than 99.48% homologous, and the consensus sequences of 3 different medicinal mushrooms were more than 97.80% homologous. Restriction analysis revealed no useful restriction sites for 6-bp recognition enzymes for distinguishing the 3 sequences from one another, but some distinctive restriction patterns were recognized by the 4-bp recognition enzymes AccII and HhaI. This analysis was also confirmed by PCR-RFLP experiments on medicinal mushrooms.