Potential Specificity Between Mycorrhizal Fungi Isolated from Widespread Dendrobium spp. and Rare D. huoshanense Seeds.

In nature, orchid seed germination and seedling development depend on compatible mycorrhizal fungi. Mycorrhizal generalist and specificity affect the orchid distribution and rarity. Here, we investigated the specificity toward fungi in the rare D. huoshanense by mycorrhizal fungal isolation and symbiotic germination in vitro. Twenty mycorrhizal fungal strains were isolated from the roots of adult Dendrobium spp. (six and 12 strains from rare D. huoshanense and widespread D. officinale, respectively, and two strains from D. nobile and D. moniliforme, respectively) and 13 strains belong to Tulasnellaceae and seven strains belong to Serendipitaceae. Germination trials in vitro revealed that all 20 tested fungal strains can stimulate seed germination of D. huoshanense, but only nine strains (~ 50%) can support it up to the seedling stage. This finding indicates that generalistic fungi are important for early germination, but only a few can maintain a symbiosis with host in seedling stage. Thus, a shift of the microbial community from seedling to mature stage probably narrows the D. huoshanense distribution range. In addition, to further understand the relationship between the fungal capability to promote seed germination and fungal enzyme activity, we screened the laccase and pectase activity. The results showed that the two enzymes activities of fungi cannot be directly correlated with their germination-promoting activities. Understanding the host specificity degree toward fungi can help to better interpret the limited geographic distribution of D. huoshanense and provides opportunities for in situ and ex situ conservation and reintroduction programs.

Phenolics, Flavonoids Content and Antioxidant Activities of Tuber indicum at Different Maturity Stages.

The Chinese black truffle Tuber indicum (Ascomycota, Pezizales) is an ectomycorrhizal fungus forming hypogeous edible ascocarps. As a famous wild edible mushroom in the world, this species also attracted an increasing interest in their chemical composition and pharmacological activity. In this study, the total phenolic content, total flavonoid content and antioxidant activities of Tuber indicum collected from July to November at different maturity stages in China were analyzed. Our results showed that T. indicum collected in July (immature stage) possessed the highest amount of flavonoids (3.89 mg/g dw) and the highest ABTS.+ scavenging activity (EC50 =3.73 mg/ml). In addition, those samples collected in August (moderate mature stage) contained the highest phenolics content (4.78 mg/g dw), the highest DPPH⋅ radical scavenging activity (EC50 =3.73 mg/ml) and ferric reducing activity power (243.63 µmol FeSO4 /g). The study reveals T. indicum in the early maturity stage yield significantly higher content of phenolics and flavonoids and possessed stronger antioxidant activity than those collected in other months. This study provided important data for understanding the relationship between maturity stages and truffle formation and evaluating the quality of Chinese black truffle at different maturity.

Symbiotic and Asymbiotic Germination of Dendrobium officinale (Orchidaceae) Respond Differently to Exogenous Gibberellins.

Seeds of almost all orchids depend on mycorrhizal fungi to induce their germination in the wild. The regulation of this symbiotic germination of orchid seeds involves complex crosstalk interactions between mycorrhizal establishment and the germination process. The aim of this study was to investigate the effect of gibberellins (GAs) on the symbiotic germination of Dendrobium officinale seeds and its functioning in the mutualistic interaction between orchid species and their mycobionts. To do this, we used liquid chromatograph-mass spectrometer to quantify endogenous hormones across different development stages between symbiotic and asymbiotic germination of D. officinale, as well as real-time quantitative PCR to investigate gene expression levels during seed germination under the different treatment concentrations of exogenous gibberellic acids (GA3). Our results showed that the level of endogenous GA3 was not significantly different between the asymbiotic and symbiotic germination groups, but the ratio of GA3 and abscisic acids (ABA) was significantly higher during symbiotic germination than asymbiotic germination. Exogenous GA3 treatment showed that a high concentration of GA3 could inhibit fungal colonization in the embryo cell and decrease the seed germination rate, but did not significantly affect asymbiotic germination or the growth of the free-living fungal mycelium. The expression of genes involved in the common symbiotic pathway (e.g., calcium-binding protein and calcium-dependent protein kinase) responded to the changed concentrations of exogenous GA3. Taken together, our results demonstrate that GA3 is probably a key signal molecule for crosstalk between the seed germination pathway and mycorrhiza symbiosis during the orchid seed symbiotic germination.

[Molecular cloning and prokaryotic expression of a type II ribosome inactivating protein from Polyporus umbellatus].

A type Ⅱ ribosome inactivating protein (RIP) gene was cloned from Polyporus umbellatus sclerotia by RT-PCR method. The full open reading frame cDNA sequence of this gene was 873 bp in length and encoded a 290-aa protein with a molecular weight of 32.33 kDa and an isoelectric point of 5.58. Multiple sequence alignment revealed that the deduced amino acids possessed conserved domains of RICIN superfamily protein. A neighbor joining phylogenetic analysis suggests that PuRIP was closely related to RIP in Marasmius oreades. Real time PCR results showed that this gene expressed in all tested tissues of P. umbellatus. Meanwhile, the expression of this gene was significantly up-regulated in the part infected by Armillaria mellea. This result suggested that this PuRIP might played important role with potential biotic stress tolerance of P. umbellatus. Otherwise, we successfully constructed the pET15b-PuRIP plasmid, produced and purified the His-PuRIP fusion protein, which would provide the basic material for polyclonal antibody preparation and gene function research.

[Molecular cloning and characterization of two kind of heat-shock protein gene from Polyporus umbellatus].

With RT-PCR approaches, the full-length cDNA of two heat shock protein genes were cloned from total RNA of the Polyporus umbellatus sclerotium. The full open reading frame cDNA sequence of the Hsp90 was 2 091 bp, encoding 696 amino acid residues with a predicted molecular mass of 78.9 kDa. The full open reading frame cDNA sequence of the Hsp70 was 1 944 bp, encoding 647 amino acid residues with a predicted molecular mass of 70.5 kDa. The Hsp90 and Hsp70 protein contained the conservative structure domain, respectively. Phylogenetic analysis showed that Hsp90 and Hsp90 from Trametes versicolor were clustered into one group, Hsp70 and Hsp70 from Fistulina hepatica were clustered into one group. Real-time PCR analysis showed that, the expression of Hsp90 and Hsp70 in the infected part by Amillariella mellea was upregulated. The expression profiling of Hsp90 and Hsp70 showed same patterns underbiotic stress. The results indicate that these two genes may play an important role in response to Amillariella mellea infection.

Diversity Analysis of Polyporus umbellatus in China Using Inter-simple Sequence Repeat (ISSR) Markers.

Polyporus (P.) umbellatus, an endangered medicinal fungus in China, is distributed throughout most areas of the country. Thirty-seven natural P. umbellatus samples collected from 12 provinces in China were subjected to the inter-simple sequence repeat (ISSR) assay to investigate the genetic diversity within and among the 11 natural populations. Nine ISSR primers selected from 100 primers produced 88 discernible DNA bands, with 46 being polymorphic. The frequency of polymorphism varied from 19.57 to 93.48% with an average of 61.26% across all populations. At the population level, the within-population variance was much greater (92.04%) than the between-population variance (7.96%) as revealed by analysis of molecular variance. Eleven P. umbellatus populations were grouped into two major clusters, and the clustering pattern displayed four groups using the unweighted pair-group method with an arithmetic mean dendrogram. Principal coordinate analysis further indicated that the genetic diversity of P. umbellatus strains was unevenly distributed and displayed a clustered distribution pattern instead. Within these clusters, subgrouping (Henan and Hubei) and cluster II (Jilin and Heilongjiang) related to the geographic distribution were evident. The present study provides the first global overview of P. umbellatus diversity analysis in China, which may open up new opportunities in comparative genetic research on this medicinal fungus in other countries.

[Molecular cloning and characterization of four small GTPase genes from medicinal fungus Polyporus umbellatus].

Four small GTPase genes which may be relative to sclerotial development were firstly cloned from medicinal fungus Polyporus umbellatus using rapid amplification of cDNA end PCR (RACE) method. The results showed that full-length cDNA of PuRhoA was 698 bp contained 585 bp ORF, which was predicted to encode a 194 amino acid protein with a molecular weight of 21.75 kD with an isoelectric point (pI) of 6.44; the full length cDNA of PuRhoA2 was 837 bp in length and encoded a 194 amino acid protein with a molecular weight of 21.75 kD and an isoelectric point (pI) of 6.33; the full length cDNA of Puypt1 was 896 bp in length and encoded a 204-aa protein with a molecular weight of 22.556 kD and an isoelectric point (pI) of 5.75; the full length cDNA of PuRas was 803 bp in length and encoded a 212-aa protein with a molecular weight of 23.821 kD and an isoelectric point (pI) of 5.2. There are fani acyl transferase enzyme catalytic site and myrcene-transferase enzyme catalytic site in PuRhoA1 while the PuRhoA2 only possess myrcene-transferase enzyme catalytic site. Puypt1 contains the Rab1-Ypt1 conserved domain of small GTPase family and PuRas contains the fani acyl transferase enzyme catalytic site. According to the phylogenetic analysis all these four small GTPase clustered with basidiomycete group. Quantitative real-time PCR analysis revealed that Puypt1, PuRas and PuRhoA1 transcripts were significantly higher in the beginning of sclerotial formation than that in the mycelia, whereas the transcripts levels of PuRhoA2 gene were particularly lower in sclerotia than that in mycelia, suggesting that these four genes might be involved in P umbellatus selerotial development.

[Molecular cloning and characterization of two oxidative stress related genes from medicinal fungus Polyporus umbellatus].

OBJECTIVE: To clone the NADPH gene (PuNOX) and Glyoxal oxidase gene (PuGLOX) from a medicinal fungus Polyporus umbellatus, and to carry out the bioinformatic analysis. METHODS: We used the Rapid Amplification of cDNA ends (RACE) technique to obtain the full length cDNA of these two genes. We used a series of bioinformatic tools to characterize physiochemical properties of the two deduced protein. The analyses of multiple alignment and phylogenetic trees were performed using Bioeditor and MEGA 5.0 softwares. RESULTS: The entire cDNA of PuNOX and PuGLOX were 1674 bp, 1723 bp in length and encoded a 557-amino acid protein and 515-amino acid protein with a molecular weight of 63.845 kDa and 55.891 kDa and the isoelectric point of 5.58 and 4.82, respectively. PuNOX had high identities (74 to 80%) with NADPH peroxidase from other fungus. From the evolutionary tree, PuNOX was closely related to that of Pleurotus ostreatus. PuGLOX had high identities (> 50%) with Glyoxal oxidases from various fungus. Phylogenetic tree analysis suggested that PuGLOX was closely related to that of Phanerochaete chrysosporium. CONCLUSION: Molecular characterization of the two oxidative stress related genes will be useful for further functional determination of the genes involved in the sclerotium development of Polyporus umbellatus.

[Habitat suitability assessment of medicinal Polyporus umbellatusin China based on Maxent modeling].

Geographic distribution of Polyporus umbellatus was predicted by using distribution records. Based on 42 distribution records from 12 provinces and bioclimatic data (1950-2000), georaphic distribution of P. umbellatus was modeled using Maxent. The results showed thatthe Receiver Operating Characteristic (ROC) curve analysis method was used to assess the accuracy of MAXENT model and the area under ROC curve (AUC) value of MAXENT was 0. 960 which suggested that the result of assessment was dependable. The geographic distribution pattern of were divided into three distribution block based on distribution values of 0.5-0.8: small area of Heilongjiang, Jilin, Liaoning and Hebei province, the board area of Yunnan, Guizhou and Sichuan, the southeast area of Tibet and the most area of Shanxi and Shannxi, the southeast board area of Shannxi, Gansu and Ningxia. Jackknife Test showed that average precipitation in warm seasons had the greatest contribution to the distribution gain of P. umbellatus, followed by mean temperature of driest quarter and annual mean temperature. The object suggests the potential distribution areasof P. umbellatus which is useful for the habitat conservation and introduction of P. umbellatus.

ESTs analysis of putative genes engaged in Polyporus umbellatus sclerotial development.

Polyporus umbellatus is one of the most widely used and precious medicinal fungi and the underground sclerotia are known to be with great medicinal value. However, the molecular mechanisms involved in sclerotial development are poorly understood. In the present study, we constructed a forward suppression subtractive hybridization (SSH) cDNA library of Polyporus umbellatus to identify genes expressing differently between mycelium and sclerotia. In this library, a total of 1202 clones were sequenced, assembled into 222 contigs and 524 singletons which were further searched against the NCBI nonredundant (NR) protein database (E-value cutoff, 10-5). Based on sequence similarity with known proteins, 378 sequences between mycelium and sclerotial were identified and classified into different functional categories through Gene Ontology (GO), Clusters of orthologous Groups of proteins (COGs). We have finally identified a majority of differentially expressed genes (constituting 5.6% of the present library) between the two different periods. An expression level of 32 selected expressed sequence tags (ESTs) generated from the above SSH cDNA library was studied through RT-PCR. This study provides the first global overview of genes putatively involved in Polyporus umbellatus sclerotial development and provides a preliminary basis for further functional research in terms of regulated gene expression in sclerotial production.

[Physicochemical properties of medicinal fungus Polyporus umbellatus sclerotial exudate].

This study was conducted to investigate the physicochemical properties of Polyporus umbellatus sclerotial exudate. Morphological characteristics of the sclerotia and its exudate were observed during different stages of sclerotial formation. The pH of the exudate was detected at different time during cultivation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of P. umbellatus sclerotial exudate during cultivating time. Additionally, the protein content was measured by means of BCA protein assay. Furthermore, CAT content was detected using ultraviolet absorption method. That the protein content of the exudate and CAT specific activity rose gradually during the passage of the cultivating time indicated a high level of oxidative stress during P. umbellatus sclerotial exudate formation. The results showed that the pH of the exudate increased gradually and then dropped down during sclerotial formation. That the pH of the exudate maintained the acidity state during the cultivation indirectly indicated that acidic environment would help sclerotial formation. The exudate produced gradually and was absorbed by the sclerotia itself.

Perspective and challenges of mycorrhizal symbiosis in orchid medicinal plants.

The family Orchidaceae is of the most diverse taxon in the plant kingdom, and most of its members are highly valuable herbal medicines. Orchids have a unique mycorrhizal symbiotic relationship with specific fungi for carbohydrate and nutrient supplies in their whole lifecycle. The large-scale cultivation of the medicinal plant Gastodia elata is a successful example of using mycorrhizal symbiotic technology. In this review, we adopted G. elata and Dendrobium officinale as examples to describe the characteristics of orchid mycorrhiza and mycorrhizal benefits for host plants' growth and health (e.g. biotic and abiotic stress and secondary metabolite accumulation). The challenges in applying mycorrhizal technology to the cultivation of orchid medicinal plants in the future were also discussed. This review aims to serve as a theoretical guide for the cultivation of mycorrhizal technology in medicinal orchid plants.

Insight into the nuclear distribution patterns of conidia and the asexual life cycle of Polyporus umbellatus.

P. umbellatus sclerotium is a traditional Chinese medicine that is widely utilized in China, Korea, Japan, and other countries due to its diverse medicinal activities, such as diuretic, antitumor, anticancer, and immune system enhancement effects. Conidia, which are common asexual spores in various fungi, are not universally present in Polyporus species. In this study, the asexual life cycle of P. umbellatus was elucidated. Conidia, i.e. arthorconidia, were produced by both dikaryotic and monokaryotic strains. In the dikaryotic strain, binucleate, uninucleate, and nuclei-free conidia were identified with proportions of 67.9 %, 12.4 %, and 19.7 %, respectively. Conversely, the monokaryotic strain did not produce binucleate conidia. This discrepancy suggests that binucleate spores are heterokaryons, while uninucleate spores are homokaryons. Clamp connections were observed in dikaryotic hyphae, but were absent in monokaryotic hyphae. Monokaryotic strains were obtained from conidia of the dikaryotic strain. Additionally, mating types were determined through pairing tests, and successful crossbreeding occurred between monokaryotic strains derived from conidia and basidiospores from different strains. This study introduced the first crossbreeding strategy for P. umbellatus.

Nox gene expression and cytochemical localization of hydrogen peroxide in Polyporus umbellatus sclerotial formation.

The effect of temperature shift on Polyporus umbellatus sclerotial development was investigated. Micromorphology of the sclerotia was observed by using scanning electron microscopy (SEM). The cytochemical localization of H2O2 expressed as CeCl3 deposition at the subcellular level was observed by using transmission electron microscopy (TEM). Nox gene expression in sclerotia and mycelia was detected by quantitative real-time PCR (qRT-PCR) analysis. In addition, superoxide dismutase (SOD) and catalase (CAT) specific activities increased during sclerotial development and decreased after the antioxidant diphenyleneiodonium (DPI) was used. Results indicated that the temperature shift treatment induced P. umbellatus sclerotial formation. Compared with the mycelia, the Nox gene was respectively upregulated by 10.577-, 30.984- and 25.469-fold in the sclerotia of SI, SD and SM stages respectively. During the sclerotial formation, H2O2 accumulation was observed in the cell walls or around the organelle membranes of the mycelial cells. The antioxidant DPI decreased the generation of H2O2 in mycelial cells. The specific activity of SOD and CAT levels was decreased significantly by DPI. The activity of the two antioxidant enzymes in the mycelia increased much more during sclerotial formation (p < 0.05). Oxidative stress was closely associated with sclerotial development in P. umbellatus induced by temperature shift treatment.

[Expression of recombinant human kallistatin in Pichia pastoris by high density cell culture, and its purification and characterization].

Kallistatin (Kal) is a negative acute phase endogenous protein which can inhibit tumor angiogenesis, growth and metastasis effectively. To express and purify recombinant human kallistatin (rHKal), and characterize its biological activity, P. pastoris was transformed with pPIC9-Kal/GS115 (His4) to express rHKal. The fermentation was carried out in a 7.5 L bioreactor with high density cell culture. 1%-2% methanol was added to the medium to induce the expression of rHKal. The secretion was purified with phenyl sepharose, G-25 sepharose, heparin sepharose and Sephacryl S-100 chromatography. The biological activity of purified bulk rHKal on HUVEC was evaluated with MTT and tube formation assays. The final expression of rHKal in the supernatant reached 50 mg x L(-1), the purity of bulk rHKal after purification was above 98%. A dose-dependent inhibition of rHKal on HUVEC proliferation was observed, however, a U-shaped dose-response curve of rHKal on capillary formation of HUVEC was revealed. The described protocol provides an effective means for preparing rHKal that could be used for anti-angiogenesis therapy in the future.

Transcriptome and metabolome analysis reveals stage-specific metabolite accumulation during maturity of Chinese black truffle Tuber indicum.

Tuber indicum is the most economically important member of Tuber, with the highest production and widest distribution in China. However, the overexploitation of immature ascocarps not only has driven wild resources of the species toward extinction, but also has caused enconomic losses and a decline in the reputation of T.indicum quality. In this study, stage-specific metabolites of T. indicum in relation to nutritional quality and the mechanism of their accumulations were explored by transcriptome and metabolome analysis at five harvest times, representing four maturation stages. A total of 663 compounds were identified in T. indicum ascocarps by a widely targeted metabolomic approach. Lipid compounds are the most prominent metabolites (18%) in our samples and also are higher accumulation at the immature stage than at mature stage, representing 30.16% differential accumulated metabolites in this stage. Levels of some of the amino acids, such as S-(methyl) glutathione, S-adenosylmethionine, which are known truffle aroma precursors, were increased at the mature stage. The gene expression level related to the biosynthesis of volatile organic compounds were verified by qPCR. This study contributes to the preliminary understanding of metabolites variations in T. indicum ascocarps during maturity for quality evaluation and truffle biology.

Antimicrobial activities of endophytic fungi isolated from Ophiopogon japonicus (Liliaceae).

BACKGROUND: Drug resistance in bacteria has become a global concern and the search for new antibacterial agents is urgent and ongoing. Endophytes provide an abundant reservoir of bioactive metabolites for medicinal exploitation, and an increasing number of novel compounds are being isolated from endophytic fungi. Ophiopogon japonicus, containing compounds with antibacterial activity, is a traditional Chinese medicinal plant used for eliminating phlegm, relieving coughs, latent heat in the lungs, and alleviating diabetes mellitus. We investigated the antimicrobial activities of 30 strains of O. japonicus. METHODS: Fungal endophytes were isolated from roots and stems of O. japonicus collected from Chongqing City, southwestern China. Mycelial extracts (MC) and fermentation broth (FB) were tested for antimicrobial activity using peptide deformylase (PDF) inhibition fluorescence assays and MTT cell proliferation assays. RESULTS: A total of 30 endophytic strains were isolated from O. japonicus; 22 from roots and eight from stems. 53.33% of the mycelial extracts (MC) and 33.33% of the fermentation broths (FB) displayed potent inhibition of PDF. 80% of MC and 33.33% of FB significantly inhibited Staphylococcus aureus. 70% of MC and 36.67% of FB showed strong activities against Cryptococcus neoformans. None showed influence on Escherichia coli. CONCLUSION: The secondary metabolites of endophytic fungi from O. japonicus are potential antimicrobial agents.

Identification of Hortaea werneckii Isolated from mangrove plant Aegiceras comiculatum based on morphology and rDNA sequences.

Hortaea werneckii is a black yeast-like ascomycetous fungi associated with the human superficial infection tinea nigra, which commonly occurs in tropical and subtropical countries. Now, this fungus has been found in the halophilic environment all over the world and recognized as a new model organism in exploring the mechanisms of salt tolerance in eukaryotes. During a survey of endophytic fungi of mangrove forest at South China Sea, two isolates of H. werneckii were recovered from medicinal plant of Aegiceras comiculatum. The isolates were identified by morphological characters and phylogenetic analyses (e.g., ITS rDNA, LSU rDNA and translation elongation factor EF1α). Some physiological tests such as thermotolerance, acid tolerance (pH) and NaCl tolerance as well as pathogenicity test in vitro for the strains of Hortaea were performed. It is the first report that H. werneckii was isolated from medicinal plant of A. comiculatum in south sea of China as the endophytic fungi.

Antimicrobial activity of endophytic fungi isolated from Dendrobium species in southwestern China.

OBJECTIVE: To isolate and characterize endophytic fungi from seven Dendrobium species, and detect their antimicrobial activities. METHOD: Fungal endophytes were isolated by strictly sterile sample preparation and fungal identification methods were based on their ITS ribosomal DNA (ITS rDNA gene) sequences. The agar well diffusion method was then employed to evaluate the antimicrobial activity against six pathogenic organisms and the phylogenetic tree of active isolates was constructed by the MEGA. RESULT: Ninety-eight endophytic fungi obtained from seven Dendrobium spp., and among them twenty-four isolates, representing 11 genera and 14 species, displayed anti-microbial activities. The phylogenetic assay based on ITS-rDNA showed that 24 active isolates were sorted to 7 taxonomic orders: Hypocreales, Sordariales, Capnodiales, Eurotiales, Botryosphaeriales, Xylariales and Mucorales. The results of antimicrobial activity assay revealed that 1.02%, 10.2%, 18.4%, 1.02%, 1.02% and 10.2% of fermentation broths of 98 isolates displayed significant antimicrobial activities against E. coli, B. subtilis, S. aureus, C. albicans, C. neoformans and A. fumigatus, respectively. Four strains DL-R-3, DL-S-6, DG-R-10 and DN-S-1 displayed strong and broad antimicrobial spectrum. CONCLUSION: Endophytic fungi associated with Dendrobium species have fungal diversity, and possess diverse antimicrobial activity.

Acetylome analysis of acetylation providing new insight into sclerotial generation in medicinal fungus Polyporus umbellatus.

Sclerotium-forming fungi are ecologically diverse and possess notable pathogenic or medicinal properties. The sclerotial generation mechanism is still elusive though Polyporus umbellatus sclerotia are typical Traditional Chinese Medicine with diuretic and antitumor effects. Protein acetylation displays a crucial role in several biological processes, but the functions of acetylation in this valuable fungus are unknown at present. In this study, acetylome of P. umbellatus was studied using nano LC-Triple TOF mass spectrometry system following immune-affinity-based enrichment. Totally, 648 acetylated sites in 342 proteins were identified and nine motifs were found to be conserved in P. umbellatus including KacY, KacA, KacL, KacG, MacS, MacA, RacA, RacL, and RacG. Acetylated proteins taken part in types of biological processes, particularly to those in biological processes associated with reactive oxygen species (ROS) metabolism. Inhibitors complement tests were carried out to verify the role of ROS in acetylation modification. It was concluded that oxidative stress regulated sclerotial generation via proteins acetylation in P. umbellatus. The present study presents new insight into the essential roles of acetylation in sclerotial formation, which may also be applicable for other sclerotium-forming fungi.