Chunlan Wang

Isolation and Identification of Endophytic Fungi in Roots of Nine Holcoglossum Plants (Orchidaceae) Collected from Yunnan, Guangxi, and Hainan Provinces of China

Holcoglossum is one of the smaller genera of Orchidaceae, mainly distributed in southwest China. Some members of this genus as well as H. rupestre and H. flavescens are endemic and rare Chinese orchids. As far as we know, little work has been done concerning the relationships between the Holcoglossum plants and endophytic microorganisms. In this study, 46 culturable fungal endophytes were isolated and identified from roots of nine Holcoglossum plants collected from Yunnan, Guangxi, and Hainan provinces of China based on molecular techniques. The results showed that all strains belonged to four classes, i.e., Sordariomycetes (41.30%), Dothideomycetes (36.96%), Agaricomycetes (17.39%), Leotiomycetes (4.35%). Thirty-six strains were identified at the genus level, including Alternaria, Cladosporium, Clonostachys, Colletotrichum, Cosmospora, Cryptosporiopsis, Cylindrocarpon, Didymella, Epulorhiza (Anamorphic Tulasnella), Fusarium, Myrmecridium, Leptosphaeria, Paraconiothyrium, Phomopsis, Pyrenochaeta, and Stephanonectria. Fusarium and Epulorhiza (Anamorphic Tulasnella) were the dominant fungal endophytes. Some orchids mycorrhizal fungi as well as Tulasnella calospora and Epulorhiza sp. were found in roots. This is the first report concerning endophytic fungi from Holcoglossum plants (Orchidaceae), suggesting that endophytic fungi in Holcoglossum plants are very abundant.

Discrimination of the rare medicinal plant Dendrobium officinale based on naringenin, bibenzyl, and polysaccharides.

The aim of this study was to establish a method for discriminating Dendrobium officinale from four of its close relatives Dendrobiumchrysanthum, Dendrobium crystallinum, Dendrobium aphyllum and Dendrobium devonianum based on chemical composition analysis. We analyzed 62 samples of 24 Dendrobium species. High performance liquid chromatography analysis confirmed that the four low molecular weight compounds 4′,5,7-trihydroxyflavanone (naringenin), 3,4-dihydroxy-4′,5-dime-thoxybibenzyl (DDB-2), 3′,4-dihydroxy-3,5′-dimethoxybibenzyl (gigantol), and 4,4′-dihydroxy-3,3′,5-trimethoxybibenzy (moscatilin), were common in the genus. The phenol-sulfuric acid method was used to quantify polysaccharides, and the monosaccharide composition of the polysaccharides was determined by gas chromatography. Stepwise discriminant analysis was used to differentiate among the five closely related species based on the chemical composition analysis. This proved to be a simple and accurate approach for discriminating among these species. The results also showed that the polysaccharide content, the amounts of the four low molecular weight compounds, and the mannose to glucose ratio, were important factors for species discriminant. Therefore, we propose that a chemical analysis based on quantification of naringenin, bibenzyl, and polysaccharides is effective for identifying D. officinale.

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

BackgroundDrug 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.MethodsFungal 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.ResultsA 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.ConclusionThe secondary metabolites of endophytic fungi from O. japonicus are potential antimicrobial agents.

Sclerotial Formation of Polyporus umbellatus by Low Temperature Treatment under Artificial Conditions

Background Polyporus umbellatus sclerotia have been used as a diuretic agent in China for over two thousand years. A shortage of the natural P. umbellatus has prompted researchers to induce sclerotial formation in the laboratory. Methodology/Principal Finding P. umbellatus cultivation in a sawdust-based substrate was investigated to evaluate the effect of low temperature conditions on sclerotial formation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of wild P. umbellatus sclerotia and mycelia and sclerotia grown in low-temperature treatments. In addition, reactive oxygen species (ROS) content, expressed as the fluorescence intensity of mycelia during sclerotial differentiation was determined. Analysis of ROS generation and sclerotial formation in mycelia after treatment with the antioxidants such as diphenyleneiodonium chloride (DPI), apocynin (Apo), or vitamin C were studied. Furthermore, macroscopic and microscopic characteristics of sclerotial differentiation were observed. Sclerotia were not induced by continuous cultivation at 25°C. The polysaccharide content of the artificial sclerotia is 78% of that of wild sclerotia. In the low-temperature treatment group, the fluorescent intensity of ROS was higher than that of the room temperature (25°C) group which did not induce sclerotial formation all through the cultivation. The antioxidants DPI and Apo reduced ROS levels and did not induce sclerotial formation. Although the concentration-dependent effects of vitamin C (5–15 mg mL−1) also reduced ROS generation and inhibited sclerotial formation, using a low concentration of vitamin C (1 mg mL−1) successfully induced sclerotial differentiation and increased ROS production. Conclusions/Significance Exposure to low temperatures induced P. umbellatus sclerotial morphogenesis during cultivation. Low temperature treatment enhanced ROS in mycelia, which may be important in triggering sclerotial differentiation in P. umbellatus. Moreover, the application of antioxidants impaired ROS generation and inhibited sclerotial formation. Our findings may help to provide new insights into the biological mechanisms underlying sclerotial morphogenesis in P. umbellatus.

Complete assignments of 1H and 13C NMR data of three new dihydrophenanthrofurans from Pleione yunnanensis

Three new dihydrophenanthrofurans, pleionesins A–C (1–3), together with two known dihydrophenanthrenes (4–5) were isolated from the tubers of Pleione yunnanensis (Rolfe). The complete 1H and 13C NMR spectra assignments of these compounds were carried out using 1D and 2D NMR experiments (1H, 13C, selective 1D NOE, HSQC and HMBC). Copyright

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.

Oxalic acid and sclerotial differentiation of Polyporus umbellatus

The present investigation aimed to uncover the effects of exogenous oxalic acid during the sclerotial formation of Polyporus umbellatus, with an emphasis on determining the content of the endogenic oxalic acid in the fungus. To this end, the oxalic acid content of the vegetative mycelia, sclerotia, culture mediums and sclerotial exudate were measured using High Performance Liquid Chromatography (HPLC). Furthermore, the lipid peroxidation was estimated by detecting thiobarbituric bituric acid reactive substances (TBARS). The results showed that the exogenous oxalic acid caused a delay in sclerotial differentiation (of up to 9 or more days), suppressed the sclerotial biomass and decreased the lipid peroxidation significantly in a concentration-dependent manner. Oxalic acid was found at very low levels in the mycelia and the maltose medium, whereas it was found at high levels in the mycelia and sucrose medium. After sclerotial differentiation, oxalic acid accumulated at high levels in both the sclerotia and the sclerotial exudate. Oxalic acid was therefore found to inhibit P. umbellatus sclerotial formation.

Shancigusins E-I, five new glucosides from the tubers of Pleione yunnanensis

Five new glucosides, shancigusins E‐I (1–5) were isolated from the tubers of Pleione yunnanensis (Rolfe) together with 18 known compounds. The structures of these compounds were determined by extensive analyses of their spectroscopic data. Copyright

Highly specific separation for antitumor Spiropreussione A from endophytic fungal [Preussia sp.] fermentation broth by one-step macroporous resins AB-8 treatment

It is attractive to pharmaceutical works to seek useful material from endophytic fungi. Spiropreussione A (SA) which is isolated from endophytic fungus Preussia sp. is a novel anti-tumor product. Since previous preparation method cannot provide enough SA, and considering the large volume of broth and low concentration of the target product, macroporous resins were introduced to separate SA in our study. Four kinds of macroporous resins ADS-8, H103, X-5 and AB-8 were applied for separating SA, and AB-8 was selected as the optimal resin according to its performances through static and dynamic measurements. HPLC was used to analyze SA in all samples. Under optimal conditions, the specific SA adsorption capacity of AB-8 resin was 15.23mg/g, and the purity increased by 2.5-fold from 35.0% in broth to 90.0% in eluent with 70.0% recovery yield by a one-step treatment. Conclusively, our study achieved the goal of separating and purifying SA in high efficiency, and offered references for further fermentation works.

SWATH label-free proteomics analyses revealed the roles of oxidative stress and antioxidant defensing system in sclerotia formation of Polyporus umbellatus

Understanding the initiation and maturing mechanisms is important for rational manipulating sclerotia differentiation and growth from hypha of Polyporus umbellatus. Proteomes in P. umbellatus sclerotia and hyphae at initial, developmental and mature phases were studied. 1391 proteins were identified by nano-liquid chromatograph-mass spectrometry (LC-MS) in Data Dependant Acquisition mode, and 1234 proteins were quantified successfully by Sequential Window Acquisition of all THeoretical fragment ion spectra-MS (SWATH-MS) technology. There were 347 differentially expressed proteins (DEPs) in sclerotia at initial phase compared with those in hypha, and the DEP profiles were dynamically changing with sclerotia growth. Oxidative stress (OS) in sclerotia at initial phase was indicated by the repressed proteins of respiratory chain, tricarboxylic acid cycle and the activation of glycolysis/gluconeogenesis pathways were determined based on DEPs. The impact of glycolysis/gluconeogenesis on sclerotium induction was further verified by glycerol addition assays, in which 5% glycerol significantly increased sclerotial differentiation rate and biomass. It can be speculated that OS played essential roles in triggering sclerotia differentiation from hypha of P. umbellatus, whereas antioxidant activity associated with glycolysis is critical for sclerotia growth. These findings reveal a mechanism for sclerotial differentiation in P. umbellatus, which may also be applicable for other fungi.