"Mini-community" simulation revealed the differences of endophytic fungal communities between the above- and below-ground tissues of Ephedra sinica Stapf.

The microecology of endophytic fungi in special habitats, such as the interior of different tissues from a medicinal plant, and its effects on the formation of metabolites with different biological activities are of great importance. However, the factors affecting fungal community formation are unclear. This study is the first to utilize "mini-community" remodeling to understand the above phenomena. First, high-throughput sequencing technology was applied to explore the community composition and diversity of endophytic fungi in the above-ground tissues (Ea) and below-ground tissues (Eb) of Ephedra sinica. Second, fungi were obtained through culture-dependent technology and used for "mini-community" remodeling in vitro. Then, the effects of environmental factors, partner fungi, and plant tissue fluid (internal environment) on endophytic fungal community formation were discussed. Results showed that environmental factors played a decisive role in the selection of endophytic fungi, that is, in Ea and Eb, 93.8% and 25.3% of endophytic fungi were halophilic, respectively, and 10.6% and 60.2% fungi were sensitive to high temperature (33 °C), respectively. Meanwhile, pH had little effect on fungal communities. The internal environment of the plant host further promoted the formation of endophytic fungal communities.

The composition characteristics of endophytic communities and their relationship with metabolites profile in Ephedra sinica under wild and cultivated conditions.

Ephedra sinica is one of the most famous Chinese medicinal plants. The insufficient supply of wild resources has led to the increased use of cultivated products. However, the related medicinal quality differs significantly. Although the influence of external environment on the quality of E. sinica has been studied, the impact of endophytic microbes on it remains vague. This study characterized differential metabolites and microbial community compositions in wild and cultivated E. sinica by combining metabolomics with microbiomics, and explored the effect of endophytes on the formation of differential metabolites further. The results showed that the difference in quality between wild and cultivated E. sinica was mainly in the productions of alkaloids, flavonoids, and terpenoids. The associated endophytes had special compositional characteristics. For instance, the distribution and abundance of dominant endophytes varied between wild and cultivated E. sinica. Several endophytes had significant or highly significant correlations with the formations of ephedrine, pseudoephedrine, D-cathinone, methcathinone, coumarin, kaempferol, rhamnetin, or phenylacetic acid. This study will deepen our understanding of the plant-endophyte interactions and provide a strategy for the quality control of E. sinica products.

Transcriptional Regulatory Mechanism of Differential Metabolite Formation in Root and Stem of Ephedra sinica.

Ephedra sinica, a well-known Chinese medicinal plant, is characterized as having the opposite medicinal effect among its root and stem. However, there is a lack of understanding to differentiate the active components present in the root and stem of E. sinica, as well as the molecular mechanisms underlying the formation of the differential compounds, which has significantly hampered the further development and utilization of E. sinica resource. In this study, forty-five differential metabolic markers are affiliated to alkaloids, flavonoids, terpenoids, and organic acids between root and stem of E. sinica, and sixty genes of key enzymes are involved in their biosynthesis distributed in metabolic pathway branches such as phenylalanine metabolism, flavonoid biosynthesis and phenylpropane biosynthesis, based on combination non-targeted metabolome with transcriptome technologies. The finding revealed that the expression activity changes of these enzyme genes had a direct impact on the distinction of differential metabolic markers in the root and stem of E. sinica. This study will help to understand the molecular mechanism of the differentiation and biosynthesis of the primary active metabolites in the root and stem of E. sinica, providing a theoretical foundation for its quality control and promotion in cultivation.

Endophytic fungi and their bioactive secondary metabolites in medicinal leguminosae plants: nearly untapped medical resources.

There are many species of Chinese traditional leguminosae family plants that are well known for their medicinal applications, such as Astragalus membranaceus, Catsia tora, Glycyrrhiza uralensis, Sophora flavescens and Albacia acacia. Their unique bioactive composition and internal phenological environment contribute to the formation of specific and unique endophytic fungal communities, which are important resources for new compounds used in a variety of pharmacological activities. Nonetheless, they have not been systematically studied. In the last decade, nearly 64 genera and thousands of species of endophytic fungi have been discovered from leguminosae plants, as well as 138 secondary metabolites (with 34 new compounds) including flavonoid, alkaloids, phenol, anthraquinone, macrolide, terpenoid, phytohormone and many more. These were shown to have diverse applications and benefits, such as antibacterial, antitumor, antioxidative, immunoregulatory and neuroprotective properties. Here, we provide a summarized overview with the aim of raising awareness of endophytic fungi from medicinal leguminosae plants and providing a comprehensive review of the discoveries of new natural products that may be of medicinal and pharmaceutical importance.

A review of the phytochemistry and pharmacological activities of Ephedra herb.

Ephedra herb is a traditional Chinese medicine with a long history. Conventionally, it was used as a folk phytomedicine in many ancient medical books and traditional prescriptions. Up to date, a variety of specific ingredients have been found in Ephedra herb, mainly including alkaloids, flavonoids, tannins, polysaccharides, organic acids, volatile oils, and many other active compounds. These components from Ephedra herb account for its use as the accurate treatment of cold, cough, cardiovascular and immune system disease, cancer, microbial infection, and other diseases. Moreover, with the fast development of novel chemistry and medicine technology, new chemical constituents and pharmacological effects of Ephedra herb are increasingly identified, demonstrating their great potential for various diseases treatment. Therefore, further detailed understanding and investigation of this ancient herb will offer new opportunities to develop novel therapeutics. This study systematically reviews its progress of phytochemistry, traditional and modern pharmacology based on research data that have been reported, aiming at providing useful insight for commercial exploitation, further study and precision medication of Ephedra herb in future.

Correlation in Chemical Metabolome and Endophytic Mycobiome in Cynomorium songaricum from Different Desert Locations in China.

Cynomorium songaricum Rupr. is a valuable food and medicinal plant with functions, such as an increase in sexual function, mainly attributed to its complex secondary metabolites. However, the effect of internal microbes on metabolite production in C. songaricum is still largely unclear. In this study, the relationship between endophytes and differential secondary metabolites in C. songaricum from seven major producing regions of China were explored based on established methods of metabolomics and high-throughput sequencing. The results showed that there were 13 different marker metabolites, seven shared fungal OTUs, and numerous unshared OTUs among C. songaricum distributed at different locations in China and identified significant correlations between metabolites and endophytic fungi. Our study revealed that endophytic fungi may be one possible factor that can affect the plant secondary metabolite composition.

Secondary Metabolite Accumulation Associates with Ecological Succession of Endophytic Fungi in Cynomorium songaricum Rupr.

Cynomorium songaricum Rupr. is a rare root-parasitic plant distributed in the desert ecosystem. Little is known about the role of endophytes in accumulation of metabolites in C. songaricum. Here, the correlations between the seven active components (total sugars, flavonoids, protocatechuic acid, catechins, tannins, gallic acid, and ursolic acid) and the endophytic fungi of C. songaricum were investigated, and their causal relationships are discussed further. The results showed that the accumulation of these components and the assembly of endophytic fungi changed with different plant developmental stages. Diverse relationships including positive and negative correlation were found among chemicals and endophytic fungal operational taxonomic units based on correlation coefficient matrices, which demonstrated that the accumulation of secondary metabolites in C. songaricum is closely related to the endophytic fungal community composition. These results present new opportunities to deeply understand plant-fungal symbioses and secondary metabolite productions.

Partitioning of Fungal Endophyte Assemblages in Root-Parasitic Plant Cynomorium songaricum and Its Host Nitraria tangutorum.

Endophytic fungi are an integral part and even seen as host organs of plant, influencing physiology, ecology, and development of host plants. However, little is known about micro-ecosystems and functional interactions of endophytic fungi in root-parasitic interactions of Cynomorium songaricum and its host Nitraria tangutorum. Here, distribution and dynamics of endophytic fungi were objectively investigated in their associations with C. songaricum and N. tangutorum based on mycobiome studies using high-throughput sequencing. Results suggest that endophytic fungi may be exchanged between C. songaricum and its host N. tangutorum probably through haustorium, connection of xylem and phloem in the vascular system. The similarity of endophytic fungal composition between C. songaricum and parasitized N. tangutorum was 3.88% which was significantly higher than the fungal similarity of 0.10% observed between C. songaricum and non-parasitized N. tangutorum. The similarities of fungal community in parasitized N. tangutorum were much closer to C. songaricum than to the non-parasitized N. tangutorum. The composition of endophytic fungi in these associations increased in progressive developmental stages of C. songaricum from sprouting to above ground emergence, and decreased subsequently probably due to host recognition and response by fungi. However, the shared fungal operational taxonomic units (OTUs) increased among interactions of C. songaricum with parasitized and non-parasitized N. tangutorum. Studies of bioactivity on culturable endophytic fungi showed that isolates such as Fusarium spp. possess the ability to promote seed germination of C. songaricum. Our study reports for the first time the special ecological system of endophytic fungi in C. songaricum and its host N. tangutorum. Overall, we hypothesize that a deeper understanding of the sharing, movement, and role of endophytic fungi between root-parasitic plant and its host may lead to finding alternative approaches to help increase the output of ethno-pharmacologically important medicinal plants.

[Interaction between endophytes and host plant and the role of endophytes in genuineness analysis of medicinal plant].

Research of plant development and metabolism has drawn lots of attention with the fast development of science of mycorrhizal biology, molecular biology and metabonomics technology. It has become one of hot fields in the study of endophytes and plant, which would affect plant 's metabolite composition. This would provide opportunity for appraising and modifying traits to medicinal plant, and would also perfect the tranditional standpoint on forming reason of medicinal plant genuineness. Here we provide a review of theory and mechanism, research and application of interaction between plant and endophyte. This review may enhance understanding of medicinal plant, and evaluating the quality of herbs in production.

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This is the first report of the distribution and genetic relationships of endophytic fungi from the parasitic plant Cynomorium songaricum and its host Nitraria tangutorum. Endophytic fungi from the root of natural N. tangutorum, parasitic plant C. songaricum and its host N. tangutorum were isolated by tissue culture, and they were identified by morphology combined with molecular barcoding based on ITS-rDNA sequence. The isolation rates, colonization rates, isolation frequency, diversity index, evenness index, similarity coefficient and genetic relationships among fungal taxa were estimated by phylogenetic analysis, and differences in fungal endophyte distribution were investigated. The results showed that a total of 49 isolates were obtained belonging to 18 different taxa. 95.9% of these taxa were in Ascomycota, and the remaining was in Basidiomycota (4.1%). The isolation rate and colonization rate of endophytic fungi were 15.3% and 25.0%, respectively. The Shannon biodiversity index was the highest in the root of natural N. tangutorum at 2.13. The simila-rity coefficient was highest between the stem of C. songaricum and the flower of C. songaricum at0.50. Fusarium was the dominant genus in N. tangutorum, while Penicillium was the primary genus in C. songaricum. The differential distribution of fungal taxa between N. tangutorum and C. songaricum suggested that the parasitic relationship influences the endophytic fungal community.

Stimulation of dragon's blood accumulation in Dracaena cambodiana via fungal inoculation.

Dragon's blood is a rare and precious traditional medicine used by different cultures since ancient times. However, studies on enhancing the rapid accumulation of dragon's blood in Dracaena cambodiana and determining its formation mechanism are unavailable. In this study, the activities of two fungi, namely, BJDC01 and BJDC05, and their effect on promoting the accumulation of five main compositions of dragon's blood in D. cambodiana were investigated for the first time. Results of field tests conducted for ten months indicated that the contents of Loureirin D, 4,4'-dihydroxy-2'-dimethoxychalcone, Loureirin A and Loureirin B in two fungal-inoculated materials were 1.67 to 2.85 times greater than those of natural samples, and thus were significantly higher than those of the control groups. The content of 4,4'-dihydroxy-2'6'-dimethoxydihydrochalcone in each fungal-inoculated sample was close to that of the natural sample, and was more than twice of each of the control group. By combining the results of morphological characterizations, both BJDC01 and BJDC05 can stimulate the accumulation of the compositions of dragon's blood. This stimulation may be considered as a defense response of D. cambodiana tree against the invasion of foreign fungi. Thus, this study provides a potential way of producing dragon's blood via the inoculation of two fungal elicitors.

Biotransformation of ursolic acid by Syncephalastrum racemosum CGMCC 3.2500 and anti-HCV activity.

Microbial transformation of ursolic acid (UA, 3ß-hydroxy-urs-12-en-28-oic acid, 1) by filamentous fungus Syncephalastrum racemosum CGMCC 3.2500 was conducted. Five metabolites 3ß, 7ß, 21ß-trihydroxy-urs-12-en-28-oic acid (2); 3ß, 21ß-dihydroxy-urs-11-en-28-oic acid-13-lactone (3); 1ß, 3ß, 21ß-trihydroxy-urs-12-en-28-oic acid (4); 3ß, 7ß, 21ß-trihydroxy-urs-1-en-28-oic acid-13-lactone (5); and 21-oxo-1ß, 3ß-dihydroxy-urs-12-en-28-oic acid (6) were afforded. Elucidation of the structures of these metabolites was primarily based on 1D and 2D NMR and HR-MS data. Metabolite 2 was a new compound. In addition, the anti-HCV activity of compounds 1-6 was evaluated.

Biotransformation of ursolic acid by an endophytic fungus from medicinal plant Huperzia serrata.

Endophytic fungi were used not only for their producing bioactive products but also for their ability to transform natural compounds. An endophytic fungus, isolated from medicinal plant Huperzia serrata, was identified as Umbelopsis isabellina based on the internal transcribed spacer of ribosomal DNA (rDNA-ITS) region. It was used to transform ursolic acid (1), a pentacyclic triterpene. Incubation of ursolic acid with U. isabellina afforded three products, 3ß-hydroxy-urs-11-en-28,13-lactone (2), 3ß,7ß-dihydroxy-urs-11-en-28,13-lactone (3), 1ß,3ß-dihydroxy-urs-11-en-28,13-lactone (4). Although product 2 was a known compound, it was first obtained by microbial transformation. Products 3 and 4 were new compounds. The structural elucidation of the three compounds was achieved mainly by the 1D- and 2D-NMR, MS, IR data. The endophytic fungus U. isabellina can hydroxyate the C12-C13 double bond at position 13 of ursolic acid 1 and form a five-member lactone effectively. In the meantime, this fungus can also introduce the hydroxyl group at C-1 or C-7 of ursolic acid 1.

Multihydroxylation of ursolic acid by Pestalotiopsis microspora isolated from the medicinal plant Huperzia serrata.

The structural modification of ursolic acid by an endophytic fungus Pestalotiopsis microspora, isolated from medicinal plant Huperzia serrata was reported for the first time. The structure diversity was very important for the SAR study of ursolic acid and its derivatives. Incubation of ursolic acid 1 with P. microspora afforded four metabolites: 3-oxo-15α, 30-dihydroxy-urs-12-en-28-oic acid (2), 3ß, 15α-dihydroxy-urs-12-en-28-oic acid (3), 3ß, 15α, 30- trihydroxy-urs-12-en-28-oic acid (4) and 3,4-seco-ursan-4,30-dihydroxy-12-en-3,28-dioic acid (5). All products were new compounds and their structures elucidation was mainly based on the spectroscopic data.

Antitumor and antimicrobial activities of endophytic fungi from medicinal parts of Aquilaria sinensis.

The purpose of this study was to isolate and characterize endophytic fungi from the stem tissue which can produce fragrant ingredients in Aquilaria sinensis (also called agarwood) to determine their antitumor and antimicrobial activities. Twenty-eight fungal endophytes were isolated from agarwood by strict sterile sample preparation and were classified into 14 genera and 4 taxonomic classes (Sordariomycetes, Dothideomycetes, Saccharomycetes, and Zygomycetes) based on molecular identification. Of the 28 isolates, 13 (46.4%) showed antimicrobial activity against at least one of the test strains by the agar well diffusion method, and 23 isolates (82.1%) displayed antitumor activity against at least one of five cancer cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The diameters of inhibition zones of YNAS07, YNAS14, HNAS04, HNAS05, HNAS08, and HNAS11 were equal to or higher than 14.0 mm against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, B. subtilis, Aspergillus fumigatus, and B. subtilis, respectively. The inhibition rates of YNAS06, YNAS08, and HNAS06 were not less than 60% to 293-T, 293-T, and SKVO3 cells, respectively. These results suggest that the endophytic fungi associated with agarwood will provide us with not only useful micro-ecological information, but also potential antimicrobial and antitumor agents.

Endophytic fungi from Dragon's blood specimens: isolation, identification, phylogenetic diversity and bioactivity.

Endophytic fungi from Dragon's blood specimens of different locations of China were characterized taxonomically and investigated concerning their antimicrobial and antitumor activity against six pathogenic microbes and five tumor cells. A total of 49 endophytic fungi were obtained from Dragon's blood materials of Dracaena spp., 18 taxa were represented by 43 (87.8%) isolates and only six (12.2%) isolates were unknown. Twenty (40.8%) of the isolates displayed antimicrobial activity against at least one pathogenic microorganism. Three isolates YNDC07, BJDC06 and BJDC09 displayed significant antimicrobial activities against Staphylococcus aureus, Cryptococcus neoformans and Aspergillus fumigates, respectively. The results of antitumor activity by the MTT assay revealed that 26.5%, 69.4%, 48.9%, 6.1% and 42.9% of isolate fermentation broths displayed growth inhibition on HepG2 cells, SKVO3 cells, MCF7 cells, HL-60 cells and 293-T cells, respectively. HNDC09 and HNDC10 showed very strong antitumor activity against MCF7 and 293-T, respectively. The results showed that endophytic fungi in Dragon's blood samples were valuable in screening antitumor and antimicrobial bioactivity agents.