[Inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction].

To determine the inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction,the protein-protein interaction between human immunodeficiency virus type 1( HIV-1) integrase and lens epithelial growth factor p75 protein( LEDGF/p75) was used as a target. The homogeneous time-resolved fluorescence( HTRF) technique was used in the inhibitory activity assay. The results showed that eight endophytic fungi with anti-IN-LEDGF/p75 interaction activity were screened out from fifty-three strains with different morphological characteristic. Among them,106 strain showed strong inhibitory activity against HIV-1 IN-LEDGF/p75 interaction with IC50 value of 5. 23 mg·L-1,and was identified as a potential novel species of Magnaporthaceae family by the analyses of ITS-rDNA,LSU and RPB2 sequences data. This study demonstrated that potential natural active ingredients against the HIV-1 IN-LEDGF/p75 interaction exist in the endophytic fungi of D. versipellis. These results may provide available candidate strain resources for the research and development of new anti-acquired immunodeficiency syndrome drugs.

Propioniciclava sinopodophylli sp. nov., isolated from leaves of Sinopodophyllum hexandrum (Royle) Ying.

A Gram-reaction-positive, facultatively anaerobic, rod-shaped and non-motile bacterial strain, designated TEYR-7T, was isolated from the leaves of Sinopodophyllum hexandrum collected from the Qinling Mountains in Shaanxi Province, northwest China. Growth of strain TEYR-7T occurred at 15-37 °C (optimum, 28-30 °C), at pH 6.0-9.0 (optimum, pH 7.0) and in the presence of 0-3 % (w/v) NaCl (optimum, 0-1 %). Propionate and acetate were produced from glucose fermentation. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TEYR-7T was a member of the phylum Actinobacteria, exhibiting the highest sequence similarity to Propioniciclava tarda DSM 22130T (94.3 %). The only respiratory quinone detected in strain TEYR-7T was menaquinone MK-9(H4) and the major cellular fatty acids (>10 %) were anteiso-C15 : 0 and C16 : 0. The polar lipid profile consisted of phosphatidylglycerol, diphosphatidylglycerol, two unidentified glycolipids, an unidentified phospholipid and three unidentified lipids. The genomic DNA G+C content was 71.2 mol%. meso-Diaminopimelic acid was detected in the peptidoglycan. On the basis of data from the present polyphasic taxonomic study, strain TEYR-7T is considered to represent a novel species of the genus Propioniciclava, for which the name Propioniciclava sinopodophylli sp. nov. is proposed. The type strain is TEYR-7T (=CCTCC AB 2015257T=KCTC 33808T).

Paenibacillus qinlingensis sp. nov., an indole-3-acetic acid-producing bacterium isolated from roots of Sinopodophyllum hexandrum (Royle) Ying.

A novel indole-3-acetic acid-producing bacterium, designated TEGT-2T, was isolated from the roots of Sinopodophyllum hexandrum collected from the Qinling Mountains in shaanxi province, northwestern China, and was subjected to a taxonomic study by using a polyphasic approach. Cells of strain TEGT-2T were Gram-stain-positive, strictly aerobic, endospore-forming rods and motile by means of peritrichous flagella. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TEGT-2T was a member of the genus Paenibacillus, exhibiting the highest sequence similarity to Paenibacillus pectinilyticus KCTC 13222T (97.9 %), Paenibacillus frigoriresistens CCTCC AB 2011150T (97.3 %), Paenibacillus ferrarius CCTCC AB 2013369T (96.9 %) and Paenibacillus alginolyticus NBRC 15375T (96.5 %). The only menaquinone detected was MK-7, and the major fatty acid was anteiso-C15 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phospholipids, an unidentified aminolipid and two unidentified lipids. meso-Diaminopimelic acid was detected in the peptidoglycan. The DNA G+C content was 46.6 mol%. DNA-DNA relatedness values for strain TEGT-2T with respect to its closest phylogenetic relatives Paenibacilluspectinilyticus KCTC 13222T and Paenibacillus. frigoriresistens CCTCC AB 2011150T were lower than 40 %. Based on the phenotypic, phylogenetic and genotypic data, strain TEGT-2T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus qinlingensis sp. nov. is proposed. The type strain is TEGT-2T (=CCTCC AB 2015258T=KCTC 33806T).

Paenibacillus sinopodophylli sp. nov., a siderophore-producing endophytic bacterium isolated from roots of Sinopodophyllum hexandrum (Royle) Ying.

A Gram-stain-positive, strictly aerobic, rod-shaped, motile and endospore-forming bacterial strain, designated TEGR-3T, was isolated from the roots of Sinopodophyllum hexandrum collected from the Qinling Mountains in Shaanxi Province, China. Strain TEGR-3T produced siderophores and hydrolysed aesculin, starch and CM-cellulose. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TEGR-3T was a member of the genus Paenibacillus, exhibiting the highest sequence similarity to Paenibacillus endophyticus LMG 27297T (97.3 %) and Paenibacillus castaneae DSM 19417T (97.3 %). MK-7 was the only menaquinone detected and anteiso-C15 : 0 and C16 : 0 were the major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, two unidentified aminophospholipids, two unidentified phospholipids and an unidentified lipid. The cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The DNA G+C content was 45.2 mol%. DNA-DNA relatedness values for strain TEGR-3T with respect to its closest phylogenetic relatives Paenibacillus endophyticus LMG 27297T and Paenibacillus castaneae DSM 19417T were lower than 40 %. Based on the phenotypic, phylogenetic and genotypic data, strain TEGR-3T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus sinopodophylli sp. nov. is proposed. The type strain is TEGR-3T (=CCTCC AB 2016047T=KCTC 33807T).

Endophytic Fungus from Sinopodophyllum emodi (Wall.) Ying that Produces Podophyllotoxin.

The aryltetralin lactone podophyllotoxin, which exhibits pronounced antineoplastic activity, is used as the precursor of the following three clinical anticancer drugs: Etoposide™, Etopophos™ and Teniposide™. The natural occurrence of this arylnaphthalene lignan is scarce and unable to meet the ever-rising demand in the medical industry. Thus, developing alternative sources for the production of podophyllotoxin is extremely urgent. This is the first report of the production of podophyllotoxin from endophytic Alternaria tenuissima isolated from Sinopodophyllum emodi (Wall.) Ying. The identification of podophyllotoxin was performed using high-performance liquid chromatography and liquid chromatography-mass spectrometry (MS)-MS and confirmed by comparison with authentic standards.

[Community composition and diversity of endophytic fungi from roots of Sinopodophyllum hexandrum in forest of Upper-north mountain of Qinghai province].

High throughput sequencing technology is also called Next Generation Sequencing (NGS), which can sequence hundreds and thousands sequences in different samples at the same time. In the present study, the culture-independent high throughput sequencing technology was applied to sequence the fungi metagenomic DNA of the fungal internal transcribed spacer 1(ITS 1) in the root of Sinopodophyllum hexandrum. Sequencing data suggested that after the quality control, 22 565 reads were remained. Cluster similarity analysis was done based on 97% sequence similarity, which obtained 517 OTUs for the three samples (LD1, LD2 and LD3). All the fungi which identified from all the reads of OTUs based on 0.8 classification thresholds using the software of RDP classifier were classified as 13 classes, 35 orders, 44 family, 55 genera. Among these genera, the genus of Tetracladium was the dominant genera in all samples(35.49%, 68.55% and 12.96%).The Shannon's diversity indices and the Simpson indices of the endophytic fungi in the samples ranged from 1.75-2.92, 0.11-0.32, respectively.This is the first time for applying high through put sequencing technol-ogyto analyze the community composition and diversity of endophytic fungi in the medicinal plant, and the results showed that there were hyper diver sity and high community composition complexity of endophytic fungi in the root of S. hexandrum. It is also proved that the high through put sequencing technology has great advantage for analyzing ecommunity composition and diversity of endophtye in the plant.

Meroterpenoids and isoberkedienolactone from endophytic fungus Penicillium sp. associated with Dysosma versipellis.

Seven meroterpenoids and five small-molecular precursors were isolated from Penicillium sp., an endophytic fungus from Dysosma versipellis. The structures of new compounds, 11beta-acetoxyisoaustinone (1) and isoberkedienolactone (2) were elucidated based on analysis of the spectral data, and the absolute configuration of 2 was established by TDDFT ECD calculation with satisfactory match to its experimental ECD data. Meroterpenoids originated tetraketide and pentaketide precursors, resepectively, were found to be simultaneously produced in specific fungus of Penicillium species. These compounds showed weak cytotoxicity in vitro against HCT-116, HepG2, BGC-823, NCI-H1650, and A2780 cell lines with IC 50 > 10 micromol x L(-1).

[Microscopic observation on mycorrhiza of rare herb Dysosma versipellis].

Endophytic fungi played an important role in the growth of its host plant. To investigate the mycorrhizal characteristics and the distribution of fungi in the root, an endangered wild plant-Dysosma versipellis was collected and observed by electron microscope. The results showed that the host was closely associated with endophytic fungi. The fungi were mainly distributed in the epidermis and cortex. The aseptate and septate fungi with swollen hyphae were observed in some cell of the cortex. The result provides a reference for the study of mycorrhizal structure of Dysosma genus and the interaction between the fungi and its host.

Novel tandem biotransformation process for the biosynthesis of a novel compound, 4-(2,3,5,6-tetramethylpyrazine-1)-4'-demethylepipodophyllotoxin.

According to the structure of podophyllotoxin and its structure-function relationship, a novel tandem biotransformation process was developed for the directional modification of the podophyllotoxin structure to directionally synthesize a novel compound, 4-(2,3,5,6-tetramethylpyrazine-1)-4'-demethylepipodophyllotoxin (4-TMP-DMEP). In this novel tandem biotransformation process, the starting substrate of podophyllotoxin was biotransformed into 4'-demethylepipodophyllotoxin (product 1) with the demethylation of the methoxyl group at the 4' position by Gibberella fujikuroi SH-f13, which was screened out from Shennongjia prime forest humus soil (Hubei, China). 4'-Demethylepipodophyllotoxin (product 1) was then biotransformed into 4'-demethylpodophyllotoxone (product 2) with the oxidation of the hydroxyl group at the 4 position by Alternaria alternata S-f6, which was screened out from the gathered Dysosma versipellis plants in the Wuhan Botanical Garden, Chinese Academy of Sciences. Finally, 4'-demethylpodophyllotoxone (product 2) and ligustrazine were linked with a transamination reaction to synthesize the target product 4-TMP-DMEP (product 3) by Alternaria alternata S-f6. Compared with podophyllotoxin (i.e., a 50% effective concentration [EC(50)] of 529 µM), the EC(50) of 4-TMP-DMEP against the tumor cell line BGC-823 (i.e., 0.11 µM) was significantly reduced by 5,199 times. Simultaneously, the EC(50) of 4-TMP-DMEP against the normal human proximal tubular epithelial cell line HK-2 (i.e., 0.40 µM) was 66 times higher than that of podophyllotoxin (i.e., 0.006 µM). Furthermore, compared with podophyllotoxin (i.e., log P = 0.34), the water solubility of 4-TMP-DMEP (i.e., log P = 0.66) was significantly enhanced by 94%. For the first time, the novel compound 4-TMP-DMEP with superior antitumor activity was directionally synthesized from podophyllotoxin by the novel tandem biotransformation process developed in this work.

Cytotoxic activities of endophytic fungi isolated from the endangered, Chinese endemic species Dysosma pleiantha.

Eleven strains of endophytic fungi which habitat in an endangered, Chinese endemic medicinal plant, Dysosma pleiantha (Hance) Woodson, were isolated and tested for their cytotoxic activity using the brine shrimp lethality bioassay. Six isolates were found to exhibit some cytotoxic activity. Extracts of F1273, F1276, and F1280, which were identified as Trichoderma citrinoviride, Chaetomium globosum and Ascomycete sp., in particular, showed most potent activity with LC50 values of 4.86, 7.71, and 14.88 microg/ml, respectively. These results indicate that endophytic fungi of Dysosma pleiantha could be a promising source for antitumour agents.