A total of 127 strains of endophytic fungi were isolated from roots, branches and leaves of Huperzia serrata. These strains were identified into 19 genera based on morphological characters and ribosomal DNA (rDNA) sequence analysis, there into Penicillium, Aspergillus and Podospora were dominant populations in H. serrata. From analysis results we found some endophytic fungi showed a certain degree of tissue preference. The isolation rate and colonization rate of stems were both larger than those of leaf and roots. After testing the acetylcholinesterase (AChE) inhibitory activity of these endophytic fungi, a total of 39 endophytic fungi belonging to 15 genera showed AChE inhibition. Eleven endophytic fungi showed potent AChE inhibition, 7 of which were isolated from leaf. The research not only provided theoretical basis for developing and utilizing the resources of endophytic fungi in H. serrata but also showed a new path for searching medicines resource which has AChE inhibitory activity.
Cholinesterase Inhibitors ; pharmacology ; Fungi ; classification ; isolation & purification ; Huperzia ; microbiology

Xanthocillin is a unique natural product with an isonitrile group and shows remarkable antibacterial activity. In this study, the genome of an endophytic fungus Penicillium chrysogenum MT-40 isolated from Huperzia serrata was sequenced, and the gene clusters with the potential to synthesize xanthocillin analogues were mined by local BLAST and various bioinformatics analysis tools. As a result, a biosynthetic gene cluster (named for) responsible for the biosynthesis of xanthocillin analogues was identified by further heterologous expression of the key genes in Aspergillus oryzae NSAR1. Specifically, the ForB catalyzes the synthesis of 2-formamido-3-(4-hydroxyphenyl) acrylic acid, and the ForG catalyzes the dimerization of 2-formamido-3-(4-hydroxyphenyl) acrylic acid to produce the xanthocillin analogue N, N'-(1, 4-bis (4-hydroxyphenyl) buta-1, 3-diene-2, 3-diyl) diformamide. The results reported here provide a reference for further discovery of xanthocillin analogues from fungi.
Penicillium chrysogenum/genetics* ; Huperzia/microbiology* ; Acrylates ; Multigene Family

OBJECTIVETo study the effects of soil microbes of habitats, endophytic fungi and root surface fungi of Huperzia serrata on rooting rate of the cuttings and on several physiological and biochemical indexes.

METHODThe cuttings of H. serrata were planted on soil I of primary habitat, the soil II had been inoculated endophytic fungi and root surface fungi, the soil III had been auto-claved, rooting rate was examined, and soluble protein and soluble sugar contents, activities of PPO and POD, flavonoids and huperzine A contents were determined.

RESULTCompared with the H. serrata that were planted on soil III, soil I and soil II were found to increase the rooting rate by 10% and 16%, soil II increased the soluble protein contents of stem of H. serrata (P < 0.05), Soil I increased the soluble sugar contents of leaves and stem of H. serrata (P < 0.05), soil I and Soil II increased the flavonoids contents of H. serrata (P < 0.05).

CONCLUSIONSoil microbes from primary habitat, endophytic fungi and root surface fungi promote rooting, they also increase plant metabolism level of H. serrata.

Carbohydrate Metabolism ; Flavonoids ; metabolism ; Huperzia ; growth & development ; metabolism ; microbiology ; Plant Proteins ; chemistry ; metabolism ; Plant Roots ; growth & development ; metabolism ; microbiology ; Soil Microbiology ; Solubility

OBJECTIVETo study the effects of soil microbes of habitats on the cuttings rooting of Huperzia serrata and its mechanism.

METHODThe cuttings of H. serrata were planted on the primary habitat soil and on the soil autoclaved, rooting rates were examined respectively. Changes of contents of phenolic compounds (catechin, quercetin, chlorogenic acid, ferulic acid) and indoleacetic acid (IAA) , and changes of activities of polyphenoloxidase (PPO) and peroxydase (POD) during the cuttings rooting were determined.

RESULTSoil microbes of primary habitat could increase the rooting rate and the number of roots, could increase contents of catechin, chlorogenic acid, ferulic acid and IAA during rooting, and could affect activities of PPO and POD.

CONCLUSIONDuring the rooting higher contents of catechin, chlorogenic, ferulic acid and IAA caused by soil microbes of primary habitat are important factor for rooting rate.

Catechol Oxidase ; metabolism ; Flavonoids ; metabolism ; Huperzia ; growth & development ; metabolism ; microbiology ; Indoleacetic Acids ; metabolism ; Peroxidase ; metabolism ; Plant Roots ; growth & development ; metabolism ; microbiology ; Plants, Medicinal ; growth & development ; metabolism ; microbiology ; Soil Microbiology

A total of 27 endophytic fungal strains were isolated from Huperzia serrata,which were richly distributed in the stems and leaves while less distributed in roots. The 27 strains were identified by Internal Transcribed Spacer( ITS) r DNA molecular method and one of the strains belongs to Basidiomycota phylum,and other 26 stains belong to 26 species,9 general,6 families,5 orders,3 classes of Ascomycota Phylum. The dominant strains were Colletotrichum genus,belonging to Glomerellaceae family,Glomerellales order,Sordariomycetes class,Ascomycota Phylum,with the percentage of 48. 15%. The inhibitory activities of the crude extracts of 27 endophytic fungal strains against acetylcholinesterase( ACh E) and nitric oxide( NO) production were evaluated by Ellman's method and Griess method,respectively. Crude extracts of four fungi exhibited inhibitory activities against ACh E with an IC50 value of 42. 5-62. 4 mg·L~(-1),and some fungi's crude extracts were found to inhibit nitric oxide( NO) production in lipopolysaccharide( LPS)-activated RAW264. 7 macrophage cells with an IC50 value of 2. 2-51. 3 mg·L~(-1),which indicated that these fungi had potential anti-inflammatory activities.The chemical composition of the Et OAc extract of endophytic fungus HS21 was also analyzed by LCMS-IT-TOF. Seventeen compounds including six polyketides,four diphenyl ether derivatives and seven meroterpenoids were putatively identified.
Acetylcholinesterase ; Animals ; Anti-Inflammatory Agents ; isolation & purification ; pharmacology ; Ascomycota ; chemistry ; classification ; isolation & purification ; Cholinesterase Inhibitors ; isolation & purification ; metabolism ; Endophytes ; classification ; isolation & purification ; Huperzia ; microbiology ; Mice ; RAW 264.7 Cells

OBJECTIVETo screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata.

METHODEndophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics.

RESULTFungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium.

CONCLUSIONEndophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.

Acetylcholinesterase ; metabolism ; Acremonium ; genetics ; metabolism ; Cholinesterase Inhibitors ; isolation & purification ; metabolism ; Chromatography, Thin Layer ; DNA, Fungal ; chemistry ; genetics ; DNA, Ribosomal ; chemistry ; genetics ; Diazonium Compounds ; metabolism ; Fungi ; classification ; genetics ; metabolism ; Huperzia ; microbiology ; Hydrolysis ; Naphthaleneacetic Acids ; metabolism ; Phylogeny ; RNA, Ribosomal, 18S ; classification ; genetics ; RNA, Ribosomal, 5.8S ; classification ; genetics ; Sequence Analysis, DNA