Streptomyces albipurpureus sp. nov., a novel actinobacterium with antimicrobial activity isolated from the rhizospheric soil of Fritillaria cirrhosa D. Don.

A novel actinobacterium, designated strain CWNU-1T, was isolated from the rhizospheric soil of Fritillaria cirrhosa D. Don and examined using a polyphasic taxonomic approach. The organism developed pale blue aerial mycelia that was simply branched and terminated in open or closed coils of three or more volutions on International Streptomyces Project 3 agar. Spores were ellipsoidal to cylindrical with wrinkled surfaces. The strain showed high 16S rRNA gene sequence similarity to Streptomyces kurssanovii NBRC 13192T (98.8 %), Streptomyces xantholiticus NBRC 13354T (98.7 %) and Streptomyces peucetius JCM 9920T (98.6 %). The phylogenetic result based on 16S rRNA gene and genome sequences clearly demonstrated that strain CWNU-1T formed an independent phylogenetic lineage. On the basis of orthologous average nucleotide identity, CWNU-1T was most closely related to Streptomyces inusitatus NBRC 13601T with 79.3 % identity. The results of the digital DNA-DNA hybridization analysis also indicated low levels of relatedness with other species, as the highest value was observed with S. inusitatus NBRC 13601T (25.3 %). With reference to phenotypic characteristics, phylogenetic data, orthologous average nucleotide identity and digital DNA-DNA hybridization results, strain CWNU-1T was readily distinguished from its most closely related strains and classified as representing a novel species, for which the name Streptomyces albipurpureus sp. nov. is proposed. The type strain is CWNU-1T (=CGMCC 4.7758T=MCCC 1K07402T=JCM 35391T).

[Correlation between rhizospheric microorganisms distribution and alkaloid content of Fritillaria taipaiensis].

In order to reveal the relationship between the amount of soil microorganisms and the quality of Fritillaria taipaiensis, both cultivated and wild F. taipaiensis were collected from Chongqing, Wuxi at different stages of their growth as objects of the research. The mycorrhizal infection rate and colonization intensity, peimisine and total alkaloid content in bulbs, the amount of microorganisms and biomass carbon content in rhizospheric soil were all determined using common methods. The results showed that the typical arbuscular-vesicle roots were formed after the AM fungi infected the F. taipaiensis roots which were collected from different origins. The mycorrhizal infection rates were ranged from 78.74% to 98.68% and the colonization intensities were ranged from 13.29% to 37.06%. The rhizospheric microorganisms of F. taipaiensis showed abundant resources. The distribution rule of them in the rhizospheric soil was as follows: the amount of bacteria>the amount of actinomycetes>the amount of fungi. The rhizospheric bacteria, decomposition inorganic phosphorus bacteria, decomposition organic phosphorus bacteria, actinomycetes amount and the total number of microbes increased first and then decreased with the increase of years, while decomposition potassium bacteria showed decreasing trend and fungi showed gradual increasing trend. The soil microbial flora content in the soil changed from "bacterial type" with a high fertility to "fungal type" with a low fertility. The mass fraction of peimisine and total alkaloid content increased first and then decreased with the increase of over the years, the same trend of culturable rhizosphere soil bacteria and actinomycetes indicated that the growth years affected the quality of soil and medicinal materials on different levels. Therefore, the diversity of microbial communities in rhizosphere soil reduced with the increase of years leading to the continuous cropping obstacles and the destruction of medicinal quality of F. taipaiensis.

Exopolysaccharides from the fungal endophytic Fusarium sp. A14 isolated from Fritillaria unibracteata Hsiao et KC Hsia and their antioxidant and antiproliferation effects.

Exopolysaccharides (EPSs) are high-molecular-weight carbohydrates with a wide range of biophysiological activities, such as antioxidant activity, immunostimulatory activity, antitumor activity, hepatoprotective activity, and antifatigue effects. In the present work, two water-soluble EPSs, namely, A14EPS-1 and A14EPS-2, were isolated and purified from the fungal endophytic strain A14 using ethanol precipitation, DEAE-cellulose ion exchange chromatography and Sepharose G-150 gel filtration chromatography. A14EPS-1 (∼2.4 × 104 Da, the major fraction) was mainly composed of mannose, rhamnose, glucose, galactose, xylose and arabinose with a molar ratio of 0.31:0.55:10.00:0.34:0.03:0.06. The major monosaccharide of A14EPS-1 was pyranose, which was connected by α-glycosidic linkages. And the side chains of A14EPS-1 may be composed of rhamnose, arabinose, glucose and galactose; moreover, the backbone of A14EPS-1 may be composed of rhamnose, xylose, arabinose and glucose. A14EPS-2 (∼0.5 × 104 Da) was mainly composed of mannose, rhamnose, glucose, galactose, xylose and arabinose in a ratio of 0.16:0.88:10.00:0.39:0.06:0.06. Pyranose was observed in both the α- and ß-configurations in A14EPS-2, and the α configuration was dominant. In addition, the results of the bioactivity assays indicated that both A14EPS-1 and A14EPS-2 had moderate antioxidant activity in vitro, and A14EPS-2 showed a moderate antiproliferation effect on human hepatocellular carcinoma HepG2 cells.

Fungal endophyte-derived Fritillaria unibracteata var. wabuensis: diversity, antioxidant capacities in vitro and relations to phenolic, flavonoid or saponin compounds.

Diverse fungal endophytes are rich fungal resources for the production of an enormous quantity of natural products. In the present study, 53 fungal endophytes were isolated from the bulbs of Fritillaria unibracteata var. wabuensis (FUW). Of these, 49 strains were identified and grouped into 17 different taxa, and priority was conferred to the Fusarium genus. All fungal fermented filtrates displayed antioxidant activities. The DPPH activity, total antioxidant capacities (ABTS), reduction power (FRAP), total phenolic content (TPC), total flavonoid content (TFC) and total saponin content (TSC) were evaluated using petroleum ether, ethyl acetate, n-butyl alcohol and ethanol fractions extracted from five representative fungal cultures. The last three fractions showed more potent antioxidant activity than the first fraction. Significant positive correlations were found between the compositions (TPC, TFC and TSC) and antioxidant capacities (DPPH, ABTS and FRAP). In addition, multifarious natural antioxidant components were identified from the fungal extracts, including gallic acid, rutin, phlorizin, 2,4-di-tert-butylphenol and 2,6-di-tert-butyl hydroquinone; these were determined preliminarily by TLC-bioautography, HPLC and GC-MS analysis. This study showed abundant fungal resources in FUW. Phenolics, flavonoids and saponins are crucial bioactive constituents in these abundant fungal endophytes and can be viewed as new potential antioxidant resources.

[Physicochemical property and antioxidant activity of exopolysaccharide produced by endophytic fungal Fusarium redolens 6WBY3 isolated from Fritillaria unibracteata var. wabuensis].

Objective: To study the exopolysaccharide (EPS) of endophytic fungal Fusarium redolens 6WBY3 isolated from Fritillaria unibracteata var. wabuensis (FUW) including its antioxidant activities. Methods: We isolated the EPS from the culture medium of strain 6WBY3 using the methods of degreasing with organic solvent, precipitation with ethanol, decoloration with macroporous resins, deproteinization with protease in combination with sevag reagent, desalination with dialysis and separation with DEAE-cellulose ion exchange chromatography. Then, we analyzed the EPS fractions using high-performance gel-permeation chromatography with evaporative light scattering detector (HPGPC-ELSD), ultra violet (UV) spectra, scanning electron microscope (SEM), PMP precolumn derivatization with high performance liquid chromatography (PMP-HPLC), fourier transform infrared (FT-IR) spectroscopy and 1H-nuclear magnetic resonance (1H NMR) methods. We evaluated the antioxidant activity of the EPS using DPPH and ABTS radical scavenging activities and iron ion chelating ability methods. Results: We obtained two homogeneous EPSs, namely 6WBY3EPS-3 and 6WBY3EPS-4, with the molecular weight (Mw) of 17.41×106 and 8.84×105 Da, respectively. 6WBY3EPS-3 was composed of mannose, glucose and galactose in a molar ratio of 8.16:4.96:10.00, while 6WBY3EPS-4 was composed of mannose, rhamnose, glucose and galactose in a molar ratio of 8.08:1.71:6.32:10.00. The results of SEM showed that 6WBY3EPS-3 was the irregular multilateral body, and 6WBY3EPS-4 was rules of quadrilateral body. The results of FT-IR and 1H NMR analysis exhibited that 6WBY3EPS-3 was acidic polysaccharose with abundant galactofuranose, mannofuranose and a few α-D-glucopyranose, while 6WBY3EPS-4 possessed pyranose ring mainly. Those two EPSs had anomeric hydrogen with α-(main) and ß-glycosidic configuration. Furthermore, the results of antioxidant activity suggested that both 6WBY3EPS-3 and 6WBY3EPS-4 had a weak DPPH radical scavenging ability, a moderate ABTS radical scavenging activity and a moderate iron ion chelating effect. Conclusion: The two EPSs from the endophytic fungus 6WBY3 were firstly obtained and investigated. Our investigation indicated that the EPS from 6WBY3 had the application potential as an antioxidant in medicine and food industries. In addition, this work can also provide theory reference for improving development of EPS from other endophytic fungi.

[Screening, identification and antimicrobial activity of alkaloid produced by endophytic actinomycetes from Fritillaria unibracteata in western Sichuan plateau].

To explore the resource of endophytic actinomycete in Fritillaria unibracteata, and alleviate the shortage of F. unibracteata resource, using F. unibracteata as experimental materials which growth in the western Sichuan plateau and cut its healthy bulb. Pure culture, insert, TLC and Oxford cup were applied to observe the mycelial morphology, research the ability of producing alkaloid and its antibacterial activity. Totally, 14 endophytic actinomycete strains were isolated by using Gao culture media. Based on the color reaction, 5 typical strains were selected for producing alkaloid. Through the TLC technique, all strains produced 2 obvious alkaloids spots. Antibacterial activity determination showed that the antimicrobial effects of 2 strains is prominent, the diameter up to 11 mm.16S rRNA gene sequence comparison analysis showed that 5 strains belonging to the Streptomyces. The alkaloids produced by endophytic actinomycetes are not related to F. unibracteata, but its fermentation liquid has antibacterial effect, it is worthy of further study.

Fusarium redolens 6WBY3, an endophytic fungus isolated from Fritillaria unibracteata var. wabuensis, produces peimisine and imperialine-3ß-D-glucoside.

The major biological active ingredients of Bulbus Fritillariae cirrhosae (BFC) are steroidal alkaloids, such as peimisine, imperialine-3ß-D-glucoside, and peimine. The bulbus of Fritillaria unibracteata var. wabuensis (FUW) was officially recorded in the National Pharmacopoeia of China (2010 edition) as one of the sources of BFC because of its positive therapeutic effects and few side effects. The endophytic fungus strain 6WBY3 was isolated from the fresh bulbus of FUW that had been cultivated for six years. Based on morphological methods and the phylogenetic analysis of internal transcribed spacer (ITS) sequences, this strain was identified as Fusarium redolens. Using color reaction analysis, high performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD), and mass spectrometry (MS), it was demonstrated that F. redolens 6WBY3 could produce peimisine and imperialine-3ß-D-glucoside, similar to its host plant. The yields of peimisine and imperialine-3ß-D-glucoside were 16.0 µg·l(-1) and 18.8 µg·l(-1), respectively, in one week of culture. These results indicate that F. redolens 6WBY3 is a promising candidate for the large scale production of peimisine and imperialine-3ß-D-glucoside. In addition, the results from the strain 6WBY3 lay the foundation for further study into the mechanism of Fritillaria alkaloids biosynthesis in fungi.

Peimisine and peiminine production by endophytic fungus Fusarium sp. isolated from Fritillaria unibracteata var. wabensis.

Steroidal alkaloids, as the major biologically active components in Bulbus Fritillariae, possess a variety of toxicological and pharmacological effects on humans. The objective of this work was to determine whether endophytic fungi isolated from fresh bulbs of Fritillaria unibracteata var. wabensis can produce one or more alkaloids like its host plant. Four classical reagents including Wagner's, iodine-potassium iodide, Mayer's and improved Dragendorff's were used for primary screening. Then thin-layer chromatography (TLC) and high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) were employed to identify the fermentation products of the selected strains. The results showed that extract from one stain (WBS007) has positive reactions in process of primary screening. A further TLC scan and HPLC-ELSD showed that strain WBS007 had two components with the same TLC relative front (Rf) value and HPLC retention time (RT) as authentic peimisine and peiminine. In addition, strain WBS007 was identified as Fusarium sp. based on phylogenetic analysis of ITS sequences. Thus, strain WBS007 produced the bioactive ingredient peimisine and peiminine, as does its host plant, and could be used for the production of peimisine and peiminine by fermentation.

[Differences of fungal diversity and structure in rhizosphere of Fritillaria thunbergii from different provenances].

To explore the mechanism of soil microbial ecology, the differences of fungal diversities in rhizosphere of different provenances of Fritillaria thunbergii were analyzed. The diversities and compositions of rhizo-fungi of the samples were analyzed by using DGGE and 454 pyrosequencing. DGGE results showed the Shannon index of Ninbo provenance planted in Ninbo was the highest one. And its dominant fungi were Ascomycota, Deuteromycota and Zygomycota. Except the same fungi, every provenance planted in Ninbo had its own special ones. From the 454 pyrosequencing, the fungal diversity in Panan producing was the highest which was similar with DGGE result. Among the ten phylum detected in its rhizosoil, Fungi_incertae_sedis, Ascomycota, Mucoromycotina, Basidiomycota and Chytridiomycota almost amounted to 90% of the whole community. The fungal types and amounts in Panan were more than those in Ninbo indicating the differences between producing areas and the advantage of macro genome sequencing. There were 10 phyla, 29 families, 28 genus and 159 species of fungi in Panan provenance, 6 phyla, 20 families, 19 genus, 136 species in Ninbo provenance, 8 phyla, 37 families, 47 genus, 289 species in Nantong provenance and 7 phyla, 25 families, 24 genus, 102 species in the bulk soil. Some genus such as Dothidea, Capnobotryella and Conidiobolus were only existed in Nantong provenance, while Pyrenochae- ta, Glomus and Pseudonectria were only in Panan provenance, which implied these species could grew because F. thunbergii influenced the existence of fungi. Experiments of provenance and producing area of F. thunbergii showed that the fungal diversity of indigenous provenance was higher than that of exotic provenance and each provenance had unique fungal species in the rhizosphere, which indicated that the diversity and structure was shaped cooperatively by the species and soil type. These fungal species are interacted with the soil-rhizhosphere-microbe microecological system, which in turn influence the growth of F. thunbergii.

Okibacterium fritillariae gen. nov., sp. nov., a novel genus of the family Microbacteriaceae.

Okibacterium fritillariae gen. nov., sp. nov. (type strain VKM Ac-2059T = IFO 16404T) is proposed for aerobic, oxidase- and catalase-positive, coryneform bacteria isolated from seeds of Fritillaria ruthenica Wikstr. and Clematis recta L. Strains of the new genus form a distinct branch within the phylogenetic cluster composed of members of the family Microbacteriaceae and are characterized by B-type peptidoglycan containing amino acids glycine, glutamate, homoserine, alanine and lysine, the glycolyl type of muramic acid, the major menaquinones MK-10 and MK-11, the principal phospholipids phosphatidylglycerol and diphosphatidylglycerol, and a DNA G+C content of approximately 67 mol %.