[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.

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.

[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.