The Search On Diversity Of Endophytic Fungi Associated With Kadsura Angustifolia And Their Fermentation

Kadsura angustifolia (Lem.) A.C.Smith (Schisandraceae) is a perennial evergreen woody liana with worthy medicines in Yunnan province, China. Its canes have the pharmacological action of promoting blood circulation and dissolving stasis, which are used as a traditional medicine to treat fractures and menstrual irregularities. Modern phytovhemical studies in past decades have shown that this species is a rich source of lignans and triterpenoids, which contains various beneficial pharmacological activities such as anti-inflammatory, anti-oxidation, anti-tumor, anti-HIV activities, etc. Kadsura angustifolia is a kind of folk medicine in Yunan province, China, but there is no report about endophytic fungi of Kadsura angustifolia. Fungal endophytes is a group of fungi that inhabit plant tissues during the whole or part of their life cycle, and without causing visible disease symptoms (i.e. they are asymptomatic) at the time of observation. They can be found colonizing the tissues of all plants. Accumulating evidence has demonstrated that endophytic fungi are hyperdiverse and abundant groups. Furthermore, the mutualism between endophytic fungi and their host plants through years of coevolution forces their coexistence in long term. Many studies indicated that fungal endophytes in the medicinal plants could produce the same secondary metabolites of pharmacological activities as their hosts and some novel compounds. Therefore, fungal endophytes have become an important reservoir to get a large number of active products.In this paper, the main work was to isolate and characterize the fungal isolates in K. angustifolia to determine the biodiversity, distribution of endophytes and their biocatalytic activities and to elucidate the secondary metabolites. This work will provide some basic knownledge to understand the relationship between K. angustifolia and endophytic fungi, the origin and evolution of fungal isolates, and to exploit some bioactive components of K. angustifolia.Fungal endophytes were isolated from stems and roots of K. angustifolia at Xichou and Maguan of Yunnan province with surface-sterilization method. A total of 426 fungal isolates (170 isolates/ Xichou stem,84 isolates/Xixhou root,172 isolates/Maguan root) were recovered from the segments of plant samples. Among them,21 isolates were classified into Mycelia Sterilia (4.93%), which do not produce spores in PDA medium.405 isolates were grouped into 41 genera based on morphological characteristics. Among those detected, Verticillium and Penicillium were dominant genera with relative frequencies 16.43% and 11.27%, respectively. The abundance, richness, Shannon-Weiner diversity index, Simpson diversity index, and evenness of endophytic assemblages were obviously dependent on tissues and sampling sites. In term of tissue types, a total of 254 isolates grouped into 33 taxa were recovered from Xichou samples, of which,14 were found in both stems and roots,11 were exclusively obtained from stems,8 were only recovered from roots. In term of localities, the number of isolates isolated from different localities was 84 and 172 isolates at Xichou and Maguan sites, respectively. The isolated strains at Xichou were grouped into 22 genera and at Maugan belonged to 30 genera.From 134 morphospecies selected,39 showed significant biocatalytic activities using thin layer chromatography (TLC). Based on morphological characterizations and the sequence analysis of the internal transcribed spacer (ITS1-5.8S-ITS2), the 39 fungal isolates were identified as species belonging to the genera Colletotrichum, Eupenicillium, Fusarium, Hypoxylon, Penicillium, Phomopsis, Trametes, Trichoderma, Umbelopsis, Verticillium and Xylaria. Of these, we selected one strain Eupenicillium brefeldianum SWUKD2.0300 with remarkable biotransformative activities to ferment K. angustifolia. Two compounds were obtained from secondary metabolites by silica gel column chromatography, Sephadex LH-20 column chromatography, etc. and further identified as brefeldin A and 12-methoxy-brefeldin A, respectively, on the basis of spectral data analyses. To the best of my knowledge,12-methoxy-brefeldin A is a new compound of macrolide.The optimal condition was obtained for producing extracellular brefeldin A of E. brefeldianum SWUKD2.0300 by orthogonal experiment. The optimal condition was as follows:potato 200g/L, sucrose 20.0g/L, CuSO40.02g/L、CaCO3 5g/L、KH2PO4 1.0g/L、MgSO4·7H2O 2.0g/L,pH=7, culture medium volume 100mL/250mL. Cultures were incubated at 170 rpm at 2 ℃ for 8 days. The biggest production of brefeldin A was 200.0mg/L. Additionally, E. brefeldianum SWUKD2.0300 could grow well in solid media such as Sabouraud, PDA, Martin, Carrot and Czapek, and could hydrolyze cellulose and pectin.The results indicated that a large number of fungal communities existed in K. angustifolia, and some have host component-transforming activities. It could provide an interesting niche on the hunt for novel biocatalysts.