Endophytic Fungi From Scapania Verrucosa Heeg. And Their Bioactive Metabolites

Endophytes are referred to fungi colonizing in healthy plant tissues without causing overt symptoms in or apparent injury to the host. Research results in recent years show that endophytes can produce some metabolites same or similar to those from the host plant, and metabolites with the novel structure and specific bioactivity. In this paper, we selected Scapania verrucosa Heeg., which grows in special environment as our materials, to isolate and identify its endophytes, and screen the bioactive strains from them. Then, we isolated and elucidated the secondary metabolites of three bioactive strains. The main results are as follows:1. Taking five different separating culture mediums, a total of 49 endophytic fungi were isolated from Scapania verrucosa Heeg. by tissue isolation method.2. After morphology observation, 10 strains of endophytic fungi with coiniophore were identified. They were subjected to Penicillum spp. and Chaetomium spp.. The rest strains (75.6%) without coindiophore were identified by PCR method to recognize the sequence of the rDNA ITS regions. In comparison with similar taxa, the sequence was grouped together.Forty-nine fungal strains in 7 genus(Hypocrea sp., Penicillium sp., Tolypocladium sp., Chaetomium sp., Xylaria sp., Nemania sp., Creosphaeria sp.) were classified, identified, and belonged to 2 classes(Sordariomycetes, Eurotiomycetes), 4 orders(Hypocreales, Eurotiales, Sordiriales, Xylariales) and 5 families (Hypocreaceae, Trichocomaceae, Clavicipitaceae, Chaetomiaceae, Xylariaceae) by morphological and molecular method. Chaetomium sp. and Creosphaeria sp. were dominate genus in endophytes from Scapania verrucosa which occupy 18.36 and 18.36 % of the all strains, respectively.3. The bioassay of the ethyl acetate extract of the fermentation broths of the 49 endophytes from Scapania verrucosa was studied. Biological model Pyricularia oryzae was efficiently applied to the primary screening of the antifungal and antitumor in vitro agents from the secondary metabolites of the microorganisms. Forty (81.6 %) strains of endophytes from S. verrucosa could induce morphological abnormality of P. oryzae. Antimicrobial activity against four fungi and cytotoxic effect against two cancer cell lines of 8 endophpytes extracts with better anti-P. oryzae activity were studied further. The results showed that endophytes from S. verrucosa exhibited antifungal activity and cytotoxic effect in some degree. 8 endophytes all had obvious antifungal activity on Cryptococcus neoformans and Trichophyton rubrum. The strain T11 and T35 could effectively inhibit all the tested fungi. The antitumor study in vitro showed that 8 strains displayed cytotoxic effect against one or more sensitive tumor cell lines and had stronger cytotoxic effect against HL-60 cell. Endophytes from S. verrucosa have antifungal activity and cytotoxic effects, and could be a potential resource for the development of antifungal and antitumor agent.4. A comparison of the constituents of the ether extracts between S. verrucosa and its endophytic fungus Chaetomium fusiforme was investigated by gas chromatography-mass spectrometry (GC/MS). 59 compounds were found in S. verrucosa. and 49 ones were characterized, while the number of identified constituents of the cultured endophyte extract was 28. The components of S. verrucosa were predominantly sesquiterpenes, whilst its endophytic fungus C. fusiforme produced alkanes, ketones, phenols and esters mainly. Although the extracts of S. verrucosa and its endophyte showed little correlation in chemical composition, both of them demonstrated antifungal and antitumor activities. The fungal strain T37 produced prolific secondary metabolites much differently from its host plant.5. The components of the ethyl acetate extract of active strains T37,T31 and T35 were isolated by bioassay-guided methods. Fifty chemical compounds were obtained by normal pressure column chromatography and TLC. Based on the data of spectral (MS, 1H-NMR, 13C-NMR) and comparison with related literatures, the chemical structures of the ten are clearly explained, which may be ergosterol, ergosterol peroxideone, mannitol, 2,3-dimethoxy-5-methylphenol, methyl curvulinate, curvulin, O-methylcurvulinic acid, sterigmatocystin, and two new unusual alkaloid compounds named Chaetofusins A and Chaetofusins B. Identification of other five components needs to be studied further.Endophytes from S. verrucosa have antifungal activity and cytotoxic effects, and produce metabolites with the novel structure and bioactivity. They could be a potential resource for natural medicine.