| Plant endophytic fungi are referred to fungi which spends the whole or part of its life cycle colonizing inter-and/or intra-cellularly inside the healthy tissue of the host plant, typically causing no apparent symptoms of disease. Previous studies have shown that endophytes can produce some metabolites same or similar to those from the host plants, and metabolites with the novel structure and specific bioactivities. Chloranthus, a genus of the Chloranthaceae, which is mainly distributed in eastern and southern Asia, were applied in Chinese folk medicine for their antitumor, antifungal, and anti-inflammatory activities. So far,15species were found thoughout the world. Among them,13species and5varieties have detected in the southern part of China. However, there have no reports on the endophytic fungi from the genus Chloranthus up till now. In this paper, we selected Chloranthus multistachys, a famous traditional Chinese medicine, to investigate the species, distributions, bioactivities and secondary metabolites of the representative fungal endophytes colonized inside the roots, stems and leaves. The main results are discussed as follows:1. Systemically study the fungal endophyte distribution and biodiversity of C. multistachys. A total of65fungal endophytes were isolated from roots, stems and leaves of C. multistachys by tissue isolation method and three kinds of medium.29strains were from stems, which occupy45%in65strains;27strains were from leaves, which occupy42%;9strains were from roots, which occupy14%. After morphological observation and5.8S rDNA-ITS sequence analysis,65fungal strains were identified, which belonged to2subdivisions,3families,6orders,6classes and10genus: Fusarium sp., Cephalosporium sp., Gliocladium sp., Cordana sp., Rhizoctonia sp., Coryneum sp., Colletotrichum sp., Phomopsis sp., Cryptosporiopsis sp., Pestalotiopsis sp.. Among them, Fusarium sp., Cephalosporium sp. and Coryneum sp. were dominate genus in endophytes from C. multistachys which occupy15.4%,13.8%,13.8%of the all strains, respectively. The results showed great diversity of the fungal endophytes in C. multistachys and the amounts, species and distribution of endophytic fungal community varied in different parts of C. multistachys.2. The antagonistic activity of the fermentation products from65endophytic fungi against7pathogens (G. Cingnlata, A.brassicae, P.digitatum, B. Cinerea,P. capsici, V.alboatrum, E.turcicum) was investigated. The results showed that the inhibition rates of the fermentation products agianist at least one pathogenic fungi more than75%were43strains which occupy66.2%in65strains; at least3pathogenic fungi more than30%were39strains which occupy60%; at least7pathogenic fungi more than30%were9strains which occupy13.8%. Among them, the fermentation filtrates of CL39and CL61have better antagonistic activity to7pathogens. The results showed that the strains of CL39and CL61were broad-spectrum and stronger antifungal activity.3. The characteristics of morphology and analysis of5.8S rDNA-ITS sequences of the strains CL39and CL61suggested that strain CL39was identified as Fusarium sp., strain CL61was identified as Pestalotiopsis sp..4. Strains CL39and CL61with antimicrobial activity was fermented in liquid CZA culture medium. Six compounds were obtained from acetyl acetate extract of CL39and CL61by silica gel column chromatography, Sephadex LH-20column chromatography, thin layer preparation chromatography. On the basis of spectral data analyses (’H-NMR,13C-NMR,’H-’H COSY, DEPT, HMBC, HMQC) and relevant references,6compounds were elucidated as:compounds CL391and CL392, cycloeucalenol,(-)-(7S,8R)-dihydrodehydrodiconifery alcohol,12R,15-dihydroxylabda-8(17),13E-dien-19-oic acid and β-ecdysterone. Among them, compounds CL391and CL392are two new chemical components.The results showed that the endophytic fungi isolated from C. multistachys have broad-spectrum antifungal activity and produce metabolites with the novel structure. They could be a potential resource for natural medicine. |
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