Studies Of Endophytic Fungi Forsythia Isolation And Identification Of Fermentation Conditions

Endophytic fungi are microorganisms that live inside the tissues and organs of healthy plants during their entire or part of life cycle without causing discernible manifestation of disease. Studies have shown that, endophytic fungi not only can promote the synthesis and accumulation of some metabolites in their host plants, but also can produce identical or similar biological active constituents as their hosts during the long-term symbiosis and interplay between endophytic fungi and their hosts. Therefore, as a special microorganism resource with potential pharmaceutical value, the endophytic fungi from medicinal plants have become important source for the natural active products.Forsythia suspensa (Thunb.) Vahl is a deciduous shrub with perennial rootstock belong to Oleaceae and Forsythia, and mainly distributed in Shanxi, Shaanxi and Henan of our country. The air-dried fruits of F. suspensa are traditional Chinese medicine in China, which has long been used for gonorrhea, erysipedas and pyrexia. Studies have shown that, F. suspensa has various biological activities, such as antimicrobial, anti-inflammatory, antiviral, antioxidant, antipyretic, etc. In this paper, endophytic fungi from the medicinal plants F. suspensa were studied, and the results are as follows.(1) A total of24strains of endophytic fungi were isolated from the healthy stems, leaves and fruits of F. suspensa with routine separation methods of microorganism, and9from stems,5from leaves,10from fruits, the fungi code were J1-J9, Y1-Y5, G1-G10respectively.(2) Antimicrobial activities of fermentation products of all the fungal endophytes were detected by filter paper method against3bacterial strains, Staphylococcus aureus26003, Bacillus subtitles A99and Escherichia coli A70. The results showed that, among the24fungal isolates,20isolates displayed inhibitory effect on at least1bacterium, the isolates whose inhibition zone diameters more than10mm, group of fermentation broth had11, group of mycelia had19, and G5, G7, G10showed stronger antibacterial activities.(3) Phillyrin-producing endophytic fungi were screened from the antibacterial active isolates by TLC and HPLC, the potential fungal isolates were further analyzed by HPLC-MS. TLC and HPLC results showed that, the mycelia extract of isolate G10had the same Rf value and retention time as authen4tic phillyrin, HPLC-MS found that isolate G10had a ion fragment peak identical with the molecular ion peak of authentic phillyrin, the results indicated that the isolate G10could produce the same active component phillyrin as its host.(4) The isolate G10was identified as Colletotrichum gloeosporioides based on morphological characteristics and ITS sequence analysis.(5) The fermentation medium component and culture condition of isolate G10were optimized. Through the comparison of several common fungal culture medium, bean sprouts medium was choosed as the best basal medium for mycelial growth and active ingredients accumulation of isolate G10. Through the single factor analysis of culture medium component, glucose and yeast extract were proved to be the optimum C source and the optimum N source respectively. Further optimizing for ferment condition were done by orthogonal optimization, and the best ferment condition for mycelial growth and phillyrin accumulation of isolate G10was proved to be that:based on the bean sprouts medium, glucose5.0%, yeast extract0.25%, pH6.0. Through comparison, the mycelial dry weight and phillyrin yield of isolate G10in the optimized medium have increased by33.33%and19.15%respectively.