Isolation and antimicrobial effects of endophytic fungi from Edgeworthia chrysantha

Ten fungal strains isolated from Edgeworthia chrysantha , one of traditional medicinal plants in China, were evaluated their antimicrobial activities against three human pathogens, Escherichia coli, Staphyloccocus aureus and Candida albicans , and two phytopathogens, Rhizoctonia cerealis and Colletotrichum gloeosporioides . The results indicated that most ethyl acetate extracts of fermentation broth of these fungal endophytes had stronger antimicrobial activities than their fermentation broth. Among these endophytic strains, both fermentation broth and the ethyl acetate extract of strain D showed the strongest inhibitory effects on all pathogens. Strains 5-19 and BZ also exhibited potent antibacterial activities. However, other strains had weak or no antimicrobial effect. This was the first report on the isolation and antimicrobial effects of endophytic fungi from E. chrysantha .


Introduction
Endophyte, an important member of plant microbial community, has been shown to be a prolific producer of bioactive secondary metabolites (Zhang et al., 2006).As we know, medicinal plants have been used for centuries as remedies to treat human diseases.A great number of valuable therapeutic agents from medicinal plants had been isolated and identified from their derived endophytic microbes, such as taxol, camptothecin, vinblastine (Zhang et al., 2014).Therefore, chemical investigation of medicinal plant-derived endophyte not only contributes to the protection of plant resources, but also provides an alternative way to look for natural leading compounds and/or sustainably supply natural medicines.
Edgeworthia chrysantha Lindl.(E.papyrifera S. et Z., Thymelaeaceae), widely distributed in eastern Asia, is used to make paper in Korea and Japan.Its alabastrum is the succedaneum of traditional Chinese medicine "Buddleja officinalis Maxim (Chinese name: Mi Meng Hua)" used to treat swelling of eye, ophthalmalgia, delacrimation, nephelium of eye and nocturnal emission (Baba et al., 1989;Hashimoto et al., 1991;Baba et al., 1990).Pharmacological study showed that E. chrysantha has many biological effects, such as anticoagulated blood, antimicrobial, anti-inflammation and antioxidant activities.However, there is not any report on biology and chemistry of endophytic microbe associated with this plant.

Material and Methods
The healthy plant of E. chrysantha was obtained from Zhaohui campus of Zhejiang University of Technology (Hangzhou, China) and used for endophyte isolation within 48 hours after harvest.Five testing pathogenic strains, including three human pathogens, Escherichia This article was downloaded by you on: Jul 26, 2015 coli, Staphyloccocus aureus, Candida albicans, and two phytopathogens, Rhizoctonia cerealis, Colletotrichum gloeosporioides, were purchased from China Center for Type Culture Collection.All chemicals used in this project were of analytical grade.
Isolation of endophytic strain: Endophytic fungal strains from E. chrysantha were isolated according to our reported procedure (Zhang et al., 2012).All these fungal strains were transferred into potato dextrose agar slants followed by storing at 4ºC.

Preparation of fermentation broth and its ethyl acetate extract:
Each fungal strain was cultured on potato dextrose agar at 28ºC for 7 days.Then a balanced amount of fungal colony was transferred to culture broth in a 500 mL erlenmeyer flask, which contained 200 mL sterilized potato dextrose broth, followed by shaking at 150 rpm for 15 days at 28ºC.After fermentation, the mycelium and the culture broth were separated by centrifugation at 4,500 rpm for 15 min at 4ºC.About 2 mL fermentation broth of each endophytic fungus was filtered with a bacterial filter (0.22 µm) and preserved at 4ºC until antimicrobial assay.The residual fermentation broth was extracted twice with 400 mL ethyl acetate (Merck).Then the upper solvent was evaporated at 25ºC in vacuum to yield the ethyl acetate extract, which possibly had antimicrobial secondary metabolites.Each afforded extract was kept in a vacuum drier for 3 days and dissolved in dimethyl sulfoxide (DMSO, Merck) before bioassay.The final concentration of each ethyl acetate extract had two levels, including 1, 10 mg/mL.

Antibacterial test:
The antibacterial activity was carried out using disc diffusion method (Taylora et al., 1995).E. coli and S. aureus were separately transferred into two 500-mL Erlenmeyer flasks, which contained 200 mL sterilized nutrition agar, and incubated at 37ºC on a rotary shaker at 150 rpm for 24 hours.Firstly, 5 mL melt water agar medium was evenly poured into petri dishes (Ф = 9 cm).Next, 200 µL seed liquid was added to fresh nutrition agar medium and mix well.Thirdly, the same amount of fresh nutrition agar medium was poured on the solidified water agar medium and the testing plate with double medium for bioassay was prepared.After 5 holes (Ф = 6 mm) were equidistantly drilled on inoculated media, a piece of standard sterile filter paper (Ф = 6 mm) was put in one hole followed by adding 100 μL fermentation broth or ethyl acetate extract of endophytic fungal strain.Ampicillin sodium (30 µg/ disk, Amresco) was used as the positive control while DMSO and sterilized water were the negative controls.Then the plates were incubated at 37ºC for 48 hours.All tests were performed in triplicate and the anti-bacterial activity was expressed as the average value of inhibition diameters (mm).

Antifungal test:
The antifungal assay was also carried out using disc diffusion method.Three fungal blocks of C. albicans, R. cerealis and C. gloeosporioides were separately transferred into three 500-mL Erlenmeyer flasks, each flask had 200 mL sterilized potato dextrose broth.The seed liquid was prepared after incubation at 28ºC on a rotary shaker at 150 rpm for 3 days.The testing plate with double medium for bioassay was made using the same approach described above.Amphotericin B (30 µg/disk, Sigma-Aldrich) was used as the positive control and the pure DMSO or sterilized water was the negative control.The diameter of inhibition zone (in mm) was measured to assess anti-fungal activity.All tests were carried out in triplicate.

Discussion
During the long co-evolution of endophytes and their host plants, endophytes have adapted themselves to their special microenvironments by genetic variation, including uptake of some plant DNA into their own genomes (Germaine et al., 2004).This could have led to the ability of certain endophytic microbes to biosynthesize some 'phytochemicals' originally from their host plants (Zhang et al., 2014).A growing evidence suggests that endophytic microbes are widely distributed in plants on the earth and have abundant biodiversity.Numerous chemical investigations indicate that endophyte is one of rich sources of bioactive natural products.These functional compounds would effectively accelerate new drug discovery and contribute to the development of natural products chemistry.

1
School of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310 014, P. R. China; 2 College of Life and Environmental Sciences, Hangzhou Normal University, 310 036, P. R. China.