Bioprospecting of Novel and Bioactive Metabolites from Endophytic Fungi Isolated from Rubber Tree Ficus elastica Leaves
1Institute of BioPharmaceutical Research, Liaocheng University,P.R China, 2School of Life Sciences, Liaocheng University, P.R China, 3State key laboratory of bioactive seaweed substances, Qingdao brightmoon seaweed Group Co Ltd,PR ChinaCorrespondence to:
J. Microbiol. Biotechnol. 2019; 29(5): 731-738
Published May 28, 2019
Copyright © The Korean Society for Microbiology and Biotechnology.
Endophytic fungi are generally regarded as the fungal microorganisms colonizing the internal tissues of healthy plants without causing any apparent negative effects. These fungi are ubiquitously found in most plant species studied so far and exist in various tissues of host plants, such as roots, stems, leaves, flowers, fruits and seeds . Complex interactions exist between endophytic fungi and host plants , and many endophytic fungi are considered as beneficial for their hosts in many ways, including promoting host growth and nutrient gain , as well as enhancing host resistance to phytopathogens, pests or abiotic stress .
In long-term symbioses with their host plants, many endophytes could produce bioactive secondary metabolites to exert positive influence on their hosts . It has been surmised that endophytic fungi and host plants have similar biosynthesis pathways to produce secondary metabolites due to horizontal gene transfer, especially after the discovery of paclitaxel (taxol) in the endophytic fungus
Over 2000 species of higher plants make latex, though only a few laticiferous plants have been exploited commercially, such as
Methods and Materials
Sample Collection and Isolation of Endophytic Fungi
Fresh leaves of
Molecular Identification of the Strains
Total DNA was extracted from each strain following the protocol described by Ding
Fermentation and Extract Preparation
The endophytic fungal strains were cultured using PDA solid medium at 25°C for 5 days. Subsequently, 5 mm diameter plugs with adhering mycelia were added to 250 ml flasks containing 100 ml of potato dextrose broth (PDB) medium. All cultures were grown under shaking condition at 180 rpm and 25°C for 7 days. Each test contained five replicates for each strain.
The fermentation extracts were processed following the protocols established by Ding
Metabolite Fingerprint Analysis
The extracts were analyzed in a HPLC system (Waters Inc., USA), which contained a model 1525 pump, a model 2489 UV detector, and a HPLC column (Pack ODS-A, 250 × 4.6 mm, 5 μm, YMC Co., Ltd., Japan). The gradient increased from 10 to 100% MeOH over 30 min and was retained at 100% for 10 min.
Purification and Identification of Natural Products
Large-scale culture (10 L) and extract preparation of the strain Lcu-Fe1712 were performed in PDB liquid medium using the method mentioned above. 2.7 g of the EtOAc extract was gained and separated by silica gel vacuum liquid chromatography using CH2Cl2-MeOH (20:1) to give five fractions (Fractions 1 to 5). Fraction 2 was further separated by Sephadex LH-20 chromatograph eluted with CH2Cl2-MeOH (1:1) and then on a semi-preparative HPLC column (Pack ODS-A, 250 × 10 mm, 5 μm, YMC Co., Ltd.) eluted with MeOH-H2O (80:20, 3 ml/min) to provide compound 4 (5.6 mg,
The structures of the compounds were elucidated from extensive MS and NMR. High-resolution electrospray ionization MS (HRESI-MS) spectra were measured on a Micromass EI-4000 Autospec-Ultima-TOF (Micromass communication Inc., UK). NMR spectra were recorded on a Varian 500 spectrometer (Varian Medical Systems Inc., USA) using tetramethylsilane as an internal standard, and chemical shifts were recorded as δ values.
Antimicrobial activity of the extracts were evaluated by the well diffusion method. The five microorganisms indicated were the bacteria
Isolation, Identification and Bioactivity of the Endophytic Fungi
A total of 42 endophytic fungi were isolated from healthy leaves of
The antimicrobial activity of the EtOAc extracts of endophytic fungi isolated from
Purification, Structural Identification and Bioactivities of the Metabolites from Strain Fes1712
Due to its great antimicrobial activity, the endophyte strain Fes1712 was selected for further chemical investigation using its EtOAc extract. Two new isocoumarin derivatives (1 and 2), together with three known compounds (3–5) were isolated from the fermentation extract. Structures of these compounds were determined using MS analyses and NMR methods (Fig. 1).
Chemical structures of isolated compounds 1–5 from T. harzianumFes1712.
Compound (1) was isolated as a white solid powder and had the molecular formula C13H14O7 as determined by the HRESI-MS peak ([M + Na]+ at
Key COSY and HMBC correlations of 1 and 2.
Compound (2) was obtained as a white powder with the same molecular formula as that of 1 (Fig. S9). Similarly, the 1D and 2D NMR data of 2 (Table 3, Figs. S10-S15) showed that it shared the same isocoumarin skeleton as 1. The differences between the observed compounds 1 and 2 were the substitutional positions of hydroxyl groups in the butanetriol residue, shown as the replacement of the 1,2,3-butanetriol group in 1 by a 1,1,2-butanetriol group (δC 35.3/δH 3.03, 2.83, CH2-9; δC 72.3/δH 4.38, CH-10; δC 75.1/δH 5.92, CH-11) in 2. However, due to lack of samples, the configurations of 1 and 2 were not determined.
In addition, the other three known compounds (3–5) were also isolated. By comparison with the published spectroscopic data in the literature (Figs. S16-S21), their structures were identified as 5-hydroxy-3-hydroxymethyl-2-methyl-7-methoxychromone (3), 5-hydroxy-2,3-dimethyl-7-methoxychromone (4), lichexanthone (5), respectively.
The antimicrobial activity of the purified compounds in this study were further investigated (Table 4). Compounds 1 and 2 exhibited growth inhibitory activity against
In this study, the diversity characterization and bioactivities of cultivable fungi isolated from fresh leaves of
Three strains of
Strain Fes1712, identified as
According to the bioactivity results, the chemical constituents extracted from fermentation of
Isocoumarin and its derivatives are widely distributed in various bioresources and have been shown to possess a series of biological activities due to the combination with different functional residues . Engelmeier
Both chromone and xanthone compounds have been reported to appear in the fermentation extract of
This work was supported by funding obtained from Key Research & Development Project of Shandong Province [no. 2018YYSP008], Natural Science Foundation of Shandong Province [no. ZR2017BB077, no. ZR2018BH043], the Open Foundation of the State Key Laboratory of Bioactive Seaweed Substances [no. SKL-BASS1705] and Taishan Scholar Foundation of Shandong Province.
Conflict of interest
The authors have no financial conflicts of interest to declare.
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