Articles Service
Research article
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 China
Correspondence to:J. Microbiol. Biotechnol. 2019; 29(5): 731-738
Published May 28, 2019 https://doi.org/10.4014/jmb.1901.01015
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
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 [1]. Complex interactions exist between endophytic fungi and host plants [2], and many endophytic fungi are considered as beneficial for their hosts in many ways, including promoting host growth and nutrient gain [3], as well as enhancing host resistance to phytopathogens, pests or abiotic stress [4].
In long-term symbioses with their host plants, many endophytes could produce bioactive secondary metabolites to exert positive influence on their hosts [5]. 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.
Bioactive Assays
Antimicrobial activity of the extracts were evaluated by the well diffusion method. The five microorganisms indicated were the bacteria
Results
Isolation, Identification and Bioactivity of the Endophytic Fungi
A total of 42 endophytic fungi were isolated from healthy leaves of
-
Table 1 . Endophytic fungi isolated from
Ficus elastica as identified by ITS sequences.Strain number Closest BLAST match [GenBank accession number] Query coverage (%) Identity (%) No. of bp analyzed Identification [GenBank accession number] Fes1714 Penicillium aeneum [KP016812] 100 100 516 Penicillium aeneum Fes1701 Penicillium chrysogenum [JN851002] 100 99 523 Penicillium chrysogenum Fes1711 Penicillium funiculosum [GQ337426] 100 100 517 Penicillium funiculosum Fes1703 Penicillium variabile [HQ288049] 100 99 535 Penicillium variabile Fes1707 Scytalidium lignicola [MH863583] 99 93 528 Scytalidium sp. Fes1712 Trichoderma harzianum [KM078037] 100 100 551 Trichoderma harzianum Fes1702 Zasmidium anthuriicola [MH863035] 100 99 315 Zasmidium anthuriicola
The antimicrobial activity of the EtOAc extracts of endophytic fungi isolated from
-
Table 2 . Bioactivities of the metabolites from endophytic fungi associated with
Ficus elastica .Strain number Antimicrobial activity: (mm)a B. subtilis C. albicans E. coil P. aeruginosa S. aureus Fes1701 - 9.6 ± 2.0 - - 10.3 ± 0.7 Fes1702 - - - - - Fes1703 - – – – – Fes1707 - – – – – Fes1711 - 10.4 ± 1.7 16.5 ± 1.6 – 15.9 ± 2.5 Fes1712 - 9.0 ± 1.2 18.5 ± 2.2 9.1 ± 2.2 15.7 ± 1.7 Fes1714 - – 10.7 ± 3.3 – 9.2 ± 2.5 Chloramphenicolb 17.9 ± 1.1 – 26.1 ± 0.8 9.7 ± 1.6 21.0 ± 1.5 Fluconazoleb – 20.1 ± 0.8 – – – aAntimicrobial activity was estimated by the inhibitory zone (mm) to five indicator microorganisms. The diameter of the inhibition zone: >15 mm is intense (bold), 9–15 mm is medium, <9 mm is low/no activity and not shown. Indicator microorganisms:
B. subtilis, Bacillus subtilis CMCC 63501;C. albicans, Candida albicans CMCC 98001;E. coil, Escherichia coli CMCC 44102;P. aeruginosa, Pseudomonas aeruginosa CMCC 10104;S. aureus, Staphylococcus aureus CMCC 26003. The crude extracts at concentration of 1 mg/ml were used in the evaluation of antimicrobial activities.bAntibacterial chloramphenicol (0.1 mg/ml) and antifungal fluconazole (0.1 mg/ml) were used as positive controls.
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).
-
Fig. 1.
Chemical structures of isolated compounds 1–5 from T. harzianum Fes1712.
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
-
Table 3 . 1H and 13C NMR data of Compounds 1 and 2 (500 MHz in CDCl3); δ in p.p.m.,
J in Hz.Position Compound (1) Compound (2) δC δH δC δH 1 165.1( s )165.9 ( s )3 150.8 ( s )152.8 ( s )4 108.4 ( d )6.55 ( s )106.4 ( d )6.57 ( s )4a 137.7( s )138.8 ( s )5 103.0 ( d )6.41 ( s )100.9 ( d )6.44 ( d ,J =2.10)6 167.0 ( s )166.5 ( s )7 101.6 ( d )6.54 ( s )100.2 ( d )6.47 ( s )8 163.89 ( s )162.5 ( s )8a 100.4( s )99.2 ( s )9 57.7( d )4.66 ( d ,J =8.85)35.3 ( t )3.03 ( dd ,J =14.90, 3.4)2.83 ( dd ,J =14.90, 9.15)10 71.2 ( d )4.49 ( m )72.3 ( d )4.38 ( m )11 47.2 ( t )4.05 ( d ,J =11.80)75.1 ( d )5.92 ( d ,J =3.4)3.96 ( d ,J =11.75)6-OCH3 55.8 ( q )3.87 ( s )55.0 ( q )3.89 ( s )
-
Fig. 2.
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
-
Table 4 . MIC values of the isolated compounds evaluated against the tested microorganisms.
Compound Strains, MIC (μg/ml) B. subtilis C. albicans E. coil P. aeruginosa S. aureus 1 >256 256 32 128 128 2 >256 256 32 128 128 3 >256 128 256 256 >256 4 >256 256 >256 >256 >256 5 128 >256 256 128 128 Chloramphenicol 16 – 4 64 8 Fluconazole – 16 – – – Antimicrobial activity was estimated by the inhibitory zone to five indicator microorganisms. The >9 mm diameter of the inhibition zone indicated that the test compound in the corresponding concentration has inhibitory activity. Indicator microorganisms:
B. subtilis, Bacillus subtilis CMCC 63501;C. albicans, Candida albicans CMCC 98001;E. coil, Escherichia coli CMCC 44102;P. aeruginosa, Pseudomonas aeruginosa CMCC 10104;S. aureus, Staphylococcus aureus CMCC 26003.
Discussion
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 [28]. Engelmeier
Both chromone and xanthone compounds have been reported to appear in the fermentation extract of
Supplementary Material
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
Acknowledgments
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.
References
- Gouda S, Das G, Sen SK, Shin HS, Patra JK. 2016. Endophytes: a treasure house of bioactive compounds of medicinal importance.
Front. Microbiol. 7 : 1538. - Saikkonen K, Faeth SH, Helander M, Sullivan TJ. 1998. Fungal endophytes: a continuum of interactions with host plants.
Annu. Rev. Ecol. Syst. 29 : 319-343. - Rudgers JA, Fischer S, Clay K. 2010. Managing plant symbiosis: fungal endophyte genotype alters plant community composition.
J. Appl. Ecol. 47 : 468-477. - Rodriguez RJ, Woodward CJ, Redman RS. 2012. Fungal influence on plant tolerance to stress, pp. 155-163.
In: Southworth D (ed),Biocomplexity of plant-fungal interactions . Wiley-Blackwell, Oxford. - Kumar S, Kaushik N. 2013. Endophytic fungi isolated from oil seed crop
Jatropha curcas produces oil and exhibit antifungal activity.PLoS One 8 : e56202. - Soliman SSM, Trobacher CP, Tsao R, Greenwood JS, Raizada MN. 2013. A fungal endophyte induces transcription of genes encoding a redundant fungicide pathway in its host plant.
BMC Plant Biol. 13 : 93. - Stierle A, Strobel G, Stierle D. 1993. Taxol and taxane production by
Taxomyces andreanae , an endophytic fungus ofPacific yew .Science 260 : 214-216. - Subbulakshmi GK, Thalavaipandian A, Bagyalakshmi RV, Rajendran A. 2012. Bioactive endophytic fungal isolates of
Biota orientalis (L) Endl.,Pinus excelsa Wall. andThuja occidentalis L.Int. J. Adv. Life Sci. 4 : 9-15. - Debbab A, Aly AH, Proksch P. 2011. Bioactive secondary metabolites from endophytes and associated marine derived fungi.
Fungal Divers. 49 : 1-12. - Kharwar RN, Mishra A, Gond SK, Stierle A, Stierle D. 2011. Anticancer compounds derived from fungal endophytes: their importance and future challenges.
Nat. Prod. Rep. 28 : 1208-1228. - Maheshwari R. 2016. Fungi: experimental methods in biology, 2nd ed. CRC Press, New York.
- Siler DJ, Cornish K. 1993. A protein from
Ficus elastica rubber particles is related to proteins fromHevea brasiliensis andParthenium argentatum .Phytochemistry 32 : 1097-1102. - Solis MJL, Yurkov A, Cruz TE, Unterseher M. 2015. Leaf-inhabiting endophytic yeasts are abundant but unevenly distributed in three
Ficus species from botanical garden greenhouses in Germany.Mycol. Progress 14 : 1019. - Ding Z, Li L, Che Q, Li D, Gu Q, Zhu T. 2016. Richness and bioactivity of culturable soil fungi from the Fildes Peninsula, Antarctica.
Extremophiles 20 : 425-435. - Sharma N, Kushwaha M, Arora D, Jain S, Singamaneni V, Sharma S,
et al . 2018. New cytochalasin fromRosellinia sanctae-cruciana , an endophytic fungus ofAlbizia lebbeck .J. Appl. Microbiol. 125 : 111-120. - Frisvad JC, Smedsgaard J, Larsen TO, Samson RA. 2004. Mycotoxins, drugs and other extrolites produced by species in
Penicillium subgenusPenicillium .Stud. Mycol. 49 : 201-241. - Uzma F, Mohan CD, Hashem A, Konappa NM, Rangappa S, Kamath PV,
et al . 2018. Endophytic fungi - alternative sources of cytotoxic compounds: a review.Front. Pharmacol. 9 : 309. - Lin ZL, Lu ZY, Zhu TJ, Fang YC, Gu QQ, Zhu WM. 2008. Penicillenols from
Penicillium sp. GQ-7, an endophytic fungus associated withAegiceras corniculatum .Chem. Pharm. Bull. 56 : 217-221. - Malhadas C, Malheiro R, Pereira JA, Pinho PG, Baptista P. 2017. Antimicrobial activity of endophytic fungi from olive tree leaves.
World J. Microbiol. Biotechnol. 33 : 46. - Reino JL, Guerrero RF, Hernández-Galán R, Collado IG. 2008. Secondary metabolites from species of the biocontrol agent
Trichoderma .Phytochem. Rev. 7 : 89-123. - Ding G, Wang H, Li L, Song B, Chen H, Zhang H,
et al . 2014. Trichodermone, a Spiro-cytochalasan with a Tetracyclic Nucleus (7/5/6/5) Skeleton from the Plant Endophytic FungusTrichoderma gamsii .J. Nat. Prod. 77 : 164-167. - Zhou P, Wu Z, Tan D, Yang J, Zhou Q, Zeng F,
et al . 2017. Atrichodermones A-C, three new secondary metabolites from the solid culture of an endophytic fungal strain,Trichoderma atroviride .Fitoterapia 123 : 18-22. - Shi XS, Wang DJ, Li XM, Li HL, Meng LH, Li X,
et al . 2017. Antimicrobial polyketides fromTrichoderma koningiopsis QA-3, an endophytic fungus obtained from the medicinal plantArtemisia argyi .RSC Adv. 7 : 51335-51342. - Vinale F, Marra R, Scala F, Ghisalberti EL, Lorito M, Sivasithamparam K. 2006. Major secondary metabolites produced by two commercial
Trichoderma strains active against different phytopathogens.Lett. Appl. Microbiol. 43 : 143-148. - Liu K, Yang YB, Miao CP, Zheng YK, Chen JL, Chen YW,
et al . 2016. Koningiopisins A-H, polyketides with synergistic antifungal activities from the endophytic fungusTrichoderma koningiopsis .Planta. Med. 82 : 371-376. - Pu X, Qu X, Chen F, Bao J, Zhang G, Luo Y. 2013. Camptothecin-producing endophytic fungus
Trichoderma atroviride LY357: isolation, identification, and fermentation conditions optimization for camptothecin production.Appl. Microbiol. Biotechnol. 97 : 9365-9375. - Leylaie S, Zafari D. 2018. Antiproliferative and antimicrobial activities of secondary metabolites and phylogenetic study of endophytic
Trichoderma species from Vinca plants.Front. Microbiol. 9 : 1484. - Hill RA. 1986. Naturally occurring isocoumarins.
Prog. Chem. Org. Nat. Prod. 49 : 1-78. - Engelmeier D, Hadacek F, Hofer O, Lutz-Kutschera G, Nagl M, Wurz G,
et al . 2014. Antifungal 3-Butylisocoumarins fromAsteraceae-Anthemideae .J. Nat. Prod. 67 : 19-25. - Thongbai B, Surup F, Mohr K, Kuhnert E, Hyde KD, Stadler M. 2013. Gymnopalynes A and B, chloropropynyl-isocoumarin antibiotics from cultures of the basidiomycete
Gymnopus sp.J. Nat. Prod. 76 : 2141-2144. - Kornsakulkarn J, Thongpanchang C, Lapanun S, Srichomthong K. 2009. Isocoumarin glucosides from the scale insect fungus
Torrubiella tenuis BCC 12732.J. Nat. Prod. 72 : 1341-1343. - Krupke OA, Castle AJ, Rinker DL. 2003. The North American mushroom competitor,
Trichoderma aggressivum f.aggressivum , produces antifungal compounds in mushroom compost that inhibit mycelial growth of the commercial mushroomAgaricus bisporus .Mycol. Res. 107 : 1467-1475. - Jeerapong C, Phupong W, Bangrak P, Intana W, Tuchinda P. 2015. Trichoharzianol, a new antifungal from
Trichoderma harzianum F031.J. Agric. Food. Chem. 63 : 3704-3708. - Qin XY, Yang KL, Wang CY, Shao CL. 2014. Secondary metabolites of the zoanthid-derived fungus
Trichoderma sp. TA26-28 collected from the south China sea.Chem. Nat. Compd. 50 : 961-964. - Liang XR, Miao FP, Song YP, Guo ZY, Ji NY. 2016. Trichocitrin, a new fusicoccane diterpene from the marine brown alga-endophytic fungus
Trichoderma citrinoviride cf-27.Nat. Prod. Res. 30 : 1605-1610. - Wang L, Zhou HB, Frisvad JC, Samson RA. 2004.
Penicillium persicinum , a new griseofulvin, chrysogine and roquefortine C producing species from Qinghai province, China.Antonie Van Leeuwenhoek 86 : 173-179. - Yang JX, Qiu SX, She ZG, Lin YC. 2013. A new xanthone derivative from the marine fungus
Phomopsis sp. (No. SK7RN3G1).Chem. Nat. Compd. 49 : 31-33. - Ango YP, Kapche GDWF, Kuete V, Mapitse R, Yeboah SO, Ngadjui BT. 2016. Three new derivatives and others constituents from the roots and twigs of
Trilepisium madagascariense DC.Helv. Chim. Acta 99 : 642-649. - Fru CG, Sandjo LP, Kuete V, Liermann JC, Schollmeyer D, Yeboah SO,
et al . 2013. Omphalocarpoidone, a new lanostane-type furano-spiro-γ-lactone from the wood ofTridesmostemon omphalocarpoides Engl. (Sapotaceae ).Phytochem. Lett. 6 : 676-680. - Pettit GR, Zhang Q, Pinilla V, Herald DL, Doubek DL, Duke JA. 2004. Isolation and structure of gustastatin from the Brazilian nut tree
Gustavia hexapetala .J. Nat. Prod. 67 : 983-985. - Wansi JD, Wandji J, Waffo AFK, Ngeufa HE, Ndom JC, Fotso S,
et al . 2006. Alkaloids fromOriciopsis glaberrima Engl. (Rutaceae ).Phytochemistry 67 : 475-480.
Related articles in JMB
