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Antimicrobial activities of endophytic fungi obtained from the arid zone invasive plant Opuntia dillenii… Ratnaweera, Pamoda B; de Silva, E. D; Williams, David E; Andersen, Raymond J Jul 10, 2015

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RESEARCH ARTICLE Open AccessAntimicrobial activities of endophytic fungiobtained from the arid zone invasive plantOpuntia dillenii and the isolation ofequisetin, from endophytic Fusarium sp.Pamoda B. Ratnaweera1,2,3, E. Dilip de Silva1*, David E. Williams2 and Raymond J. Andersen2AbstractBackground: Opuntia dillenii is an invasive plant well established in the harsh South-Eastern arid zone of Sri Lanka.Evidence suggests it is likely that the endophytic fungal populations of O. dillenii assist the host in overcoming bioticand abiotic stress by producing biologically active metabolites. With this in mind there is potential to discover novelnatural products with useful biological activities from this hitherto poorly investigated source. Consequently,an investigation of the antimicrobial activities of the endophytes of O. dillenii, that occupies a unique ecologicalniche, may well provide useful leads in the discovery of new pharmaceuticals.Methods: Endophytic fungi were isolated from the surface sterilized cladodes and flowers of O. dillenii usingseveral nutrient media and the antimicrobial activities were evaluated against three Gram-positive and twoGram-negative bacteria and Candida albicans. The two most bioactive fungi were identified by colony morphologyand DNA sequencing. The secondary metabolite of the endophyte Fusarium sp. exhibiting the best activity wasisolated via bioassay guided chromatography. The chemical structure was elucidated from the ESIMS and NMRspectroscopic data obtained for the active metabolite. The minimum inhibitory concentrations (MICs) of theactive compound were determined.Results: Eight endophytic fungi were isolated from O. dillenii and all except one showed antibacterial activitiesagainst at least one of the test bacteria. All extracts were inactive against C. albicans. The most bioactive funguswas identified as Fusarium sp. and the second most active as Aspergillus niger. The structure of the majorantibacterial compound of the Fusarium sp. was shown to be the tetramic acid derivative, equisetin. The MIC’sfor equisetin were 8 μg mL−1 against Bacillus subtilis, 16 μg mL−1 against Staphylococcus aureus and MethicillinResistant Staphylococcus aureus (MRSA).Conclusions: O. dillenii, harbors several endophytic fungi capable of producing antimicrobial substances withselective antibacterial properties. By producing biologically active secondary metabolites, such as equisetinisolated from the endophytic Fusarium sp., the endophytic fungal population may be assisting the host tosuccessfully withstand stressful environmental conditions. Further investigations on the secondary metabolitesproduced by these endophytes may provide additional drug leads.Keywords: Endophytic fungi, Invasive plants, Opuntia dillenii, Equisetin, Arid ecosystem, Fusarium sp.* Correspondence: dilip@chem.cmb.ac.lk1Department of Chemistry, University of Colombo, Colombo 03, Sri LankaFull list of author information is available at the end of the article© 2015 Ratanweera et al. This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Ratnaweera et al. BMC Complementary and Alternative Medicine  (2015) 15:220 DOI 10.1186/s12906-015-0722-4BackgroundEndophytic fungi are a diverse group of microorganismsthat thrive asymptomatically in the healthy tissues of thehost. Many of these endophytes are known to biosyn-thesis a plethora of bioactive secondary metabolites thatmay assist the host in protection and survival againstpathogenic and insect attacks, stress tolerance and dis-ease resistance [1, 2]. Moreover, some of these com-pounds have been proven to be useful as leads for noveldrug discovery [3–5]. Thus, endophytes and their sec-ondary metabolites not only play an important ecologicalrole but also positively impact the field of medicine.The arid zone ecosystem in South-East Sri Lanka is asmall area extending only 20–35 Km inland to the northof Hambantota [6]. The area is characterized by limitedrainfall of less than 1250 mm per year and mean annualtemperatures of 27–30 °C with 5–7 months per yearwith a little or no rain [7]. A combination of dry sandysoils of high salinity and dry winds has made this environ-ment unique and harsh when compared to other ecosys-tems in Sri Lanka. This area is mainly dominated bysedges, grasses, thorny shrubs including cactus species.Trees are less common in this arid zone ecosystem [6, 7].The plants growing in this area are adapted to this harshenvironmental setting.Invasive plants are non-native plants introduced to aspecific area and have a tendency to spread causingharm to the native biodiversity with consequent damageto the human economy and/or human health [8]. Inva-sive species are a growing concern in Sri Lanka [9].Opuntia dillenii is a plant in the Cactaceae family intro-duced to Sri Lanka in the mid nineteenth century andhas now become a well-established invasive plant in theBundala National Park, a RAMSAR wetland site, in theSouth-Eastern arid zone of Sri Lanka, and is consideredto be a national threat [10, 11]. O. dillenii has becomehighly competitive over the native plants in the area andhas thus successfully adapted to both the abiotic and bi-otic stress conditions of the harsh environment. Sincetolerance to biotic stress has been correlated with endo-phytic fungal natural products, it is likely that O. dilleniiwould be a rich source of endophytic fungi producingchemically diverse and biologically active secondary me-tabolites enhancing the host allelopathic effects and alsoproviding protection against phytopathogenic microbes[12, 13]. However, investigations into the endophyte sta-tus of invasive plants in arid zone ecosystems, the sec-ondary metabolites produced and the antimicrobialactivities of these metabolites are limited. Therefore theantibacterial producing potential of endophytic fungifrom O. dillenii collected in an unique ecological nicheappeared to be an attractive target of investigation.In this context, the objective of our study is to investi-gate the antimicrobial activities of the endophytic fungifrom O. dillenii and to access for the potential produc-tion of bioactive secondary metabolites that may serveas leads for novel drug discovery. The current article re-ports the isolation of endophytic fungi from the invasiveplant O. dillenii from a Sri Lankan arid zone, the anti-microbial properties of the organic extracts obtained, thebioassay-guided isolation and structure elucidation ofthe principal antimicrobial secondary metabolite alongwith the minimum inhibitory concentrations (MIC) ob-served against a number of pathogenic micro-organisms.MethodsIsolation and antimicrobial screening of endophytic fungiHealthy cladodes and flowers of the invasive plant O.dillenii were collected from the Bundala National Parkin the South-East arid zone of Sri Lanka on July 2013.The samples were tightly sealed in polythene bags underhumid conditions and kept at room temperature. Theplant was identified using a detailed guide [14] and con-firmed by comparing with the voucher specimen No.12687 at the National Herbarium, Royal Botanical Garden,Peradeniya, Sri Lanka. The isolation of the fungal endo-phytes commenced within 24 hours of collection.Prior to isolation of endophytes the plant material wassurface sterilized. The cladodes and flowers were washedthoroughly with water for 10 and 3 minutes respectively,immersed in 70 % ethanol for 1–2 minute, 5.25 % So-dium hypochlorite for 2–5 minutes and again 70 % etha-nol for 30–60 seconds [15]. The time used for thesurface sterilization for each solvent used differedslightly depending on the texture of the plant material.To finish, the surface sterilized plant parts were washedwith sterilized distilled water and allowed to dry inside alaminar flow cabinet. Small pieces of tissue were cutfrom the surface sterilized plant material and placed ondishes with potato dextrose agar (PDA), starch yeastpeptone agar (SYP), yeast peptone dextrose agar (YPD),malt agar (ME) and malt peptone dextrose agar (MEA)media. The endophytic fungi that immerged from thetissues were transferred on to new PDA dishes and se-quential sub culturing was done until pure cultures wereobtained. Each pure fungal culture was grown on fivefreshly prepared PDA dishes and after 14–21 days de-pending on the growth of the fungus, the mycelium plusthe medium was cut into small pieces and extracted with200 mL of ethyl acetate for 24 hours. The ethyl acetatewas filtered and the filtrate evaporated under reducedpressure.The resulting residues were screened for antimicrobialactivity against Gram positive Bacillus subtilis (UBC344), Staphylococcus aureus (ATCC 43300), MethicillinResistant Staphylococcus aureus (MRSA, ATCC 33591),Gram negative Escherichia coli (UBC 8161), Pseudo-monas aeruginosa (ATCC 27853) and the pathogenicRatnaweera et al. BMC Complementary and Alternative Medicine  (2015) 15:220 Page 2 of 7fungus Candida albicans (ATCC 90028) at 200 μg perdisc using the agar disc diffusion method [16].Large scale culturing and extraction of endophytic fungiEndophytic fungus that exhibited promising antimicro-bial activity was cultured in 200 medium size Petridishes (100 × 20 mm) of PDA for 17 days at roomtemperature. At the end of the incubation period thefungus together with the medium were cut into smallpieces and immersed in 1 L of ethyl acetate for 48 hoursand subsequently filtered through cotton wool. Theextraction with ethyl acetate was repeated thrice. Thefiltrates were combined and the organic solvent evapo-rated under reduced pressure at room temperature.The resulting crude extract was weighed and screenedfor antimicrobial activity at 50 μg per disc to confirmthe activity.Identification of the endophytic fungiColony morphological features of the two endophyticfungi with the most promising antimicrobial activitywere recorded. Following this fungal DNA was extractedin the laboratory using a published protocol [17]. Theextracted DNA was subjected to the polymerase chainreaction (PCR) using universal primers ITS1 and ITS4.Amplified DNA was sequenced and it was comparedwith existing DNA sequences in NCBI GenBank [18] toidentify the fungi. PCR and DNA sequencing was donecommercially.Fractionation, isolation and structure elucidation of thebioactive componentThe principal bioactive component from the complexmixture of the crude extract of the major bioactive fun-gus, was obtained by bioassay guided chromatography.The crude extract (400 mg) was first fractionated onSephadex LH-20 size exclusion column chromatography(3 × 115 cm) with methanol as eluent. The resulting frac-tions were combined according to the thin layer chroma-tography (TLC) profiles and the combined fractions weretested for antimicrobial activity using bioautography. Themost active fraction (60 mg) was chromatographed onnormal phase silica (3 × 20 cm column) with step-gradientelution (methanol : dichloromethane 1 : 99 to methanol).Finally the resulting bioactive fraction was purified by C18reversed-phase high performance liquid chromatography(HPLC) using a semi-preparative column (0.94 × 25 cm)with 13:7 acetonitrile/(0.05 % trifluoroacetic acid (TFA)/water) as eluent to give 2 mg of pure compound.The structure elucidation of the isolated compound re-sulted from analysis of the nuclear magnetic resonance(NMR) and mass spectral (MS) data obtained. 1H, 13C and2D NMR data sets were obtained using a BrukerAVANCE 600-MHz spectrometer with a 5 mm cryoprobewith deuterated dimethyl sulfoxide, (DMSO-d6) as solvent.The electron spray ionization mass spectral (ESIMS) datawas obtained using Bruker Esquire-LC electrospraymass spectrometer.Antimicrobial activity of the isolated pure compoundThe bioactive compound was tested for antimicrobial ac-tivities against three Gram-positive bacteria, B. subtilis(UBC 344), S. aureus (ATCC 43300) and MRSA (ATCC33591), two Gram-negative bacteria, E. coli (UBC 8161),P. aeruginosa (ATCC 27853) and the pathogenic fungusC. albicans (ATCC 90028). The minimum inhibitoryconcentrations (MICs) were determined using brothmicro-dilution method according to National Commit-tee for Clinical Laboratory Standards with modificationusing Mueller Hinton broth as the medium [19]. Thecommercial antimicrobial agents polymyxin B, rifamycinand amphotericin were used as positive controls.Results and discussionIsolation, antimicrobial activity and identification ofendophytesIn total eight endophytic fungi, six from cladodes andtwo from flowers, were isolated from O. dillenii. The re-sults of the antimicrobial tests of the ethyl acetate extractsof laboratory cultures of the eight fungi are listed inTable 1. Only one fungus, I8, was completely inactive.Three fungi, I4, I5, and I7 were active against only one or-ganism tested while I3 and I6 were active against two testorganisms. Most promising activities were shown by I1and I2 which were active against three microorganismseach. None of the isolated endophytes were active againstC. albicans.Table 1 Antibacterial activities of the crude extracts of endophyticfungi isolated from O. dillenii at 200 μg/discSample Plant partused forthe isolationDiameter of the inhibition zone (mm)S. aureus MRSA B. subtilis E. coli P. aeruginosaI1 leaf 9 - 15 7 -I2 flower 14 14 21 - -I3 leaf - - 11 - 9I4 leaf - - 11 - -I5 leaf - - - - 9I6 flower 8 - 12 - -I7 leaf - - - 9 -I8 leaf - - - - -+Ve 35 35 28 20 20-Ve - - - - -+Ve control - Polymyxin B (30 μg/disc) for P. aeruginosa, E. coli and B. subtilis,Rifamycin (10 μg/disc) for S. aureus, MRSA and Amphotericin B (20 μg/disc)for C. albicans. -Ve control – MethanolRatnaweera et al. BMC Complementary and Alternative Medicine  (2015) 15:220 Page 3 of 7Some endophytic fungi have specific growth require-ments and some species are not adapted to grow andsporulate in artificial culture media. Sometimes the fastgrowing fungal species outcompete the slower growingspecies [20]. Therefore use of several media with differ-ent nutrient aspects may probably be an advantage inisolating a large number of endophytic fungi includingthe cryptic species from the plant tissue. In this studyfour of the fungi were isolated in PDA medium whilethree and two were isolated in SYP and YPD media, re-spectively. Of the two most active fungi, I1 was isolatedfrom both SYP and YPD media while I2 was isolatedfrom SYP medium. This media specific isolation indi-cates that endophytic fungi look for specific nutrientneeds or less competitive medium in order to spreadtheir growth from the natural source to another.The two endophytic fungi I1 and I2 were identified asAspergillus niger and Fusarium sp. respectively. Endo-phytic A. niger showed the highest colonization in thecladodes of O. dillenii while the Fusarium sp. isolatedfrom the pistil of the flowers showed the major biologicalactivity. The isolation of bioactive compounds was carriedout for the Fusarium sp. only.The Fusarium sp. had a white puffy mycelium with apeach colour pigment that secreted into the mediumwithin seven to ten days of culture growth while A. nigerproduced black spores in a concentric ring pattern onFig. 1 Endophytic a Fusarium sp. (dorsal view), b Fusarium sp. (ventral view), c Aspergillus niger (dorsal view) and d A. niger (ventral view) in cultureFig. 2 Equisetin-chemical structureTable 2 Comparison of 13C NMR data of equisetin from thepresent study (in DMSO-d6) with published data (in CDCl3) [25]C# 13C δ (ppm) for equisetin C# 13C δ (ppm) for equisetinPresentstudyPublishedvaluesPresentstudyPublishedvalues1 190.2 190.6 12 14.0 13.72 47.9 48.4 13 127.3 127.13 44.1 44.6 14 130.4 130.44 126.4 126.2 15 17.8 18.25 129.8 129.8 16 22.4 22.56 38.2 38.4 2′ 176.3 176.77 41.8 41.9 3′ 100.6 99.88 33.0 33.3 4′ 195.8 198.99 35.4 35.5 5′ 67.5 66.410 27.8 28.1 6′ 57.7 60.011 40.0 39.6 7′ 26.8 27.2Ratnaweera et al. BMC Complementary and Alternative Medicine  (2015) 15:220 Page 4 of 7PDA medium (Fig. 1). According to DNA sequence dataand blast results obtained, these fungi showed 99 % iden-tity to previously reported Fusarium sp. (GQ505759.1)and A. niger (JN561274.1) [21, 22]. In addition on thebasis of 18S ribosomal RNA gene, partial sequence; in-ternal transcribed spacer 1, 5.8S ribosomal RNA gene, andinternal transcribed spacer 2, complete sequence and 28Sribosomal RNA gene, partial sequence, the major activefungi isolated in the current study were assigned to Fu-sarium sp. and A. niger.There are a few reports of endophytic fungi and bio-logical activities from other Opuntia species [23]. How-ever other than for a preliminary survey of endophyticfungi from Australian samples of O. dillenii there arehardly any studies reporting the isolation proceduresalong with the identification of endophytic fungi andassociated biological activities from O. dillenii [24].Therefore to the best of our knowledge this is the firstinvestigation to report the antibacterial activities offungal endophytes of O. dillenii invasive plant.Isolation and structure elucidation of the activecompoundLarge scale extraction of the Fusarium sp. with ethylacetate gave 1 g of crude extract. Bioassay guided isola-tion of 400 mg of the crude extract resulted in the iso-lation of 2 mg of the active compound. The activecomponent gave a [M + H]+ ion with m/z 374 in thelow-resolution electrospray ionization mass spectrum.Analysis of 1H and 13C NMR data along with 2D NMR(COSY, HSQC, HMBC, ROESY) data revealed that thestructure of the active compound matches that of theknown tetramic acid derivative, equisetin (Fig. 2) [25]with a molecular formula of C22H31NO4 which wasconsistent with a molecular weight of 373 daltons asseen in the ESIMS. A comparison of 13C NMR valuesobtained in the present study for equisetin with thosereported in the literature is shown in Table 2. The 1Hand 13C NMR spectra of equisetin are illustrated inFig. 3.Equisetin has previously been isolated from severalspecies of Fusarium including F. equiseti from in whichwas the first report [26]. However this is the first reportof equisetin from an endophytic Fusarium sp. isolatedfrom O. dillenii flowers.Antimicrobial activity of equisetin isolated fromendophytic Fusarium sp.In the current study, as shown in Table 3, equisetin ex-hibited antibacterial activities against the Gram-positivebacteria B. subtilis, S. aureus and MRSA with MIC’s of8–16 μg mL−1 and no activity against the Gram-negativebacteria E.coli, P. aeruginosa or pathogenic fungus C.albicans. In addition to the antibacterial activities equi-setin has shown phytotoxicity and inhibited the in vitrorecombinant integrase enzyme which is necessary forHIV replication [27].Previous research has reported that invasive plants’competitive ability is enhanced by the production of sec-ondary metabolites [28]. Yang et al. [29] and Alford et al.[30] have shown that secondary metabolites released frominvasive plants directly inhibit seed germination of nativeplants while indirectly promoting the growth of invadersFig. 3 600 MHz 1H and 13C NMR spectra of equisetin in DMSO-d6Table 3 MIC values obtained for equisetin and the positivecontrolsMIC values (μg mL−1)S.aureusMRSA B.subtilisE.coliP.aeruginosaC.albicansEquisetin 16 16 8 - - -Polymixin B - - 8 4 4 -Rifamycin 0.015 0.015 - - - -Amphotericin - - - - - 0.062Ratnaweera et al. BMC Complementary and Alternative Medicine  (2015) 15:220 Page 5 of 7through the influence of nutrient cycling. SimilarlyShipunov et al. has mentioned that in the host’s invadedrange endophytes can increase the competitiveness of thehost by producing metabolites inhibitory to evolution-arily native plants [31]. This view is also supported inthe report by Aschehoug et al. [32].In the context of these host/fungal relationships, com-pared to native plants, the production of the secondarymetabolite equisetin by the Fusarium sp. isolated fromthe internal tissues of O. dillenii, may well enhance thecompetitive ability of this plant against microorganismsand perhaps increase its adaptability to withstand theharsh and biotic stress factors in its arid environment.ConclusionsThis is the first study to describe the isolation and anti-bacterial activities of endophytic fungi from O. dillenii,an invasive plant from an arid zone ecosystem. The in-vestigation has revealed that O. dillenii harbors severalendophytic fungi which are capable of producing anti-microbial substances with selective antibacterial activ-ities. The endophytic Fusarium sp. exhibited promisingactivity and the principal antimicrobial substance pro-duced by this fungus proved to be the known secondarymetabolite, equisetin. By producing such biologically ac-tive compounds, the endophytic fungal population maybe assisting the host to successfully withstand stressfulconditions and play a role in what can only be describedas the successful spread of O. dillenii to the detriment ofthe native plants in the area. The findings of this studyalso suggests that endophytes from harsh and competi-tive environments have potential to be a productivesource for the discovery of useful drug leads for innova-tive and improved pharmaceuticals.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsPBR designed the study, carried out majority of research activities, wrote themanuscript and provided financial support. EDdS participated in designingthe research, guiding PBR to carry out the research and writing themanuscript. DEW involved in elucidating the structure of the activecompound and editing the manuscript. RJA contributed collaboration to theresearch by providing spectroscopic and other advance facilities also withguidance. All authors read and approved the final manuscript.AcknowledgementsThis work was financially supported by the Higher Education for Twenty FirstCentury (HETC) project, Ministry of Higher Education, Sri Lanka, underscholarship UWU/O-ST/N3. We are grateful to Prof. RLC Wijesundera,Department of Plant Sciences, University of Colombo for the support givenin the plant and microbiology sections and also for the Forest Department,Sri Lanka for granting permission to collect plant specimens.Author details1Department of Chemistry, University of Colombo, Colombo 03, Sri Lanka.2Department of Chemistry and Earth, Ocean and Atmospheric Science,University of British Columbia, Vancouver, Canada. 3Department of Scienceand Technology, Uva Wellassa University, Badulla, Sri Lanka.Received: 22 December 2014 Accepted: 12 June 2015References1. 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Aschehoug ET, Metlen KL, Callaway RM, Newcombe G. Fungal endophytesdirectly increase the competitive effects of an invasive forb. Ecology.2012;93(1):3–8.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitRatnaweera et al. BMC Complementary and Alternative Medicine  (2015) 15:220 Page 7 of 7


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