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Isolation and characterization of trichalasin-producing endophytic fungus from Taxus baccata

Isolation and characterization of trichalasin-producing endophytic fungus from Taxus baccata Ann Microbiol (2017) 67:255–261 DOI 10.1007/s13213-017-1256-4 ORIGINAL ARTICLE Isolation and characterization of trichalasin-producing endophytic fungus from Taxus baccata 1 1 2 1 Mondem Vasundhara & Manoj Baranwal & Nallapeta Sivaramaiah & Anil Kumar Received: 20 August 2016 /Accepted: 18 January 2017 /Published online: 9 February 2017 Springer-Verlag Berlin Heidelberg and the University of Milan 2017 Abstract An endophytic fungus (strain T1) isolated from Introduction Taxus baccata was studied for the production of metabolites with anticancer and antioxidant activities. This fungus was Endophytes are ubiquitous organisms which reside in the tis- identified as Diaporthe sp. based on rDNA-internal tran- sues of plants without causing apparent symptoms of disease scribed spacer (ITS) sequence analysis. The crude extract (Bacon and White 2000). The majority of endophytes are showed cytotoxic activity against MCF-7 and HeLa cancer fungi (Strobel and Daisy 2003), and they are present in the cell lines, with IC (concentration inhibiting 50% of growth intercellular spaces of various plant parts, such as the leaf and −1 rate) values of 1058 ± 44 and 1257 ± 80 μgml ,respectively. root (Corrêa et al. 2014). Endophytes reduce damage to their The scavenging activity of fungal extract increased signifi- host plants by producing many secondary metabolites cantly with increasing concentration [IC (concentration re- (Cabezas et al. 2012). They are also able to inhibit infection −1 quired to scavenge 50% of free radicals) 482 ± 9 μgml ]. and proliferation of pathogens in the host plant directly or Ultra-high-performance liquid chromatography-quadrupole- indirectly by inducing resistance responses intrinsic to the host time of flight analysis revealed the presence of three defence (Eaton et al. 2011). Currently, endophytic fungal re- trichalasins (trichalasin E, F and H) in the crude extract of search is focused on the ability of these fungi to produce and T1 which are known to have antitumour and antioxidant ac- accumulate secondary metabolites. Several of these com- tivities. These results suggest that Diaporthe sp. has the po- pounds have biological activities of interest for application tential to be used for therapeutic purposes because of its anti- in environmental, agriculture, pharmaceutical and healthcare proliferative and antioxidant potential and also for the produc- and food industries (Suryanarayanan et al. 2009; Kharwar tion of cytochalasins. et al. 2011; Deshmukh et al. 2015). Diverse classes of chemical substances, such as steroids, xanthones, phenols, isocoumarines, perylene derivatives, qui- . . . Keywords Endophytic fungi Diaporthe Cytochalasins nones, furandiones, terpenoids, depsipeptides and cytochala- Trichalasins Antioxidant activity sins, have been isolated from endophytic fungi (Schulz and Boyle 2005). Endophytes produce a significantly higher num- ber of novel chemical structures than soil fungi, indicating that Electronic supplementary material The online version of this article endophytes are a novel source of bioactive secondary metab- (doi:10.1007/s13213-017-1256-4) contains supplementary material, which is available to authorized users. olites (Nisa et al. 2015). In this context, the secondary metab- olites produced by endophytes associated with medicinal * Mondem Vasundhara plants are important as they could be exploited for the treat- mvasundhara@thapar.edu ment of many diseases (Tejesvi et al. 2007). Several studies have shown that endophytes produce secondary metabolites of their host plants, thus raising the prospect of using them as Department of Biotechnology, Thapar University, Patiala 147004, Punjab, India alternative sources of these metabolites (Priti et al. 2009). Endophytic fungi of the genus Diaporthe have been isolat- Nano Temper Technologies, World Trade Centre, Bangalore 560055, India ed from a wide variety of plant hosts from both temperate and 256 Ann Microbiol (2017) 67:255–261 tropical regions (Dos Santos et al. 2016). Diaporthe is one of instructions and then sequenced. The ITS sequence obtained the most abundant genera of endophytic fungi and has been from this isolate has been deposited in the GenBank under reported to produce various compounds of biotechnological accession number KX355165. interest (Silva et al. 2005; Pornpakakul et al. 2007; The ITS sequence of T1 was compared to those available in Rukachaisirikul et al. 2008). Cytochalasins are a group of the GenBank database using the BLASTN algorithm. cytotoxic fungal metabolites showing a wide range of biolog- Alignment of the sequences was constructed using MAFFT ical activities, such as anticancer, antimicrobial, antiparasitic, version 7.0 (Katoh and Standley 2013) and edited with phytotoxic activities, among others (Scherlach et al. 2010; BioEdit version 5.0.6 (Hall 1999). Phylogenetic analysis on Chen et al. 2014). These metabolites have been isolated from the resulting alignment was performed using Bayesian many fungal species, including Helminthosporium sp., Inference. A Bayesian analysis was implemented in Phoma sp., Xylaria sp., Hypoxylon sp. and Rhinocladiella MrBayes v.3.2.2 with two parallel runs, each consisting of sp. (Scherlach et al. 2010). four incrementally heated Monte Carlo Markov Chains. The In the investigation reported here, we isolated an en- analysis was run using Metropolis-coupled a Markov Chain dophytic fungus from the bark of the Taxus baccata L. Monte Carlo search algorithm over 1,000,000 generations, subsp. wallichiana (Zucc.) Pilger (Himalayan Yew) and and the convergence of Bayesian analysis was observed by subsequently identified it as Diaporthe sp. strain T1 examination of the standard deviation of split frequencies of based on its internal transcribed spacer (ITS) regions of <0.01. Trees were sampled every 100th generations resulting rDNA. The biological activities, such as anticancer and in total of 10,000 trees. The first 2500 trees, representing the antioxidant activities, of ethyl acetate extracts of its cul- burn-in phase of the analysis, were discarded, and the remain- ture filtrate were studied using various bioassays. These ing 7500 trees were used to calculate posterior probabilities extracts were also analysed by liquid chromatography/ from the 50% majority rule consensus trees. tandem mass spectrometry (LC-MS/MS) to identify some key compounds belonging to the cytochalasin Fungal extract preparation group of fungal metabolites which could be responsible for these activities. Mycelial discs (diameter 5.0 mm) of actively growing culture (7 days old) were inoculated into potato dextrose broth (500 ml) and incubated at 25 ± 2 °C for 21 days in the dark Material and methods as a static culture. The cultures were harvested by filtration through four layers of cheesecloth to remove the mycelia, and Isolation and identification of endophytic fungus strain T1 the mycelial biomass thus obtained was then dried overnight (35–40 °C) and extracted with ethyl acetate for 12 h. The cul- Endophytic fungus T1 was isolated from the bark samples of ture broth was also extracted three times with an equal volume T. baccata L. subsp. wallichiana (Zucc.) Pilger collected from of ethyl acetate; the ethyl acetate fractions were then pooled and Bhadrewah (Doda district, India) according to the method driedinvacuoat35°C. Part of the residue wasdissolved in described by Garyali et al. (2013). The fungal culture was dimethyl sulfoxide and used for the bioassays. The other part of maintained on potato dextrose agar (HiMedia Laboratories the residue was dissolved in methanol and partitioned three Ltd. Mumbai, India) medium. Molecular characterization times with an equal volume of dichloromethane); the dichloro- was performed by ITS sequence analysis. Specifically, methane fractions were pooled and dried in vacuo. The residue mycelia were harvested from actively growing colonies and was used for the LC–two-dimension-MS [ultra-high-perfor- ground in liquid nitrogen. Genomic DNA was extracted from mance LC-quadrupole-time of flight analysis (UHPLC- the finely ground fungal material by the CTAB method QTOF-MS/MS)] to analyse the bioactive compounds. (Zhang et al. 2008). The quality and quantity of DNA was checked with Nanodrop 1000 spectrophotometer (Thermo Cell growth inhibition assay Fisher Scientific, Waltham, MA) and samples were stored at −20 °C until use. The ITS region of nuclear ribosomal DNA Human breast cancer cell lines (MCF-7) and human cervical was amplified with the universal primers ITS1 and ITS4 cancer cell lines (HeLa) were procured from National Centre (White et al. 1990) in a thermal cycler (Applied Biosystems, for Cell Science, Pune, India. The cells were maintained in Foster City, CA). The thermal cycling conditions applied for Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, St. the ITS region consisted of an initial denaturation for 5 min at Louis, MO) containing 10% (v/v) foetal bovine serum −1 94 °C followed by 34 cycles of 1 min at 94 °C, 1 min at 50 °C (Gibco, Thermo Fisher Scientific), 100 IU ml penicillin, −1 −1 and 1.5 min at 72 °C and a final extension of 7 min at 72 °C. 100 μgml streptomycin, and 2.5 μgml amphotericin, in PCR products were purified using QIAquick spin columns a humidified incubator with 5% CO at 37 °C. A well-known (Qiagen, Hilden, Germany) following the manufacturer’s 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium- Ann Microbiol (2017) 67:255–261 257 bromide (MTT) assay was carried out to assess the effect of added to the wells. After 72 h of incubation, the MTT assay fungal extract on the growth of the cancer cell lines. In brief, was carried out as described by Denizot and Lang (1986). approximately 2 × 10 cells per well were seeded in 96 well Paclitaxel was used as the positive control at the concentration −1 culture plate and incubated overnight. After 16 h, varying of 20 μgml . The growth inhibition rate was calculated using −1 concentrations (250–1500 μgml )of fungalextractswere the formula (OD is optical density): Inhibition rate ¼ðÞ Mean OD of control well − Mean OD of treated cell=Mean OD of untreated cell 100 in this assay was defined as the concentration of analysed by analysis of variance, and the means were com- The IC compounds that resulted in 50% inhibition of growth rate. pared by Tukey’stest at p <0.05. Antioxidant assay Results The antioxidant potential of the fungal extract was studied by Isolation and identification of fungal strain T1 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. Fungal extract (50-μl samples) of different concentrations were mixed with The PCR product of ITS-rDNA amplified with ITS1 and ITS4 150 μlof DPPH(100 μM) in methanol in the wells of a 96- −1 was 588 bp. BLAST analysis revealed 98% similarity (query well microtiter plate. Ascorbic acid (100 μgml ;50 μl) was coverage of 100%) with Diaporthe sp. Bayesian analysis of used as the positive control. The plate was incubated in the ITS sequences of different species of Diaporthe yielded a dark (45 min), and absorbance of the reaction mixture was consensus tree (Fig. 1), and the present isolate was clustered measured at 517 nm using an enzyme-linked immunosorbent with Diaporthe sp. (KC357558 and KC357559). assay reader (Infinite microplate reader; Tecan Austria GmbH, Grödig, Austria). The percentage inhibition of DPPH radical Cytotoxic effect in cancer cell lines by the fungal extract was expressed as the inhibition concen- tration (IC ) and was calculated using the formula: The fungal extract showed a cytotoxic effect against both the DPPH scavenging activityðÞ % ¼ A − A =A  100 Control Sample Control human breast cancer cell line (MCF-7) and the human cervical cancer cell line (HeLa). The cytotoxic effect become signifi- Identification of bioactive compounds by UHPLC-QTOF cantly pronounced at higher concentrations of the fungal ex- analysis tract (Fig. 2). The IC value of the extract was 1058 ± 44 and −1 1257 ± 80 μgml for the MCF-7 and HeLa cell lines, respec- −1 For the UHPLC-QTOF-MS/MS analysis, the residue was first tively. Paclitaxel (20 μgml ), an anticancer drug, was used as dissolved in methanol. Chromatographic separation was car- a positive control. It inhibited the growth of the MCF-7 and ried out using the Agilent 1200 UHPLC system equipped with HeLa cell lines by 79 ± 6 and 97 ± 2%, respectively. C18 column (Agilent Technologies, Santa Clara, CA). Samples (5 μl) were injected into the column, and elution Antioxidant activity was carried out in a gradient mode with acidified water (0.2% v/v formic acid) to 90% methanol over a period of Free radical scavenging activity was performed to determine −1 40 min at a flow rate of 0.3 ml min . High-resolution mass the antioxidant activity of Diaporthe sp. The scavenging ac- spectroscopic detection was performed using Bruker Impact tivity significantly increased with increasing concentration of QTOF mass spectrometer (Bruker Corp., Billerica, MA, oper- fungal extract (Fig. 3). The IC value (concentration of sam- ating in Top5 data-dependent mode from 100–1500 m/z with ple required to scavenge 50% of free radicals) of the crude −1 electron spray ionization, and both positive and negative ions extract was 250 ± 3.5 μgml . Ascorbic acid, the positive were detected. Smart Formula 3D™ and Fragment Explorer control, showed 84 ± 3.5% antioxidant activity. part of Data Analysis 4.2 (Bruker Corp.) were used to generate compound formulae. Identification of compounds by UHPLQTOF analysis Statistical analysis The UHPLC-QTOF- MS/MS analysis of the crude extract of Diaporthe sp. (T1) showed a signal (m/z) corresponding to All the experiments were performed in triplicate. The results three trichalasins. Figure 4 shows the mass data of these three were expressed as mean ± standard deviation. The data were compounds that eluted from the column at different times. 258 Ann Microbiol (2017) 67:255–261 Fig. 1 Bayesian tree showing the relationships between the internal transcribed spacer (ITS) sequence of Diaporthe sp. (shown in bold) and those of related species retrieved from GenBank. Numbersatnodes Posterior probability percentages (>50%) of the Bayesian analysis Peaks at m/z ratios of 402.1753, 420.1391 and 472.1511 were Discussion observed and identified as Trichalasin H, Trichalasin F) and Trichalasin E, respectively, based on the reported values of The anamorphic form (the asexual reproductive morpho- these compounds (Electronic Supplementary Material Table). logical stage) of Diaporthe is the genus Phomopsis. Members of Diaporthe/Phomopsis are either plant patho- gens or endophytic species (van Niekerk et al. 2005;Dos Fig. 2 Cytotoxic effect of Diaporthe sp. extract against human breast cancer (MCF-7) and human cervical (HeLa) cancer cell lines. Bars Mean Fig. 3 Antioxidant effect of Diaporthe sp. extracts based on free radical values, error bars standard deviation (SD). Bars with the same lowercase scavenging activity. Bars Mean values, error bars standard deviation letter within the same cell type are not significantly different at p <0.05. (SD). Bars with the same lowercase letter are not significantly different −1 −1 PT Paclitaxel (20 μgml ) used as the positive control at p <0.05. AA Ascorbic acid (100 μgml ) used as the positive control Ann Microbiol (2017) 67:255–261 259 Fig. 4 The tandem mass spectrometry profile of selected ions in the extracts of Diaporthe sp. T1 corresponding to three tricalasins: a Trichalasin H (m/z 402.1753), b Trichalasin F (m/z 420.1391), c trichalasin E (m/z 472.1511) Santos et al. 2016) and are associated with dicotyledonous Diaporthe/Phomopsis complex has been reported by Nath and monocotyledonous plants of tropical and temperate et al. (2012) and Ascêncio et al. (2014). regions (Eriksson and Vue 1998; Guo et al. 2000). The Cytochalasins are a known class of mould metabolites that Diaporthe/Phomopsis complex occurs as endophytes with exhibit a wide range of distinctive biological activities. They medicinal plants such as Taxus chinensis (Liu et al. 2009), are implicated as phytotoxins or virulence factors and exhibit Taxus globosa (Soca-Chafre et al. 2011) and some medic- antimicrobial and cytotoxic activities (Wagenaar et al. 2000). inal shrubs of India (Naik et al. 2008). Here, we report for Cytochalasins are produced by a variety of fungal genera, in- the first time an endophytic association of a Diaporthe sp. cluding Phomopsis, Aspergillus, Penicillium and Chaetomium with Taxus baccata subsp. wallichiana. (Yan et al. 2016). Structurally, cytochalasins are comprised of a Medicinal plants are a repository of endophytic fungi that highly substituted isoindolone ring with a benzyl group at the are able to produce bioactive compounds of biotechnological C-3 position and fused to an 11- to 14-member macrocyclic and medicinal interest (Vieira et al. 2012). We recovered a ring. Wagenaar et al. (2000) reported the isolation of four cy- Diaporthe species from T. baccata as an endophyte that pro- totoxic cytochalasins from a culture of the endophytic fungus duced metabolites displaying cytotoxic activity against MCF- Rhinocladiella sp., and Yan et al. (2016) identified four cyto- 7 and HeLa tumour cells. Agusta et al. (2006)isolated (−)- chalasins (phomopchalasin A, B, C and J) from the endophytic epicytoskyrin from a Diaporthe species isolated from the tea fungus Phomopsis sp. isolated from the stem of Isodon plant and found that this compound exhibited moderate cyto- eriocalyx var. laxiflora. Pornpakakul et al. (2007) isolated toxic activity against KB cells, a subline of HeLa cells, with an diaporthichalasin, a cytochrome P450 3A4 inhibitor, from an −1 IC value of 0.5 μgml . Various researchers have reported endophyte Diaporthe species isolated from Croton sublyratus. the cytotoxic activity of the extracts of Diaporthe species Chen et al. (2014) reported the production of three new cyto- against different cell lines (Lin et al. 2005; Carvalho et al. chalasins (trichalasin E, F and H) along with four analogues 2012; Casella et al. 2013). In our study, the culture filtrate of from the endophytic fungus Trichoderma gamsii residing in the Diaporthe sp. showed antioxidant activity when assayed Chinese medicinal plant Panax notoginseng. In the present using the DPPH method. The DPPH assay has been exten- study, we identified three cytochalasins (trichalasin E, F and sively used as a reliable method to measure antioxidant activ- H) from Diaporthe sp. and confirmed their identity based on ity of pure compounds (Koleva et al. 2002). Our results sug- their m/z values. The biological activities exhibited by this fun- gest that metabolites of Diaporthe sp. could serve as potential gus may be correlated with the production of these bioactive compounds (trichalasins). This is the first report of the agents in scavenging free radicals. Antioxidant activity of the 260 Ann Microbiol (2017) 67:255–261 Eriksson OE, Vue JZ (1998) Bambusicolous pyrenomycetes,anannotated occurrence of trichalasins (E, F and H) from an endophyte check-list. Myconet 1:25–78 Diaporthe sp. isolated from T. baccata subsp. wallichiana. 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Isolation and characterization of trichalasin-producing endophytic fungus from Taxus baccata

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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Abstract

Ann Microbiol (2017) 67:255–261 DOI 10.1007/s13213-017-1256-4 ORIGINAL ARTICLE Isolation and characterization of trichalasin-producing endophytic fungus from Taxus baccata 1 1 2 1 Mondem Vasundhara & Manoj Baranwal & Nallapeta Sivaramaiah & Anil Kumar Received: 20 August 2016 /Accepted: 18 January 2017 /Published online: 9 February 2017 Springer-Verlag Berlin Heidelberg and the University of Milan 2017 Abstract An endophytic fungus (strain T1) isolated from Introduction Taxus baccata was studied for the production of metabolites with anticancer and antioxidant activities. This fungus was Endophytes are ubiquitous organisms which reside in the tis- identified as Diaporthe sp. based on rDNA-internal tran- sues of plants without causing apparent symptoms of disease scribed spacer (ITS) sequence analysis. The crude extract (Bacon and White 2000). The majority of endophytes are showed cytotoxic activity against MCF-7 and HeLa cancer fungi (Strobel and Daisy 2003), and they are present in the cell lines, with IC (concentration inhibiting 50% of growth intercellular spaces of various plant parts, such as the leaf and −1 rate) values of 1058 ± 44 and 1257 ± 80 μgml ,respectively. root (Corrêa et al. 2014). Endophytes reduce damage to their The scavenging activity of fungal extract increased signifi- host plants by producing many secondary metabolites cantly with increasing concentration [IC (concentration re- (Cabezas et al. 2012). They are also able to inhibit infection −1 quired to scavenge 50% of free radicals) 482 ± 9 μgml ]. and proliferation of pathogens in the host plant directly or Ultra-high-performance liquid chromatography-quadrupole- indirectly by inducing resistance responses intrinsic to the host time of flight analysis revealed the presence of three defence (Eaton et al. 2011). Currently, endophytic fungal re- trichalasins (trichalasin E, F and H) in the crude extract of search is focused on the ability of these fungi to produce and T1 which are known to have antitumour and antioxidant ac- accumulate secondary metabolites. Several of these com- tivities. These results suggest that Diaporthe sp. has the po- pounds have biological activities of interest for application tential to be used for therapeutic purposes because of its anti- in environmental, agriculture, pharmaceutical and healthcare proliferative and antioxidant potential and also for the produc- and food industries (Suryanarayanan et al. 2009; Kharwar tion of cytochalasins. et al. 2011; Deshmukh et al. 2015). Diverse classes of chemical substances, such as steroids, xanthones, phenols, isocoumarines, perylene derivatives, qui- . . . Keywords Endophytic fungi Diaporthe Cytochalasins nones, furandiones, terpenoids, depsipeptides and cytochala- Trichalasins Antioxidant activity sins, have been isolated from endophytic fungi (Schulz and Boyle 2005). Endophytes produce a significantly higher num- ber of novel chemical structures than soil fungi, indicating that Electronic supplementary material The online version of this article endophytes are a novel source of bioactive secondary metab- (doi:10.1007/s13213-017-1256-4) contains supplementary material, which is available to authorized users. olites (Nisa et al. 2015). In this context, the secondary metab- olites produced by endophytes associated with medicinal * Mondem Vasundhara plants are important as they could be exploited for the treat- mvasundhara@thapar.edu ment of many diseases (Tejesvi et al. 2007). Several studies have shown that endophytes produce secondary metabolites of their host plants, thus raising the prospect of using them as Department of Biotechnology, Thapar University, Patiala 147004, Punjab, India alternative sources of these metabolites (Priti et al. 2009). Endophytic fungi of the genus Diaporthe have been isolat- Nano Temper Technologies, World Trade Centre, Bangalore 560055, India ed from a wide variety of plant hosts from both temperate and 256 Ann Microbiol (2017) 67:255–261 tropical regions (Dos Santos et al. 2016). Diaporthe is one of instructions and then sequenced. The ITS sequence obtained the most abundant genera of endophytic fungi and has been from this isolate has been deposited in the GenBank under reported to produce various compounds of biotechnological accession number KX355165. interest (Silva et al. 2005; Pornpakakul et al. 2007; The ITS sequence of T1 was compared to those available in Rukachaisirikul et al. 2008). Cytochalasins are a group of the GenBank database using the BLASTN algorithm. cytotoxic fungal metabolites showing a wide range of biolog- Alignment of the sequences was constructed using MAFFT ical activities, such as anticancer, antimicrobial, antiparasitic, version 7.0 (Katoh and Standley 2013) and edited with phytotoxic activities, among others (Scherlach et al. 2010; BioEdit version 5.0.6 (Hall 1999). Phylogenetic analysis on Chen et al. 2014). These metabolites have been isolated from the resulting alignment was performed using Bayesian many fungal species, including Helminthosporium sp., Inference. A Bayesian analysis was implemented in Phoma sp., Xylaria sp., Hypoxylon sp. and Rhinocladiella MrBayes v.3.2.2 with two parallel runs, each consisting of sp. (Scherlach et al. 2010). four incrementally heated Monte Carlo Markov Chains. The In the investigation reported here, we isolated an en- analysis was run using Metropolis-coupled a Markov Chain dophytic fungus from the bark of the Taxus baccata L. Monte Carlo search algorithm over 1,000,000 generations, subsp. wallichiana (Zucc.) Pilger (Himalayan Yew) and and the convergence of Bayesian analysis was observed by subsequently identified it as Diaporthe sp. strain T1 examination of the standard deviation of split frequencies of based on its internal transcribed spacer (ITS) regions of <0.01. Trees were sampled every 100th generations resulting rDNA. The biological activities, such as anticancer and in total of 10,000 trees. The first 2500 trees, representing the antioxidant activities, of ethyl acetate extracts of its cul- burn-in phase of the analysis, were discarded, and the remain- ture filtrate were studied using various bioassays. These ing 7500 trees were used to calculate posterior probabilities extracts were also analysed by liquid chromatography/ from the 50% majority rule consensus trees. tandem mass spectrometry (LC-MS/MS) to identify some key compounds belonging to the cytochalasin Fungal extract preparation group of fungal metabolites which could be responsible for these activities. Mycelial discs (diameter 5.0 mm) of actively growing culture (7 days old) were inoculated into potato dextrose broth (500 ml) and incubated at 25 ± 2 °C for 21 days in the dark Material and methods as a static culture. The cultures were harvested by filtration through four layers of cheesecloth to remove the mycelia, and Isolation and identification of endophytic fungus strain T1 the mycelial biomass thus obtained was then dried overnight (35–40 °C) and extracted with ethyl acetate for 12 h. The cul- Endophytic fungus T1 was isolated from the bark samples of ture broth was also extracted three times with an equal volume T. baccata L. subsp. wallichiana (Zucc.) Pilger collected from of ethyl acetate; the ethyl acetate fractions were then pooled and Bhadrewah (Doda district, India) according to the method driedinvacuoat35°C. Part of the residue wasdissolved in described by Garyali et al. (2013). The fungal culture was dimethyl sulfoxide and used for the bioassays. The other part of maintained on potato dextrose agar (HiMedia Laboratories the residue was dissolved in methanol and partitioned three Ltd. Mumbai, India) medium. Molecular characterization times with an equal volume of dichloromethane); the dichloro- was performed by ITS sequence analysis. Specifically, methane fractions were pooled and dried in vacuo. The residue mycelia were harvested from actively growing colonies and was used for the LC–two-dimension-MS [ultra-high-perfor- ground in liquid nitrogen. Genomic DNA was extracted from mance LC-quadrupole-time of flight analysis (UHPLC- the finely ground fungal material by the CTAB method QTOF-MS/MS)] to analyse the bioactive compounds. (Zhang et al. 2008). The quality and quantity of DNA was checked with Nanodrop 1000 spectrophotometer (Thermo Cell growth inhibition assay Fisher Scientific, Waltham, MA) and samples were stored at −20 °C until use. The ITS region of nuclear ribosomal DNA Human breast cancer cell lines (MCF-7) and human cervical was amplified with the universal primers ITS1 and ITS4 cancer cell lines (HeLa) were procured from National Centre (White et al. 1990) in a thermal cycler (Applied Biosystems, for Cell Science, Pune, India. The cells were maintained in Foster City, CA). The thermal cycling conditions applied for Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, St. the ITS region consisted of an initial denaturation for 5 min at Louis, MO) containing 10% (v/v) foetal bovine serum −1 94 °C followed by 34 cycles of 1 min at 94 °C, 1 min at 50 °C (Gibco, Thermo Fisher Scientific), 100 IU ml penicillin, −1 −1 and 1.5 min at 72 °C and a final extension of 7 min at 72 °C. 100 μgml streptomycin, and 2.5 μgml amphotericin, in PCR products were purified using QIAquick spin columns a humidified incubator with 5% CO at 37 °C. A well-known (Qiagen, Hilden, Germany) following the manufacturer’s 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium- Ann Microbiol (2017) 67:255–261 257 bromide (MTT) assay was carried out to assess the effect of added to the wells. After 72 h of incubation, the MTT assay fungal extract on the growth of the cancer cell lines. In brief, was carried out as described by Denizot and Lang (1986). approximately 2 × 10 cells per well were seeded in 96 well Paclitaxel was used as the positive control at the concentration −1 culture plate and incubated overnight. After 16 h, varying of 20 μgml . The growth inhibition rate was calculated using −1 concentrations (250–1500 μgml )of fungalextractswere the formula (OD is optical density): Inhibition rate ¼ðÞ Mean OD of control well − Mean OD of treated cell=Mean OD of untreated cell 100 in this assay was defined as the concentration of analysed by analysis of variance, and the means were com- The IC compounds that resulted in 50% inhibition of growth rate. pared by Tukey’stest at p <0.05. Antioxidant assay Results The antioxidant potential of the fungal extract was studied by Isolation and identification of fungal strain T1 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay. Fungal extract (50-μl samples) of different concentrations were mixed with The PCR product of ITS-rDNA amplified with ITS1 and ITS4 150 μlof DPPH(100 μM) in methanol in the wells of a 96- −1 was 588 bp. BLAST analysis revealed 98% similarity (query well microtiter plate. Ascorbic acid (100 μgml ;50 μl) was coverage of 100%) with Diaporthe sp. Bayesian analysis of used as the positive control. The plate was incubated in the ITS sequences of different species of Diaporthe yielded a dark (45 min), and absorbance of the reaction mixture was consensus tree (Fig. 1), and the present isolate was clustered measured at 517 nm using an enzyme-linked immunosorbent with Diaporthe sp. (KC357558 and KC357559). assay reader (Infinite microplate reader; Tecan Austria GmbH, Grödig, Austria). The percentage inhibition of DPPH radical Cytotoxic effect in cancer cell lines by the fungal extract was expressed as the inhibition concen- tration (IC ) and was calculated using the formula: The fungal extract showed a cytotoxic effect against both the DPPH scavenging activityðÞ % ¼ A − A =A  100 Control Sample Control human breast cancer cell line (MCF-7) and the human cervical cancer cell line (HeLa). The cytotoxic effect become signifi- Identification of bioactive compounds by UHPLC-QTOF cantly pronounced at higher concentrations of the fungal ex- analysis tract (Fig. 2). The IC value of the extract was 1058 ± 44 and −1 1257 ± 80 μgml for the MCF-7 and HeLa cell lines, respec- −1 For the UHPLC-QTOF-MS/MS analysis, the residue was first tively. Paclitaxel (20 μgml ), an anticancer drug, was used as dissolved in methanol. Chromatographic separation was car- a positive control. It inhibited the growth of the MCF-7 and ried out using the Agilent 1200 UHPLC system equipped with HeLa cell lines by 79 ± 6 and 97 ± 2%, respectively. C18 column (Agilent Technologies, Santa Clara, CA). Samples (5 μl) were injected into the column, and elution Antioxidant activity was carried out in a gradient mode with acidified water (0.2% v/v formic acid) to 90% methanol over a period of Free radical scavenging activity was performed to determine −1 40 min at a flow rate of 0.3 ml min . High-resolution mass the antioxidant activity of Diaporthe sp. The scavenging ac- spectroscopic detection was performed using Bruker Impact tivity significantly increased with increasing concentration of QTOF mass spectrometer (Bruker Corp., Billerica, MA, oper- fungal extract (Fig. 3). The IC value (concentration of sam- ating in Top5 data-dependent mode from 100–1500 m/z with ple required to scavenge 50% of free radicals) of the crude −1 electron spray ionization, and both positive and negative ions extract was 250 ± 3.5 μgml . Ascorbic acid, the positive were detected. Smart Formula 3D™ and Fragment Explorer control, showed 84 ± 3.5% antioxidant activity. part of Data Analysis 4.2 (Bruker Corp.) were used to generate compound formulae. Identification of compounds by UHPLQTOF analysis Statistical analysis The UHPLC-QTOF- MS/MS analysis of the crude extract of Diaporthe sp. (T1) showed a signal (m/z) corresponding to All the experiments were performed in triplicate. The results three trichalasins. Figure 4 shows the mass data of these three were expressed as mean ± standard deviation. The data were compounds that eluted from the column at different times. 258 Ann Microbiol (2017) 67:255–261 Fig. 1 Bayesian tree showing the relationships between the internal transcribed spacer (ITS) sequence of Diaporthe sp. (shown in bold) and those of related species retrieved from GenBank. Numbersatnodes Posterior probability percentages (>50%) of the Bayesian analysis Peaks at m/z ratios of 402.1753, 420.1391 and 472.1511 were Discussion observed and identified as Trichalasin H, Trichalasin F) and Trichalasin E, respectively, based on the reported values of The anamorphic form (the asexual reproductive morpho- these compounds (Electronic Supplementary Material Table). logical stage) of Diaporthe is the genus Phomopsis. Members of Diaporthe/Phomopsis are either plant patho- gens or endophytic species (van Niekerk et al. 2005;Dos Fig. 2 Cytotoxic effect of Diaporthe sp. extract against human breast cancer (MCF-7) and human cervical (HeLa) cancer cell lines. Bars Mean Fig. 3 Antioxidant effect of Diaporthe sp. extracts based on free radical values, error bars standard deviation (SD). Bars with the same lowercase scavenging activity. Bars Mean values, error bars standard deviation letter within the same cell type are not significantly different at p <0.05. (SD). Bars with the same lowercase letter are not significantly different −1 −1 PT Paclitaxel (20 μgml ) used as the positive control at p <0.05. AA Ascorbic acid (100 μgml ) used as the positive control Ann Microbiol (2017) 67:255–261 259 Fig. 4 The tandem mass spectrometry profile of selected ions in the extracts of Diaporthe sp. T1 corresponding to three tricalasins: a Trichalasin H (m/z 402.1753), b Trichalasin F (m/z 420.1391), c trichalasin E (m/z 472.1511) Santos et al. 2016) and are associated with dicotyledonous Diaporthe/Phomopsis complex has been reported by Nath and monocotyledonous plants of tropical and temperate et al. (2012) and Ascêncio et al. (2014). regions (Eriksson and Vue 1998; Guo et al. 2000). The Cytochalasins are a known class of mould metabolites that Diaporthe/Phomopsis complex occurs as endophytes with exhibit a wide range of distinctive biological activities. They medicinal plants such as Taxus chinensis (Liu et al. 2009), are implicated as phytotoxins or virulence factors and exhibit Taxus globosa (Soca-Chafre et al. 2011) and some medic- antimicrobial and cytotoxic activities (Wagenaar et al. 2000). inal shrubs of India (Naik et al. 2008). Here, we report for Cytochalasins are produced by a variety of fungal genera, in- the first time an endophytic association of a Diaporthe sp. cluding Phomopsis, Aspergillus, Penicillium and Chaetomium with Taxus baccata subsp. wallichiana. (Yan et al. 2016). Structurally, cytochalasins are comprised of a Medicinal plants are a repository of endophytic fungi that highly substituted isoindolone ring with a benzyl group at the are able to produce bioactive compounds of biotechnological C-3 position and fused to an 11- to 14-member macrocyclic and medicinal interest (Vieira et al. 2012). We recovered a ring. Wagenaar et al. (2000) reported the isolation of four cy- Diaporthe species from T. baccata as an endophyte that pro- totoxic cytochalasins from a culture of the endophytic fungus duced metabolites displaying cytotoxic activity against MCF- Rhinocladiella sp., and Yan et al. (2016) identified four cyto- 7 and HeLa tumour cells. Agusta et al. (2006)isolated (−)- chalasins (phomopchalasin A, B, C and J) from the endophytic epicytoskyrin from a Diaporthe species isolated from the tea fungus Phomopsis sp. isolated from the stem of Isodon plant and found that this compound exhibited moderate cyto- eriocalyx var. laxiflora. Pornpakakul et al. (2007) isolated toxic activity against KB cells, a subline of HeLa cells, with an diaporthichalasin, a cytochrome P450 3A4 inhibitor, from an −1 IC value of 0.5 μgml . Various researchers have reported endophyte Diaporthe species isolated from Croton sublyratus. the cytotoxic activity of the extracts of Diaporthe species Chen et al. (2014) reported the production of three new cyto- against different cell lines (Lin et al. 2005; Carvalho et al. chalasins (trichalasin E, F and H) along with four analogues 2012; Casella et al. 2013). In our study, the culture filtrate of from the endophytic fungus Trichoderma gamsii residing in the Diaporthe sp. showed antioxidant activity when assayed Chinese medicinal plant Panax notoginseng. In the present using the DPPH method. 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Journal

Annals of Microbiology – Springer Journals

Published: Feb 9, 2017

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