World J Microbiol Biotechnol DOI 10.1007/s11274-014-1686-0
ORIGINAL PAPER
Isolation and identification of an endophytic fungus Pezicula sp. in Forsythia viridissima and its secondary metabolites Jiaying Wang • Guoping Wang • Yalei Zhang • Biqiang Zheng • Chulong Zhang • Liwei Wang
Received: 26 March 2014 / Accepted: 4 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract In a survey of endophytic fungal biodiversity, an antimicrobial endophytic isolate zjwcf069 was obtained from twigs of Forsythia viridissima, Zhejiang Province, Southeast China. Zjwcf069 was then identified as Pezicula sp. through combination of morphological and phylogenetic analysis based on ITS-rDNA. Zjwcf069 here represented the first endophytic fungus in Pezicula isolated from host F. viridissima. From the fermentation broth, four compounds were obtained through silica gel column chromatography and Sephadex LH-20 under the guide of bioassay. Their structures were elucidated by spectroscopic analysis as mellein (1), ramulosin (2), butanedioic acid (3), and 4-methoxy-1(3H)-isobenzofuranone (4). Compound 4 here stood for the very first time as natural product from microbes. In vitro antifungal assay showed that compound 1 displayed growth inhibition against 9 plant pathogenic fungi, especially Botrytis cinerea and Fulvia fulva with EC50 values below 50 lg/mL. Endophytic fungi in medicinal plants were good resources for bioactive secondary metabolites.
J. Wang C. Zhang State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China G. Wang B. Zheng Zhejiang Dayang Biotechnology Group Co., Ltd, Hangzhou 311616, China Y. Zhang College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310029, China L. Wang (&) Department of Pharmaceutical Science, College of Medical Science, Hangzhou Normal University, Hangzhou 310036, China e-mail: [email protected]
Keywords Pezicula Endophyte Metabolites Antimicrobial Activity
Introduction Endophytes are microbes that reside in tissues of living plants without any apparent symptoms, which may turn pathogenic or saprophytic when circumstances become unfavorable or host plants start to senescense (Aly et al. 2011). Because of their high biodiversity (Rodriguez et al. 2009) and largely unexploited metabolic resources (Tan and Zou 2001), they have been receiving more and more attention from microbiologists and chemists (Chen et al. 2013; El-Neketi et al. 2013; Grum et al. 2013; Hoffman et al. 2013; Li et al. 2013). In our lab, fungal endophytes and relative bioactive metabolite researches occupy a big part of the time. Up to now, (3S)-3,6,7-trihydroxy-a-tetralone, a new a-tetralone derivative and cercosporamide, with strong broad-spectrum antifungal activities have been successfully obtained from endophytic Phoma sp. residing in Arisaema erubescens (Wang et al. 2012a, b), griseofulvin from Nigrospora sp. in Moringa oleifera (Zhao et al. 2012), as well as trichodermin from Trichoderma sp. in Taxus mairei (Chen et al. 2008). Forsythia viridissima has long been accepted as a traditional Chinese medicine (Lee et al. 2010). In this study, sixteen endophytic fungal isolates were obtained from twigs of F. viridissima. Among them, zjwcf069 expressed antimicrobial activities, which was thus chosen for further chemical analysis. Altogether 4 compounds were achieved from fermentation broth of zjwcf069 and identified as mellein (1), ramulosin (2), butanedioic acid (3), and 4-methoxy-1(3H)-isobenzofuranone (4), respectively.
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Bioassay was carried out and compound 1 showed significant antagonism against 9 plant pathogenic fungi.
Materials and methods Sampling, fungal isolation and purification Healthy and intact samples of F. viridissima were collected from Shiyang yard (E1208050, N278470), Wencheng, Zhejiang Province, Southeast China, which were immediately stored in plastic bags and transported back to lab in an icy box within 48 h. During fungal isolation process, plant samples were firstly brushed with tap water to get rid of those attached microbes and particles, then secondly surface-sterilized in ethanol solution (75 %, v/v) for 1 min, as well as that of sodium hypochlorite (0.5 %, w/v) for 5–10 min (depending on different tissue types), and thirdly rinsed in sterile distilled water for at least three times. Plant tissue segments of 1 cm length were placed on a Petri dish with potato dextrose agar (PDA), supplemented with ampicillin (100 lg/mL) and streptomycin sulphate (60 lg/mL) to avert bacterial growth, and then incubated at 25 °C avoiding light. Surface-sterilized plant tissues without cutting were incubated meantime as a control. Fungal tips were transferred to sub-culture on PDA as soon as they emerged until pure colonies were formed. Finally they were stored in two distinct methods, that is, on PDA slants covered with sterile liquid paraffin at room temperature and in preservation liquid (15 % v/v glycerol, 10 g/L glucose, 1 g/L yeast extract and 1 g/L casein hydrolysate) at -80 °C (Zhang et al. 2010). Identification of strain zjwcf069 Strain zjwcf069 was identified using both morphological characters and phylogenetic data. For optimal growth, PDA and chestnut extract solid medium (10 %, w/v) were used. Vegetative and reproductive growth characteristics were obtained and compared to other known species. On the other hand, mycelium from pure culture was scraped and used to extract genomic DNA according to the quick method published earlier (Chi et al. 2009). To amplify internal transcribed spacer of ribosomal nucleotide sequence (ITS rDNA), primers ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) (Mitchell et al. 1994) were used. Reaction of 50 lL consisted of 19 polymerase chain reaction (PCR) buffer, 2.5 mM Mg2?, 100 lM dNTPs, 0.5 lM each primer, 10 ng extracted DNA and 2 U Taq polymerase. PCR thermocycling steps were 94 °C for 3 min, followed by 35 cycles of 94 °C for 40 s, 56 °C for 50 s, and 72 °C for
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60 s, and the final extension 72 °C for 10 min. PCR products were purified via a DNA gel extraction kit (Axygen Incorporation, China) and then sequenced on an ABI 3730 sequencer (Applied Bio-systems, USA). Corresponding sequence was analyzed through Basic Local Alignment Search Tool (BLAST). Neighbor-joining (NJ) phylogenetic tree was constructed in MEGA 4, using 1,000 bootstrap replicates (Tamura et al. 2007). ITS-rDNA of zjwcf069 was submitted to GenBank and the accession no. is JN387907. Fermentation and metabolite isolation Zjwcf069 was cultured in 1,000-mL Erlenmeyer flasks, each containing 500 mL potato dextrose broth (PDB, 10 L in total), and incubated on a rotary shaker (180 rpm; 25 °C) avoiding light for 15 d. After filtration, fermentation broth was extracted using ethyl acetate (EtOAc) of equal volume for three times, and then evaporated to dryness (2.3 g). The extract was mixed with silica gel, dried at 50 °C, and subsequently loaded on a silica gel column (4,091,000 mm) with 210 g silica gel (200–300 mesh). For optimal separation, column was eluted with mixture liquid of petroleum ether: ethyl acetate (from 9:1 to 1:3, v/v). Components were visualized under UV254/366 and by spraying with vanillin:H2SO4:MeOH (1:3:50, v/v/v) and then heating at 110 °C. Totally 5 fractions were collected and labeled F1 to F5 accordingly, among which F1, F2 and F5 showed antifungal activities against 9 pathogenic fungi. So these fractions were purified thereafter. The bioactive fraction F1 (653 mg) were separated further by column chromatography (CC) over SephadexLH-20 through a mixture of methanol and chloroform (1:1 v/v), to afford compound 1 (584 mg). Similarly, F2 (63 mg) were subject to CC to obtain white crystal compound 2 (39 mg). F4 (595 mg) was recrystallized to give compound 3 (518 mg). F5 (231 mg) went through purification as F1 to afford compound 4 (18.7 mg) (Zhao et al. 2012). Structure analysis Mass and nuclear magnetic resonance (NMR) spectrometries were carried out to figure out the chemical structures of four metabolites. Mass spectra were obtained from a Bruker Esquire 3000plus mass spectrometer. 1H, 13C NMRs were recorded on a Bruker AMX-500 (500 MHz) NMR spectrometer taking TMS as internal standard. Chemical shifts expressed in d (ppm) and coupling constant J in Hz. Melting points were achieved on a Beijing X4 micro melting point apparatus without correction. Silica gel (200–300 mesh) for CC was purchased from Qingdao Marine Chemical Factory (Qingdao, China). So was SephadexLH-20 from Pharmacia Biotech (Sweden).
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Antifungal activity assay Compound 1 was dissolved in sterile distilled water and tested for fungistatic activity in vitro against 9 phytopathogenic fungi: Botrytis cinerea, Pythium ultimum, Fusarium oxysporium f. sp. Cucumerinum, Colletotrichum orbiculare, Verticillium dahliae, Pyricularia oryzae, Pestalotia diospyri, Sclerotinia sclerotiorum and Fulvia fulva. During the process, a series of Petri dishes containing metabolite solutions of certain concentration (1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 lg/mL) were diluted with melted PDA medium. Solution was replaced by sterile distilled water as control. Cyclopaldic acid was chosen as positive control (Wang et al. 2012a, b). Then one colony disc (5 mm in diameter) of test fungi was placed at the center of each plate. Each treatment was carried out for three times as replicates. These plates were all incubated in darkness at 25 °C. When colony in control setting reached edge, diameters of all growing colonies were measured and plotted against the respective metabolite concentration. Median effect concentration (EC50) is defined as the concentration of test compound that allows test organism to grow at 50 % of its rate under the same condition in absence of test compound (Wang et al. 2012a, b).
Results In this study, 16 isolates were isolated from twigs of F. viridissima. Among them, zjwcf069 went through further analysis. Both vegetative and reproductive characters were induced for identification (Papagianni 2004). Colony of zjwcf069 on PDA reached a diameter of 4–6 cm after 10 days growth. While on chestnut extract solid medium, colony was suborbicular with lots of aerial hyphae, which was smooth at the back, when incubated at 25 °C with alternating light and darkness (12/12 h). Hyphae were white at first, and then tinted into gray and light brown, on which water drops colored light yellow could been seen (Fig. 1a). Other morphological characteristics included:
black pycnidia, jacinth conidium groups, single-celled conidia which were colorless and cylindrical with both ends blunt or one end blunt and the other pointed. Size of conidia was 11.25–17.00 lm in length and 4.0–5.5 lm in width (Fig. 1b, c). Morphological identification has long been the basis and tradition, which is beyond doubt of great importance. ITS rDNA of zjwcf069 was also sequenced to assist precise identification (Sugita and Nishikawa 2003). Alignment with those from Genbank database resulted in several closely related sequences and along with them phylogenetic analysis was conducted. Corresponding NJ tree showed clearly that zjwcf069 fell into the group of Pezicula spp. with quite strong support (Fig. 2). Thus combined with morphology, zjwcf069 was defined as Pezicula sp. ultimately. Altogether 4 compounds were obtained via metabolite isolation from fermentation broth of zjwcf069. Thereafter structures of them were illustrated as followings (Fig. 3). Compound 1 Mellein, white crystal, molecular formula C10H10O3; m.p. 56 °C; [a]25 D-102.5(c1.0, CHCl3); ESI–MS (m/z): 178 [M ? H]?; 1H NMR (500 MHz, CDCl3 J in Hz) d: 1.53 (3H, d, J = 6.3 Hz, –CH3), 2.93 (2H, dd, J = 6.9, 1.0 Hz, H-4); 4.74 (1H, tq, J = 6.9, 6.3 Hz, H-3); 6.70 (1H, dd, J = 7.4, 1.0 Hz, H-5); 6.89 (1H, d, J = 8.4 Hz, H-7); 7.41 (1H, dd, J = 8.4, 7.4 Hz, H-6); 11.03 (1H, s, –OH). 13C NMR (125 MHz, in CDCl3) d: 20.7 (–CH3), 34.6 (C-4), 76.1 (C-3), 108.2 (C-9), 116.2 (C-7), 117.9 (C-5), 136.1 (C6), 139.2 (C-10), 162.1 (C-8), 169.9 (C-1). Both 1H NMR and 13C NMR data turned out to be consistent with those reported earlier (Islam et al. 2007). Compound 2 Ramulosin, white crystal, molecular formula C10H14O3; m.p. 118–119 °C; [a]25 D ?19 (c0.50, EtOH); ESI–MS (m/z): 183 [M ? H]?; 1H NMR (500 MHz, CDCl3, J in
Fig. 1 Colony and conidia of zjwcf069 on chestnut extract solid medium. a Colony of zjwcf069 after 10 days. Bar equals to 1 cm. b Conidia of zjwcf069. c Conidiophores of zjwcf069 after 3 weeks. Bars in both b and c represent 10 lm
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World J Microbiol Biotechnol Fig. 2 Phylogenetic NJ tree inferred from ITS rDNA sequences Bar represents 0.005 substitutions per site
Table 1 EC50s of mellein (1) and cyclopaldic acid against 9 phytopathogens Phytopathogens Botrytis cinerea
Hz) d: 1.11 (1H, m, H-5a), 1.22 (brdd, J = 13.5,11.5, H-4a), 1.33 (d, J = 6.3, 3H, –CH3), 1.45–1.69 (m, 2H, H-6), 1.77–1.90 (m, 1H, H-5b), 1.86 (ddd, 1H, J = 13.5, 2.4, 3.9, H-4b), 2.31 (m, 2H, H-7), 2.44 (ddd, J = 12.2, 3.9, 1.9, 1H, H-4a), 4.39 (ddq, J = 11.5, 2.4, 6.3, 1H, H-3), 9.33 (s, –OH); I3C NMR (125 MHz, CDCl3) d: 20.8 (C-7), 21.7 (3-CH3), 29.0 (C-6), 29.5 (C-5), 32.9 (C-4a), 37.4 (C4), 76.5 (C-3), 96.8 (C-8a), 171.8 (C-8), 174.7 (C-1). Both 1 H NMR and 13C NMR data turned out to be consistent with those reported earlier (Stierle et al. 1998). Compound 3 Butanedioic acid, colorless crystal, molecular formula C4H6O4; m.p. 187.6–188.3 °C; ESI–MS (m/z): 117 [M ? H]?; 1H NMR (500 MHZ, DMSO-d6, J in Hz) d: 2.42 (2H, s, –CH2), 12.16 (2H, s, –COOH); 13C NMR (125 MHZ, DMSO-d6) d: 28.83 (C-2,3), 173.68 (C-1,4). Both 1H NMR and 13C NMR data turned out to be consistent with those reported earlier (SDBS Information 2013).
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Cyclopaldic acid (lg/mL)
48.63
115.36
Colletotrichum orbiculare
150.90
355.71
Verticillium dahliae
163.37
216.83
Fusarium oxysporium f. sp. cucumerinum
159.09
228.15
Pyricularia oryzae
118.83
116.68
Pestalotia diospyri
161.04
[1,000
Pythium ultimum
125.36
124.14
Sclerotinia sclerotiorum
205.01
60.75
45.98
40.01
Fulvia fulva
Fig. 3 Structures of 4 compounds
Compound 1 (lg/mL)
Compound 4 4-Methoxy-1(3H)-isobenzofuranone, white crystal, molecular formula C9H8O3; m.p. 128.4–129.3 °C; ESI–MS (m/ z): 165 [M ? H]?; 1H NMR (500 MHZ, DMSO-d6) d: 3.91 (s, 3H, OCH3), 5.36 (s, 2H, H8), 7.41 (m, 3H, Ar–H); 13C NMR(125 MHZ, DMSO-d6) d: 55.75 (C–OCH3), 67.98 (C3), 115.86–153.98 (C–Ar), 170.51 (C-1). Both 1H NMR and 13C NMR data turned out to be consistent with the literature earlier (Egan et al. 2011). According to the result of antifungal bioassay, mellein (1) expressed antagonism against these 9 pathogens at diverse levels (Table 1). Among them, B. cinerea and F. fulva were the most affected fungi (EC50 \ 50 lg/mL). Compared to positive control, compound 1 has antifungal property. Compounds 2 and 4 could not afford the bioassay because of their limited yield.
Discussion Quantitive molecular phylogenetics has been increasingly popularized in recent researches accounting fungal
World J Microbiol Biotechnol
endophyte identification as traditional morphology is unstable and largely dependent on circumstances. Here in this study, high quality sequences, especially those from type species, were chosen for solid phylogenetic conclusions (Ko et al. 2011). For further taxonomic location, more molecular information is needed, like multiple-locus phylogenetics (Wang et al. 2014). This research represents the very first time to isolate Pezicula endophyte from F. viridissima which is a typical herbal medicine with bioactivities like antiphlogosi (Lee et al. 2010), antiasthma (Lee et al. 2011) and vasodilation (Iizuka et al. 2009). So far, reports on endophytes from this particular plant are limited. Up to now, several bioactive compounds have been isolated from Pezicula spp. The antibiotic equisetin derivative (Sugie et al. 2002), furofurandiones from Pezicula livida (Krohn et al. 1994) and 5 fungicidal and herbicidal metabolites reported in 1995 (Schulz et al. 1995) represent great potential in Pezicula fungi as a natural product source with diverse activities. Also cryptocandin and its related bioactive agents which have out-standing antifungal activity against both human and plant pathogens were reported to be produced by Cryptosporiopsis quercina, the imperfect stage of Pezicula cinnamomea (Strobel and Daisy 2003). 4-Methoxy-1(3H)-isobenzofuranone (4-methoxyphthalide, compound 4) participates in production of bioactive lactone derivatives (Soucy et al. 1987) as well as antitumor compound cervicarcin Marumo et al. (1964). Currently 4-methoxyphthalide (4) is artificially synthesized (Soucy 1987). However this paper represents the first one to report it as a natural metabolite. Ramulosin (2) first isolated from metabolites of a saprophytic fungus Pestalotia ramulosa (Benjamin and Stodola 1960), has potential as herbicide and fungicide, most of which is synthesized (Islam et al. 2007). However yield of either compound 4 or 2 in this research was not enough for bioassay. Though mellein (1) as a natural product was isolated from endophytic Pezicula species before (Schulz et al. 1995), the quantitive evidences for its antibiotic activity were not clearly demonstrated. Herein 9 representative phytopathogens were chosen and exact EC50 was measured as a solid indication of antifungal activity for the first time ever (Table 1). Mellein (1) expressed similar antifungal activities with cyclopaldic acid. Butanedioic acid (3) has long been an important material for organic synthesis. In this research, 16 strains were isolated from twigs of F. viridissima. Among them, Pezicula endophyte zjwcf069 went through further chemical analysis. Totally 4 compounds were achieved from fermentation broth, that is, mellein (1), ramulosin (2), butanedioic acid (3), and 4-methoxy-1(3H)-isobenzofuranone (4). Compound 4 was obtained as a natural metabolite for the first time. Corresponding bioassay was carried out and compound 1 showed
significant antagonism against B. cinerea and F. fulva (EC50 \ 50 lg/mL). Again endophyte was proven to be a promising source of natural bioactive metabolites. Acknowledgments This research was financially supported by the Zhejiang Provincial National Natural Science Foundation of China (No. LQ13H280005) and Open Research Fund Program of the Laboratory of Hangzhou Normal University (No. 2013040).
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ORIGINAL PAPER
Isolation and identification of an endophytic fungus Pezicula sp. in Forsythia viridissima and its secondary metabolites Jiaying Wang • Guoping Wang • Yalei Zhang • Biqiang Zheng • Chulong Zhang • Liwei Wang
Received: 26 March 2014 / Accepted: 4 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract In a survey of endophytic fungal biodiversity, an antimicrobial endophytic isolate zjwcf069 was obtained from twigs of Forsythia viridissima, Zhejiang Province, Southeast China. Zjwcf069 was then identified as Pezicula sp. through combination of morphological and phylogenetic analysis based on ITS-rDNA. Zjwcf069 here represented the first endophytic fungus in Pezicula isolated from host F. viridissima. From the fermentation broth, four compounds were obtained through silica gel column chromatography and Sephadex LH-20 under the guide of bioassay. Their structures were elucidated by spectroscopic analysis as mellein (1), ramulosin (2), butanedioic acid (3), and 4-methoxy-1(3H)-isobenzofuranone (4). Compound 4 here stood for the very first time as natural product from microbes. In vitro antifungal assay showed that compound 1 displayed growth inhibition against 9 plant pathogenic fungi, especially Botrytis cinerea and Fulvia fulva with EC50 values below 50 lg/mL. Endophytic fungi in medicinal plants were good resources for bioactive secondary metabolites.
J. Wang C. Zhang State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China G. Wang B. Zheng Zhejiang Dayang Biotechnology Group Co., Ltd, Hangzhou 311616, China Y. Zhang College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310029, China L. Wang (&) Department of Pharmaceutical Science, College of Medical Science, Hangzhou Normal University, Hangzhou 310036, China e-mail: [email protected]
Keywords Pezicula Endophyte Metabolites Antimicrobial Activity
Introduction Endophytes are microbes that reside in tissues of living plants without any apparent symptoms, which may turn pathogenic or saprophytic when circumstances become unfavorable or host plants start to senescense (Aly et al. 2011). Because of their high biodiversity (Rodriguez et al. 2009) and largely unexploited metabolic resources (Tan and Zou 2001), they have been receiving more and more attention from microbiologists and chemists (Chen et al. 2013; El-Neketi et al. 2013; Grum et al. 2013; Hoffman et al. 2013; Li et al. 2013). In our lab, fungal endophytes and relative bioactive metabolite researches occupy a big part of the time. Up to now, (3S)-3,6,7-trihydroxy-a-tetralone, a new a-tetralone derivative and cercosporamide, with strong broad-spectrum antifungal activities have been successfully obtained from endophytic Phoma sp. residing in Arisaema erubescens (Wang et al. 2012a, b), griseofulvin from Nigrospora sp. in Moringa oleifera (Zhao et al. 2012), as well as trichodermin from Trichoderma sp. in Taxus mairei (Chen et al. 2008). Forsythia viridissima has long been accepted as a traditional Chinese medicine (Lee et al. 2010). In this study, sixteen endophytic fungal isolates were obtained from twigs of F. viridissima. Among them, zjwcf069 expressed antimicrobial activities, which was thus chosen for further chemical analysis. Altogether 4 compounds were achieved from fermentation broth of zjwcf069 and identified as mellein (1), ramulosin (2), butanedioic acid (3), and 4-methoxy-1(3H)-isobenzofuranone (4), respectively.
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Bioassay was carried out and compound 1 showed significant antagonism against 9 plant pathogenic fungi.
Materials and methods Sampling, fungal isolation and purification Healthy and intact samples of F. viridissima were collected from Shiyang yard (E1208050, N278470), Wencheng, Zhejiang Province, Southeast China, which were immediately stored in plastic bags and transported back to lab in an icy box within 48 h. During fungal isolation process, plant samples were firstly brushed with tap water to get rid of those attached microbes and particles, then secondly surface-sterilized in ethanol solution (75 %, v/v) for 1 min, as well as that of sodium hypochlorite (0.5 %, w/v) for 5–10 min (depending on different tissue types), and thirdly rinsed in sterile distilled water for at least three times. Plant tissue segments of 1 cm length were placed on a Petri dish with potato dextrose agar (PDA), supplemented with ampicillin (100 lg/mL) and streptomycin sulphate (60 lg/mL) to avert bacterial growth, and then incubated at 25 °C avoiding light. Surface-sterilized plant tissues without cutting were incubated meantime as a control. Fungal tips were transferred to sub-culture on PDA as soon as they emerged until pure colonies were formed. Finally they were stored in two distinct methods, that is, on PDA slants covered with sterile liquid paraffin at room temperature and in preservation liquid (15 % v/v glycerol, 10 g/L glucose, 1 g/L yeast extract and 1 g/L casein hydrolysate) at -80 °C (Zhang et al. 2010). Identification of strain zjwcf069 Strain zjwcf069 was identified using both morphological characters and phylogenetic data. For optimal growth, PDA and chestnut extract solid medium (10 %, w/v) were used. Vegetative and reproductive growth characteristics were obtained and compared to other known species. On the other hand, mycelium from pure culture was scraped and used to extract genomic DNA according to the quick method published earlier (Chi et al. 2009). To amplify internal transcribed spacer of ribosomal nucleotide sequence (ITS rDNA), primers ITS1 (50 -TCCGTAGGTGAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 ) (Mitchell et al. 1994) were used. Reaction of 50 lL consisted of 19 polymerase chain reaction (PCR) buffer, 2.5 mM Mg2?, 100 lM dNTPs, 0.5 lM each primer, 10 ng extracted DNA and 2 U Taq polymerase. PCR thermocycling steps were 94 °C for 3 min, followed by 35 cycles of 94 °C for 40 s, 56 °C for 50 s, and 72 °C for
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60 s, and the final extension 72 °C for 10 min. PCR products were purified via a DNA gel extraction kit (Axygen Incorporation, China) and then sequenced on an ABI 3730 sequencer (Applied Bio-systems, USA). Corresponding sequence was analyzed through Basic Local Alignment Search Tool (BLAST). Neighbor-joining (NJ) phylogenetic tree was constructed in MEGA 4, using 1,000 bootstrap replicates (Tamura et al. 2007). ITS-rDNA of zjwcf069 was submitted to GenBank and the accession no. is JN387907. Fermentation and metabolite isolation Zjwcf069 was cultured in 1,000-mL Erlenmeyer flasks, each containing 500 mL potato dextrose broth (PDB, 10 L in total), and incubated on a rotary shaker (180 rpm; 25 °C) avoiding light for 15 d. After filtration, fermentation broth was extracted using ethyl acetate (EtOAc) of equal volume for three times, and then evaporated to dryness (2.3 g). The extract was mixed with silica gel, dried at 50 °C, and subsequently loaded on a silica gel column (4,091,000 mm) with 210 g silica gel (200–300 mesh). For optimal separation, column was eluted with mixture liquid of petroleum ether: ethyl acetate (from 9:1 to 1:3, v/v). Components were visualized under UV254/366 and by spraying with vanillin:H2SO4:MeOH (1:3:50, v/v/v) and then heating at 110 °C. Totally 5 fractions were collected and labeled F1 to F5 accordingly, among which F1, F2 and F5 showed antifungal activities against 9 pathogenic fungi. So these fractions were purified thereafter. The bioactive fraction F1 (653 mg) were separated further by column chromatography (CC) over SephadexLH-20 through a mixture of methanol and chloroform (1:1 v/v), to afford compound 1 (584 mg). Similarly, F2 (63 mg) were subject to CC to obtain white crystal compound 2 (39 mg). F4 (595 mg) was recrystallized to give compound 3 (518 mg). F5 (231 mg) went through purification as F1 to afford compound 4 (18.7 mg) (Zhao et al. 2012). Structure analysis Mass and nuclear magnetic resonance (NMR) spectrometries were carried out to figure out the chemical structures of four metabolites. Mass spectra were obtained from a Bruker Esquire 3000plus mass spectrometer. 1H, 13C NMRs were recorded on a Bruker AMX-500 (500 MHz) NMR spectrometer taking TMS as internal standard. Chemical shifts expressed in d (ppm) and coupling constant J in Hz. Melting points were achieved on a Beijing X4 micro melting point apparatus without correction. Silica gel (200–300 mesh) for CC was purchased from Qingdao Marine Chemical Factory (Qingdao, China). So was SephadexLH-20 from Pharmacia Biotech (Sweden).
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Antifungal activity assay Compound 1 was dissolved in sterile distilled water and tested for fungistatic activity in vitro against 9 phytopathogenic fungi: Botrytis cinerea, Pythium ultimum, Fusarium oxysporium f. sp. Cucumerinum, Colletotrichum orbiculare, Verticillium dahliae, Pyricularia oryzae, Pestalotia diospyri, Sclerotinia sclerotiorum and Fulvia fulva. During the process, a series of Petri dishes containing metabolite solutions of certain concentration (1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 lg/mL) were diluted with melted PDA medium. Solution was replaced by sterile distilled water as control. Cyclopaldic acid was chosen as positive control (Wang et al. 2012a, b). Then one colony disc (5 mm in diameter) of test fungi was placed at the center of each plate. Each treatment was carried out for three times as replicates. These plates were all incubated in darkness at 25 °C. When colony in control setting reached edge, diameters of all growing colonies were measured and plotted against the respective metabolite concentration. Median effect concentration (EC50) is defined as the concentration of test compound that allows test organism to grow at 50 % of its rate under the same condition in absence of test compound (Wang et al. 2012a, b).
Results In this study, 16 isolates were isolated from twigs of F. viridissima. Among them, zjwcf069 went through further analysis. Both vegetative and reproductive characters were induced for identification (Papagianni 2004). Colony of zjwcf069 on PDA reached a diameter of 4–6 cm after 10 days growth. While on chestnut extract solid medium, colony was suborbicular with lots of aerial hyphae, which was smooth at the back, when incubated at 25 °C with alternating light and darkness (12/12 h). Hyphae were white at first, and then tinted into gray and light brown, on which water drops colored light yellow could been seen (Fig. 1a). Other morphological characteristics included:
black pycnidia, jacinth conidium groups, single-celled conidia which were colorless and cylindrical with both ends blunt or one end blunt and the other pointed. Size of conidia was 11.25–17.00 lm in length and 4.0–5.5 lm in width (Fig. 1b, c). Morphological identification has long been the basis and tradition, which is beyond doubt of great importance. ITS rDNA of zjwcf069 was also sequenced to assist precise identification (Sugita and Nishikawa 2003). Alignment with those from Genbank database resulted in several closely related sequences and along with them phylogenetic analysis was conducted. Corresponding NJ tree showed clearly that zjwcf069 fell into the group of Pezicula spp. with quite strong support (Fig. 2). Thus combined with morphology, zjwcf069 was defined as Pezicula sp. ultimately. Altogether 4 compounds were obtained via metabolite isolation from fermentation broth of zjwcf069. Thereafter structures of them were illustrated as followings (Fig. 3). Compound 1 Mellein, white crystal, molecular formula C10H10O3; m.p. 56 °C; [a]25 D-102.5(c1.0, CHCl3); ESI–MS (m/z): 178 [M ? H]?; 1H NMR (500 MHz, CDCl3 J in Hz) d: 1.53 (3H, d, J = 6.3 Hz, –CH3), 2.93 (2H, dd, J = 6.9, 1.0 Hz, H-4); 4.74 (1H, tq, J = 6.9, 6.3 Hz, H-3); 6.70 (1H, dd, J = 7.4, 1.0 Hz, H-5); 6.89 (1H, d, J = 8.4 Hz, H-7); 7.41 (1H, dd, J = 8.4, 7.4 Hz, H-6); 11.03 (1H, s, –OH). 13C NMR (125 MHz, in CDCl3) d: 20.7 (–CH3), 34.6 (C-4), 76.1 (C-3), 108.2 (C-9), 116.2 (C-7), 117.9 (C-5), 136.1 (C6), 139.2 (C-10), 162.1 (C-8), 169.9 (C-1). Both 1H NMR and 13C NMR data turned out to be consistent with those reported earlier (Islam et al. 2007). Compound 2 Ramulosin, white crystal, molecular formula C10H14O3; m.p. 118–119 °C; [a]25 D ?19 (c0.50, EtOH); ESI–MS (m/z): 183 [M ? H]?; 1H NMR (500 MHz, CDCl3, J in
Fig. 1 Colony and conidia of zjwcf069 on chestnut extract solid medium. a Colony of zjwcf069 after 10 days. Bar equals to 1 cm. b Conidia of zjwcf069. c Conidiophores of zjwcf069 after 3 weeks. Bars in both b and c represent 10 lm
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World J Microbiol Biotechnol Fig. 2 Phylogenetic NJ tree inferred from ITS rDNA sequences Bar represents 0.005 substitutions per site
Table 1 EC50s of mellein (1) and cyclopaldic acid against 9 phytopathogens Phytopathogens Botrytis cinerea
Hz) d: 1.11 (1H, m, H-5a), 1.22 (brdd, J = 13.5,11.5, H-4a), 1.33 (d, J = 6.3, 3H, –CH3), 1.45–1.69 (m, 2H, H-6), 1.77–1.90 (m, 1H, H-5b), 1.86 (ddd, 1H, J = 13.5, 2.4, 3.9, H-4b), 2.31 (m, 2H, H-7), 2.44 (ddd, J = 12.2, 3.9, 1.9, 1H, H-4a), 4.39 (ddq, J = 11.5, 2.4, 6.3, 1H, H-3), 9.33 (s, –OH); I3C NMR (125 MHz, CDCl3) d: 20.8 (C-7), 21.7 (3-CH3), 29.0 (C-6), 29.5 (C-5), 32.9 (C-4a), 37.4 (C4), 76.5 (C-3), 96.8 (C-8a), 171.8 (C-8), 174.7 (C-1). Both 1 H NMR and 13C NMR data turned out to be consistent with those reported earlier (Stierle et al. 1998). Compound 3 Butanedioic acid, colorless crystal, molecular formula C4H6O4; m.p. 187.6–188.3 °C; ESI–MS (m/z): 117 [M ? H]?; 1H NMR (500 MHZ, DMSO-d6, J in Hz) d: 2.42 (2H, s, –CH2), 12.16 (2H, s, –COOH); 13C NMR (125 MHZ, DMSO-d6) d: 28.83 (C-2,3), 173.68 (C-1,4). Both 1H NMR and 13C NMR data turned out to be consistent with those reported earlier (SDBS Information 2013).
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Cyclopaldic acid (lg/mL)
48.63
115.36
Colletotrichum orbiculare
150.90
355.71
Verticillium dahliae
163.37
216.83
Fusarium oxysporium f. sp. cucumerinum
159.09
228.15
Pyricularia oryzae
118.83
116.68
Pestalotia diospyri
161.04
[1,000
Pythium ultimum
125.36
124.14
Sclerotinia sclerotiorum
205.01
60.75
45.98
40.01
Fulvia fulva
Fig. 3 Structures of 4 compounds
Compound 1 (lg/mL)
Compound 4 4-Methoxy-1(3H)-isobenzofuranone, white crystal, molecular formula C9H8O3; m.p. 128.4–129.3 °C; ESI–MS (m/ z): 165 [M ? H]?; 1H NMR (500 MHZ, DMSO-d6) d: 3.91 (s, 3H, OCH3), 5.36 (s, 2H, H8), 7.41 (m, 3H, Ar–H); 13C NMR(125 MHZ, DMSO-d6) d: 55.75 (C–OCH3), 67.98 (C3), 115.86–153.98 (C–Ar), 170.51 (C-1). Both 1H NMR and 13C NMR data turned out to be consistent with the literature earlier (Egan et al. 2011). According to the result of antifungal bioassay, mellein (1) expressed antagonism against these 9 pathogens at diverse levels (Table 1). Among them, B. cinerea and F. fulva were the most affected fungi (EC50 \ 50 lg/mL). Compared to positive control, compound 1 has antifungal property. Compounds 2 and 4 could not afford the bioassay because of their limited yield.
Discussion Quantitive molecular phylogenetics has been increasingly popularized in recent researches accounting fungal
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endophyte identification as traditional morphology is unstable and largely dependent on circumstances. Here in this study, high quality sequences, especially those from type species, were chosen for solid phylogenetic conclusions (Ko et al. 2011). For further taxonomic location, more molecular information is needed, like multiple-locus phylogenetics (Wang et al. 2014). This research represents the very first time to isolate Pezicula endophyte from F. viridissima which is a typical herbal medicine with bioactivities like antiphlogosi (Lee et al. 2010), antiasthma (Lee et al. 2011) and vasodilation (Iizuka et al. 2009). So far, reports on endophytes from this particular plant are limited. Up to now, several bioactive compounds have been isolated from Pezicula spp. The antibiotic equisetin derivative (Sugie et al. 2002), furofurandiones from Pezicula livida (Krohn et al. 1994) and 5 fungicidal and herbicidal metabolites reported in 1995 (Schulz et al. 1995) represent great potential in Pezicula fungi as a natural product source with diverse activities. Also cryptocandin and its related bioactive agents which have out-standing antifungal activity against both human and plant pathogens were reported to be produced by Cryptosporiopsis quercina, the imperfect stage of Pezicula cinnamomea (Strobel and Daisy 2003). 4-Methoxy-1(3H)-isobenzofuranone (4-methoxyphthalide, compound 4) participates in production of bioactive lactone derivatives (Soucy et al. 1987) as well as antitumor compound cervicarcin Marumo et al. (1964). Currently 4-methoxyphthalide (4) is artificially synthesized (Soucy 1987). However this paper represents the first one to report it as a natural metabolite. Ramulosin (2) first isolated from metabolites of a saprophytic fungus Pestalotia ramulosa (Benjamin and Stodola 1960), has potential as herbicide and fungicide, most of which is synthesized (Islam et al. 2007). However yield of either compound 4 or 2 in this research was not enough for bioassay. Though mellein (1) as a natural product was isolated from endophytic Pezicula species before (Schulz et al. 1995), the quantitive evidences for its antibiotic activity were not clearly demonstrated. Herein 9 representative phytopathogens were chosen and exact EC50 was measured as a solid indication of antifungal activity for the first time ever (Table 1). Mellein (1) expressed similar antifungal activities with cyclopaldic acid. Butanedioic acid (3) has long been an important material for organic synthesis. In this research, 16 strains were isolated from twigs of F. viridissima. Among them, Pezicula endophyte zjwcf069 went through further chemical analysis. Totally 4 compounds were achieved from fermentation broth, that is, mellein (1), ramulosin (2), butanedioic acid (3), and 4-methoxy-1(3H)-isobenzofuranone (4). Compound 4 was obtained as a natural metabolite for the first time. Corresponding bioassay was carried out and compound 1 showed
significant antagonism against B. cinerea and F. fulva (EC50 \ 50 lg/mL). Again endophyte was proven to be a promising source of natural bioactive metabolites. Acknowledgments This research was financially supported by the Zhejiang Provincial National Natural Science Foundation of China (No. LQ13H280005) and Open Research Fund Program of the Laboratory of Hangzhou Normal University (No. 2013040).
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