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The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose

ABSTRACT

Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46 µmol mL-1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02 µmol mL-1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.

Keywords:
2-Hexylidene-3-methylbutanedioic acid; Amazon; Anthracnose; Plant pathogen

Introduction

Anthracnose is one of the most serious plant diseases affecting different cultures worldwide. This disease is usually caused by filamentous fungi in the genus Colletotrichum or Gloeosporium, and these pathogens cause great losses and hence reduction in the quality and quantity of fruits and vegetables.11 Bailey JA, Jeger MJ, eds. Colletotrichum: Biology, Pathology and Control. Wallingford: CAB International; 1992.44 O’Connell RJ, Thon MR, Hacquard S, et al. Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses. Genetics. 2012;44:1060-1065. Anthracnose is considered the main disease of guarana (Paullinia cupana ‘sorbilis’ Mart., Sapindaceae). The pathogen infects leaves and stems, causing deformation and necrotic lesions.55 Erickson H, Correa MPF, Escobar JR. Guaraná (Paullinia cupana) as a commercial crop in Brazilian Amazonia. Econ Bot. 1984;38:273-286.,66 Bentes JLS, Barreto RW. Taxonomic reevaluation of Colletotrichum guaranicola Albuq. causal agent of guarana anthracnose. Acta Amaz. 2004;34:129-131. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides.77 Bentes JLS, Matsuoka K. Histologia da interação Colletotrichum guaranicola e Paullinia cupana var. sorbilis em clones resistente e suscetível. Fitopatol Bras. 2002;27:71-77. However, even with the use of some methods of controlling these cultures, they are not are free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment.88 Ishi H. Impact of fungicide resistance in plant pathogens on crop disease control and agricultural environment. Jarq Jpn Agric Res Q. 2006;40:205-211.

Therefore, the search for natural products with antifungal activity of agronomic relevance is an important alternative for the control of diseases such as anthracnose.99 Slusarenko AJ, Patel A, Portz D. Control of plant diseases by natural products: allicin from garlic as a case study. Eur J Plant Pathol. 2008;121:313-322. In the search for bioactive compounds, only a small fraction of the estimated fungal biodiversity has been chemically investigated,1010 Aly AH, Debbab A, Proksch P. Fifty years of drug discovery from fungi. Fungal Divers. 2011;50:3-19. and only a small number of these have already been cultured and selected for drug production.1111 Hanson JR. Chemistry of Fungi. Cambridge: The Royal Society of Chemistry; 2008.,1212 Nisa H, Kamili AN, Nawchoo IA, Shafi S, Shameem N, Bandh SA. Fungal endophytes as prolific source of phytochemicals and other bioactive natural products: a review. Microb Pathog. 2015;82:50-59. Fungi have special interest because they are known to produce several secondary metabolites with a wide range of applications.1313 Abraham W-R. Bioactive sesquiterpenes produced by fungi: are they useful for humans as well?. Cur Med Chem. 2001;8:583-606. The fungi of the genus Xylaria stand out in the production of secondary metabolites, mainly of the class of cytochalasins, isocumarins, lactones, sesquiterpenos, triterpenos and xanthones, and with the most diverse biological activities, such as anticancer, antioxidant, antimicrobial, antiinflammatory, antiviral, inhibiting HIV-1 protease, antibiotic and antitumor,1414 Singh SB, Zink D, Polishook J, et al. Structure and absolute stereochemistry of HIV-1 integrase inhibitor integric acid. A novel eremophilane sesquiterpenoid produced by a Xylaria sp. Tetrahedron Lett. 1999;40:8775-8779.,1515 Espada A, Rivera-Sagredo A, De La Fuente JM, Hueso-Rodriguez JA, Elson SW. New cytochalasins from the fungus Xylaria hypoxylon. Tetrahedron. 1997;53:6485-6492. among others.

In a previous study, a screening was done with the fractions of 16 endophytic fungi isolated from Guarana plant from Amazonia, and two Xylaria spp. were selected because its activity against the phytopathogen Colletotrichum gloeosporioides. Thus, this research aimed to evaluate the antifungal potential of secondary metabolites produced by this two endophytic Xylaria spp. isolated from guarana (P. cupana).

Experimental

General procedures

Optical rotations were taken on a Polartronic H Schmidt & Haensch digital polarimeter (1 dm cell) at 23 ºC. The 1H nuclear magnetic resonance (NMR) was recorded on a Bruker 14.1 Tesla, AVANCE III model spectrometer (operating at 600.13 MHz for hydrogen frequency) with crioprobe™. The chemical shifts are given on a δ (ppm) scale and referenced to tetramethylsilane (TMS). High-resolution electrospray ionization mass spectrometry (HR-ESI MS) was acquired by direct infusion on a Q-TOF Maxis Impact in the following conditions: capillary voltage, 4.50 kV; operating in electrospray positive mode; The ion source was set to a nebulizer pressure of 0.3 bar, a dry gas flow rate of 4 L min-1, and a dry temperature of 180 ºC; detection range: 100–700 Da with total ion count extracting acquisition. Data acquisition was performed using a Bruker Compass Data Analysis 4.2.

High-performance liquid chromatography (HPLC) was conducted using a Agilent 1100 Series UV/Vis with a quaternary pump, coupled to a UV detector MWD (Multiple Wavelength Detector), using a reversed phase column C18 (250 mm × 4.60 mm, 5 µm; Phenomenex Kinetex), with a mobile phase H2O/MeOH (80:20, v/v), flow rate 1 mL min-1 and λ = 254 nm. HPLC-grade solvents were utilized. Solid-phase extraction was carried out using silica gel cartridges of different dimensions (Phenomenex). Gel permeation chromatography was performed in a glass column filled with Sephadex LH-20 (Pharmacia). Silica gel254 (Macherey-Nagel) were used for thin-layer chromatography (TLC). Spots were detected under UV light (254 and 365 nm).

Fungal isolation

The endophytic fungi (isolates 249 and 214) were isolated from leaves of guarana plant (P. cupana) with anthracnose symptoms (necrotic lesions), collected at Manaus-AM (03º22'5), at the Experimental Farm of the Federal University of Amazonas (UFAM), and Maués-AM (57º42'0), at Fazenda Santa Helena (Ambev – American Beverage Company), in November 2010. The collected samples were leaves and branches of a total of 20 adult plants (10 of each locality). Complete intact leaves were rinsed with running water and then with distilled water. The cleaned leaves were sterilized by consecutive washes in 75% EtOH (1 min), 3% NaOCl (3 min) and 75% EtOH (30 s), and they were rinsed with sterile distilled H2O two times.1616 Pereira JO, Azevedo JL, Petrini O. Endophytic fungi of Stylosanthes: a first report. Mycologia. 1993;85:362-364.,1717 Araújo WL, Maccheroni W, Aguilar-Vildoso CI, Barroso PAV, Saridakis HO, Azevedo JL. Variability and interactions between endophytic bacteria and fungi isolated from leaf tissues of citrus rootstocks. Can J Microbiol. 2001;47:229-236. The sterilized material was cut into 8 × 12 mm and 3–4 pieces were deposited on a Petri dish containing potato dextrose agar (PDA, Kasvi™, Brazil), chloramphenicol and streptomycin (100 µg mL-1 of each) to prevent bacterial growth. Plates were incubated at 27 ºC. Petri dishes were observed daily, and the individual hypha tips of the emerging colonies were re-inoculated in new PDA plates until pure cultures were obtained. Pure cultures of the endophytes (isolates 249 and 214) were preserved by the Castellani method.1818 Castellani A. Viability of some pathogenic fungi in distilled water. Am J Trop Med Hyg. 1939;42:225-226.

Fungal identification

Pure culture of isolates 249 and 214 were first grown in malt agar 2% (in g L-1: 20 malt extract, and 15 agar, Acumedia, Neogen™) at 25 ºC for seven days. Then, fresh mycelia were harvested from and used for DNA extraction based on the CTAB method (cetyltrimethyl ammonium bromide) used in Montoya et al.1919 Montoya QV, Meirelles LA, Chaverri P, Rodrigues A. Unraveling Trichoderma species in the attine ant environment: description of three new taxa. Antonie van Leeuwenhoek. 2016;109:633-651. We amplified the ITS barcoding region using ITS4 and ITS5 primers. Amplicons were cleaned up and submitted to cycle-sequencing reactions using BigDye Terminator v. 3.1 Kit (Thermo Fischer Scientific). Bidirectional sequences were generated in ABI 3530 (Thermo Fischer Scientific) using the same primer pair.

Forward and reverse sequences were assembled in contigs in Bioedit.2020 Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 1999;41:95-98. Then, contigs were compared with homologous sequences deposited in the NCBI-Genbank.

To refine the taxonomic assignment of isolates 249 and 214, we carried out a phylogenetic analysis in MEGA v.6.0.2121 Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725-2729. For this analysis, we retrieved sequences from closet relative taxa deposited in the database. Sequences were aligned in MAFFT,2222 Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772-780. and this dataset was used to infer a phylogenetic tree under the neighbor-joining algorithm and the Kimura 2-parameters as the nucleotide substitution model. Branch support was calculated using 1000 bootstrap pseudoreplicates.

Isolation of the active compounds

The endophytic fungi 249 and 214 were separately fermented in malt extract broth (in g L-1: 20 malt extract, Kasvi™, Brazil) at 27 ºC under agitation (150 rpm) for five days (5 L; 20 mL × 250 mL batches in 500 mL Erlenmeyer flasks, for each fungal strain). The mycelium was separated from the liquid extract using vacuum filtration (filter Macherey Nagel™, MN 640 m, 7 cm) and the liquid extract was extracted three times with EtOAc (100 mL of the liquid extract to 150 mL of EtOAc). Evaporation of the solvent from the extract in vacuo gave an EtOAc extract (306.4 mg for the strain 249 and 700.0 mg for the strain 214).

The EtOAc extract from the strain 249 was chromatographed on a Sep-Pak silica gel column (5 g, Waters™). The sample was solubilized in a mixture of hexane and CH2Cl2 (100 µL) and applied to the top of the column, which was eluting successively hexane/CH2Cl2/MeOH, starting with 100 mL of 100% hexane and increasing the gradient of CH2Cl2 until 100% CH2Cl2, and then increasing the gradient of MeOH until 100%. Based on the TLC monitoring, all the fractions collected were combined in six main fractions (a–f). Bioactive fraction d (221.4 mg) was subjected to further CC fractionation over silica gel (5 g) eluted with hexane/CH2Cl2/MeOH to give five fractions (d1–d5). Bioactive fraction d1 (105 mg) was fractioned by gel filtration over Sephadex LH-20 column eluted with 100% MeOH to give five fractions (d1a–d1e), collected based on the retention time. Bioactive fraction d1b (44 mg) was purified on reverse phase HPLC using an isocratic elution H2O/MeOH (80:20) during 25 min to give four fractions (d1b1–d1b4), being the active d1b4 (retention time: 11.22 min) with 6.8 mg.

The EtOAc extract from the strain 214 was chromatographed on a Sep-Pak silica gel column (10 g, Waters®) eluting successively hexane/CH2Cl2/EtOAc/MeOH to give six fractions: 50 mL hexane: 50 mL CH2Cl2 (fraction a), 100 mL CH2Cl2 (fraction b), 50 mL CH2Cl2: 50 mL EtOAc (fraction c), 100 mL EtOAc (fraction d), 50 mL EtOAc: 50 mL MeOH (fraction e) and 100 mL MeOH (fraction f). Bioactive fraction d (375 mg), was subjected to further CC fractionation over silica gel (5 g) the same way described above to give six fractions (d1–d6). Bioactive fraction d3 (50 mL CH2Cl2: 50 mL EtOAc, 171 mg) was purified on reverse phase HPLC during 15 min using an isocratic elution H2O/MeOH (80:20) to give three fractions (d3a–d3c), being the active d3c with retention time 5.92 min (9.6 mg).

Antifungal bioassays

Disk-diffusion assay

The disk-diffusion assay (paper disk method)2323 Heatley NG. A method for the assay of penicillin. Biochem J. 1944;38:61-65. was used to determine the antifungal activity during the separation and purification procedures. Each fraction obtained from different chromatographs was solubilized in MeOH and applied (10 µL) to 6 mm diameter sterile paper disks (with 2 mg of each EtOAc extract, 1 mg of each fraction or 0.5 mg of each pure compound) and placed at one edge of the 60 mm PDA Petri dish plate (Kasvi™, Brazil). An inoculum of the pathogen (a 7 mm diameter disc removed from a new peaked plate) was added to another edge of the PDA plate. The clear inhibition zones of mycelial growth around the paper disks were measured after incubation for 7 days at 27 ºC. After this period, the inhibition percentages were calculated compared to the control using the analysis program Image J (Image Processing and Analysis in Java). This experiment was conducted with one repeat. For the negative control, 10 µL of MeOH was applied in a sterile paper disk.

In order to know the fungicidal or fungistatic action of the isolated compounds, a small part of the fungi next to the paper disk with each compound was transferred to a new plate. The action was characterized by fungicide if the fungus does not grow in the new plate, and fungistatic if growth. Colletotrichum gloeosporioides used in this study was ceded by the UFAM, Amazonas-AM, Brazil. This phytopatogenic fungal strain was isolated from Guarana leaves infected with anthracnose and is deposited in the Central de Recursos Microbianos da UNESP (CRM-UNESP) under the code CRM 1352.

Microdilution assays

Broth microdilution techniques were performed in 96-well microplates according to the guidelines of the National Committee for Clinical Laboratory Standards2424 NCCLS. National Committee for Clinical Laboratory Standards, document M27-A2; 2002. with modifications. For the assay, compound test wells (CTWs) were prepared with stock solutions of each compound in DMSO (250 mg mL-1), diluted with malt 2% medium (in g L-1: 20 malt extract, Kasvi, Brazil) to final of 250, 125, 62.5, 31.25, 15.6, 7.81, 3.90, 1.95, 0.77, 0.48 and 0.24 µg per well. An inoculum suspension of C. gloeosporioides spores was obtained from a 14 days-old culture and was added into liquid malt 2% medium (105 spores mL-1, 60 µL) to each well (final volume in the well = 100 µL, completed with liquid malt 2% medium). Spores were obtained through the fungal culture on solid malt 2% medium after 14 days. The plate was scraped, and sterile distilled water was added. This solution was filtered in sterile glass wool to separate the spores, and the counting of the spores was done in Neubauer chamber. A growth control well (containing medium, inoculum, and the highest concentration of DMSO used in a CTW, but compound-free) was included. Microplates were incubated in B.O.D. at 28 ºC and were read in a TECAN microplate reader, SUNRISE model, operated by the software Magellan v7.1, at 620 nm, in intervals of 12 h, for 144 h. The active ingredients of commercial fungicides Captan™ (N-(trichloromethylthio)ciclohex-4-ene-1,2-dicarboximide) and Score™ (difenoconazole, cis-trans-3-chloro-4-[4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl 4-chlorophenyl ether) were used as positive control in the same concentrations of the isolated compounds. The MICs were determined spectrophotometrically after the above mentioned incubation periods, according to the NCCLS guidelines,2424 NCCLS. National Committee for Clinical Laboratory Standards, document M27-A2; 2002. with modifications. For commercial fungicides and compound, the MICs were determined as the lowest concentration showing absence or equal to the initial growth compared with the growth (until 12 h) in the drug-free well.

Results

Isolation and identification of the endophytes

The strains 249 and 214 were isolated from the guarana leaves in the Amazon in two different places. After purification of the strains and based on the phylogenetic analysis, the sequences of isolates 249 and 214 clustered with sequences from other Xylaria species with high bootstrap support. Thus, here we tentatively identified these fungi as Xylaria sp. (Fig. 1). The fungal strains are deposited at the Central de Recursos Microbianos da UNESP (CRM-UNESP) under codes CRM 1915 (Xylaria sp. 214) and CRM 1916 (Xylaria sp. 249).

Fig. 1
Phylogenetic tree of Xylaria spp. 249 and 214.

Bioguided isolation of the active compound

The EtOAc extract (306 mg) from the strain 249 showed 60.39% inhibition to C. gloeosporioides. This extract was fractionated by chromatography on a silica-gel column, permeation chromatography on Sephadex LH-20 gel and reverse phase HPLC. The active compound showed 51.33% activity (code d1b4, 6.8 mg).

The EtOAc extract (700 mg) from the strain 214 showed 30.76% inhibition to C. gloeosporioides. This extract was fractionated by chromatography on a silica-gel column and reverse phase HPLC. The active compound (code d3c, 9.6 mg) showed 38.76% inhibition to C. gloeosporioides.

These compounds were identified by analysis of spectroscopic data (MS and NMR) and compared with compounds in the Dictionary of Natural Products database.2727 Mangaleswaran S, Argade NP. An efficient synthesis of (±)-piliformic acid. J Chem Soc Perkin Trans 1. 2000;19:3290-3291.

Identification of the bioactive compounds

Compound d1b4

The compound d1b4 (6.8 mg) presents an UVmax at 227.2 nm. In the high-resolution mass spectrum, molecular ions were observed at m/z 215.1268 [M+H]+, m/z 237.1103 [M+Na]+ and m/z 451.2304 [2M+Na]+, which was appropriate for the molecular formula C11H18O4 (214.1189 Da). With the data obtained in the 1H NMR spectrum, it was possible to correlate the hydrogens shifts (δ H) of d1b4 with the compound piliformic acid.2626 Chesters NCJE, O’Hagan D. Biosynthesis of the fungal metabolite, piliformic acid(2-hexylidene-3-methylsuccinic acid). J Chem Soc Perkin Trans 1. 1997;:827-834.,2727 Mangaleswaran S, Argade NP. An efficient synthesis of (±)-piliformic acid. J Chem Soc Perkin Trans 1. 2000;19:3290-3291.

The experiment of optical rotation provided [α]D 23 -0.0375 (c, 1 mg mL-1 in MeOH), allowing identify it as (-)-piliformic acid. Piliformic acid (Fig. 2-A), exists in levo, dextro and racemic form, although the absolute stereochemistry has not been determined.2727 Mangaleswaran S, Argade NP. An efficient synthesis of (±)-piliformic acid. J Chem Soc Perkin Trans 1. 2000;19:3290-3291.

Fig. 2
Structures of the piliformic acid (C11H18O4) (A) and cytochalasin D (C30H37NO6) (B) produced by Xylaria sp. 249 and Xylaria sp. 214, respectively, isolated from guarana plant.

Compound d3c

The compound d3c presents an UVmax at 217 and 286 nm. In the high-resolution mass spectrum, molecular ions were observed at m/z 508.268 [M+H]+, m/z 530.251 [M+Na]+, m/z 1037.513 [2M+Na]+, m/z 430.236 [M-OCOCH3 H2O]+, m/z 1015.5310 [2M+H]+, which was appropriate for the molecular formula C30H37NO6 (507.260 Da). With the data obtained in the 1H NMR spectrum, it was possible to correlate the hydrogens shifts (δ H) of d3c with the compound cytochalasin D.2828 Maccotta A, Valensin G, Gaggelli N, Gaggelli E. The conformation of cytochalasin D in DMSO solution from 1H and 13C NMR relaxation rates. J Chem Soc Perkin Trans 2. 1993;2:729-732.

The experiment of optical rotation provided [α]D 23 0.0192 (c, 1 mg mL-1 in MeOH), consistent with the literature, allowing identify it as (+)-cytochalasin D (Fig. 2-B).

Evaluation of the active compounds

The isolated compounds piliformic acid and cytochalasin D have fungistatic activity against C. gloeosporioides. Piliformic acid inhibited 51.33% of the mycelial growth of C. gloeosporioides by the disk diffusion method, and cytochalasin D 38.76%, both in a concentration of 500 µg disk-1. In the microdilution assay (Fig. 3) all the compounds showed antifungal activity. Captan and difenoconazole were included as positive controls (MIC of 16.63 and 0.02 µmol mL-1, respectively). Piliformic acid and cytochalasin D were active against C. gloeosporioides, with a MIC of 2.92 and 2.46 µmol mL-1, respectively. Both have a higher MIC than the active ingredient difenoconazole, but a lower MIC than captan. Commercial fungicides were chosen because they are from different chemical classes and presented distinct mechanisms of action.

Fig. 3
Data obtained in microdilution assay of the isolated compound piliformic acid (A) and cytochalasin D (B) against spores of C. gloeosporioides.

Discussion

The piliformic acid (CAS 98985-75-2), also known as 2-hexylidene-3-methylbutanedioic acid, 2-cyclohexylidene-3-methylsuccinic acid or 3-Nonene-2,3-dicarboxylic acid2525 Dictionary of Natural Products Database; 2015. http://dnp.chemnetbase.com Accessed 12.12.15.
http://dnp.chemnetbase.com...
belongs to the chemical class of the polyketides.2929 Song F, Wu SH, Zhai YZ, Xuan QC, Wang T. Secondary metabolites from the genus Xylaria and their bioactivities. Chem Biodivers. 2014;11:673-694. The biosynthesis of piliformic acid has been studied. The metabolite can be retro-biosynthetically cleaved to generate a C8 and a C3 moiety, and the C8 unit is derived directly from octanoate that originates from a fatty acid synthase (FAS) rather than from a polyketide synthase (PKS).2626 Chesters NCJE, O’Hagan D. Biosynthesis of the fungal metabolite, piliformic acid(2-hexylidene-3-methylsuccinic acid). J Chem Soc Perkin Trans 1. 1997;:827-834. The synthesis was already studied by Mangaleswaran and Argade,2727 Mangaleswaran S, Argade NP. An efficient synthesis of (±)-piliformic acid. J Chem Soc Perkin Trans 1. 2000;19:3290-3291. and it was an easy, four-step.

Piliformic acid was identified the first time by Anderson et al.3030 Anderson JR, Edwards RL, Whalley AJS. Metabolites of the higher fungi. Part 22. 2-Butyl-3-methylsuccinic acid and 2-hexylidene-3-methylsuccinic acid from xylariaceous fungi. J Chem Soc Perkin Trans 1. 1985;:1481-1485. as a metabolite produced by fungi of the family Xylariaceae: Hypoxylon (H. deustum), Poronia (P. piliformis) and Xylaria (X. polymorpha, X. longipes, X. Mali and X. hypoxylon).2626 Chesters NCJE, O’Hagan D. Biosynthesis of the fungal metabolite, piliformic acid(2-hexylidene-3-methylsuccinic acid). J Chem Soc Perkin Trans 1. 1997;:827-834. This compound is commonly found in endophytic fungi belonging to the Xylariaceae, especially in the genus Xylaria. It was already isolated from the marine-derived fungus Xylaria sp. PSU-F1003131 Rukachaisirikul V, Khamthong N, Sukpondma Y, et al. An [11]cytochalasin derivative from the marine-derived fungus Xylaria sp. PSU-F100. Chem Pharm Bull. 2009;57:1409-1411. and also from an endophytic Xylaria sp. of Pinus strobus.3232 Richardson SN, Walker AK, Nsiama TK, et al. Griseofulvin-producing Xylaria endophytes of Pinus strobus and Vaccinium angustifolium: evidence for a conifer-understory species endophyte ecology. Fungal Ecol. 2014;11:107-113. This metabolite is also produced by others fungi, as several endophytic fungi isolated from the mangrove plant Avicennia marina3333 Zhu F, Chen X, Yuan Y, Huang M, Sun H, Xiang W. The chemical investigations of the mangrove plant Avicennia marina and its endophytes. The Open Nat Prod J. 2009;2:24-32. and from an endophytic Aspergillus allahabadii BCC453353434 Sadorn C, Saepua S, Boonyuen N, et al. Allahabadolactones A and B from the endophytic fungus, Aspergillus allahabadii BCC45335. Tetrahedron. 2016;72:489-495. among others.

The cytochalasin D belongs to the chemical class of the N-containing compounds and was first reported by Aldridge and Turner,3535 Aldridge DC, Turner WB. Structures of cytochalasins C and D. J Chem Soc C. 1969;6:923-928. isolated from the fungus Metarhizium anisopliae. Subsequently this compound was isolated from other fungi, such as Hypoxylon terricola3636 Edwards RL, Maitland DJ, Whalley AJS. Metabolites of the higher fungi. Part 24. Cytochalasin N, O, P, Q, and R. New cytochalasins from the fungus Hypoxylon terricola Mill. J Chem Soc Perkin Trans 1. 1989;1:57-65. and Engleromyces goetzii.3737 Jikai L, Jianwen T, Zejun D, Zhihui D, Xianghua W, Peigui L. Neoengleromycin, a novel compound from Engleromyces goetzii. Helv Chim Acta. 2002;85:1439-1442.

There are over 60 known cytochalasins, wich were isolated from different genera of fungi, such as Phoma sp., Daldinia sp. Chalara sp., Hypoxylon spp., among others. The production of cytochalasin D has been reported by fungi in the genus Xylaria, as X. hypoxylon,1515 Espada A, Rivera-Sagredo A, De La Fuente JM, Hueso-Rodriguez JA, Elson SW. New cytochalasins from the fungus Xylaria hypoxylon. Tetrahedron. 1997;53:6485-6492.X. mellisii,3838 Pittayakhajonwut P, Suvannakad R, Thienhirun S, Prabpai S, Kongsaeree P, Tanticharoen M. An anti-herpes simplex virus-type 1 agent from Xylaria mellisii (BCC 1005). Tetrahedron Lett. 2005;46:1341-1344.Xylaria sp.,3939 Chen Z, Huang H, Chen Y, et al. New cytochalasins from the merine-derived fungus Xylaria spp. SCSIO 156. Helv Chim Acta. 2011;94:1671-1676.X. carpophila,4040 Yin X, Feng T, Li ZH, et al. Chemical investigation on the cultures of the fungus Xylaria carpophila. Nat Prod Bioprospec. 2011;1:75-80.X. cubensis,4141 Klaiklay S, Rukachaisirikul V, Sukpondma Y, Phongpaichit S, Buatong J, Bussaban B. Metabolites from the mangrove-derived fungus Xylaria cubensis PSU-MA34. Arch Pharmacal Res. 2012;35:1127-1131.X. arbuscula4242 Amarala LS, Rodrigues-Filho E, Santos CA, De Abreu LM, Pfenning LH. An HPLC evaluation of cytochalasin D biosynthesis by Xylaria arbuscula cultivated in different media. Nat Prod Commun. 2014;9:1279-1282. and Xylaria sp. NC1214, a fungal endophyte of the moss Hypnum sp.4343 Wei H, Xu Y-M, Espinosa-Artiles P, et al. Sesquiterpenes and other constituents of Xylaria sp. NC1214, a fungal endophyte of the moss Hypnum spp. Phytochemistry. 2015;118:102-108.

In our work, we discovered the fungistatic activity of piliformic acid and cytochalasin D against the plant pathogen C. gloeosporioides. Studies suggest that fungistatic agents are unable to inhibiting the morphogenetic transformation and are more likely to block growth by budding.4444 Hawser S, Islam K. Comparisons of the effects of fungicidal and fungistatic antifungal agents on the morphogenetic transformation of Candida albicans. J Antimicrob Chemother. 1999;43:411-413.

In literature, few biological activities have been related for the piliformic acid. This compound has moderate cytotoxicity against cancer cells KB e BC-14545 Chinworrungsee M, Kittakoop P, Isaka M, Rungrod A, Tanticharoen M, Thebtaranonth Y. Antimalarial halorosellinic acid from the marine fungus Halorosellinia oceanica. Bioorg Med Chem Lett. 2001;11:1965-1969. and antifungal activity in yeast genera Nadsonia, Nematospora, Rhodotorula and Saccharomyces to a concentration >100 mg L-1 in tests performed by the disk diffusion method.4646 Schneider G, Anke H, Sterner O. Xylaramide, a new antifungal compound, and other secondary metabolites from Xylaria longipes. Z Nat C. 1996;51:802-806. Piliformic acid also showed mild antibacterial activity against standard Staphylococcus aureus ATCC 25923 and methicillin-resistant strain.4747 Rukachaisirikul V, Arunpanichlert J, Sukpondma Y, Phongpaichit S, Sakayaroj J. Metabolites from the endophytic fungi Botryosphaeria rhodina PSU-M35 and PSU-M114. Tetrahedron. 2009;65:10590-10595. The activity against the plant pathogens Cladosporium cladosporioides and C. sphaerospermum in bioautography assay (with 25 and 10 µg mL-1) was also described.4848 Cafêu MC, Silva GH, Teles HL, et al. Substâncias antifúngicas de Xylaria sp., um fungo endofítico isolado de Palicourea marcgravii (Rubiaceae). Quim Nova. 2005;28:991-995.

The biological activities of cytochalasins include glucose transport, inhibition of the actin polymerization, secretion of hormones, immunosuppressive activity, antibiotic, antiviral, herbicidal and antifungal activity.4949 Betina V, Mičeková D, Nemec P. Antimicrobial properties of cytochalasins and their alteration of fungal morphology. J Gen Microbiol. 1972;71:343-349.5353 Foissner I, Wasteneys GO. Wide-ranging effects of eight cytochalasins and latrunculin A and B on intracellular motility and actin filament reorganization in characean internodal cells. Plant Cell Physiol. 2007;48:585-597. The activity of cytochalasin D against five tumor cell lines have been tested, and the IC50 ranged from 0.22 to 1.44 µM.4343 Wei H, Xu Y-M, Espinosa-Artiles P, et al. Sesquiterpenes and other constituents of Xylaria sp. NC1214, a fungal endophyte of the moss Hypnum spp. Phytochemistry. 2015;118:102-108. In agronomy, as an antifungal agent, cytochalasin D has activity against the plant pathogen Botrytis cinerea, the causal agent of gray mold on plants, at a concentration of 25 µg disc-1, in a disk diffusion method bioassay.4949 Betina V, Mičeková D, Nemec P. Antimicrobial properties of cytochalasins and their alteration of fungal morphology. J Gen Microbiol. 1972;71:343-349. The cytochalasin D was also tested against C. cladosporioides and C. sphaerospermum, the causal agents of verrucose in plants, by the by bioautography method. It inhibited the plant pathogens in a concentration of 10 and 25 µg mL-1, respectively.5151 Prasain JK, Ueki M, Stefanowicz P, Osada H. Rapid screening and identification of cytochalasins by electrospray tandem mass spectrometry. J Mass Spectrom. 2002;37:283-291. In our study, cytochalasin D was active against C. gloeosporioides with a MIC of 2.46 µmol mL-1. In the literature, there are others compounds with activity against this plant pathogen, as preussomerin EG1 and its derivatives monoacetylpreussomerin and diacetylpreussomerin EG1, with MIC at 100 µg mL-1 (0.28 µmol mL-1) by dilution in agar (radial growth-inhibitory activity).5454 Macías-Rubalcava ML, Hernández-Bautista BE, Jiménez-Estrada M, et al. Naphthoquinone spiroketal with allelochemical activity from the newly discovered endophytic fungus Edenia gomezpompae. Phytochemistry. 2008;69:1185-1196. In another study, the compounds cyclotryprostatin A and tryptoquivaline O were tested against C. gloeosporioides by microdilution test and showed MIC of 50 µg mL-1 (0.12 µmol mL-1) and 100 µg mL-1 (0.18 µmol mL-1), respectively.5555 Li X-J, Zhang Q, Zhang A-L, Gao J-M. Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities. J Agric Food Chem. 2012;60:3424-3431. The compound dehydrozaluzanin exhibited activity at 30 mM.5656 Wedge DE, Galindo JCG, Macías FA. Fungicidal activity of natural and synthetic sesquiterpene lactone analogs. Phytochemistry. 2000;53:747-757. The isolated compounds piliformic acid and cytochalasin D have MIC values of 2.92 and 2.46 µmol mL-1.

The mechanism of action of cytochalasin D involves its binding capacity to actin filaments and therefore prevents polymerization and elongation. Therefore affect cell morphology and inhibit cellular processes such as cell division, which becomes some cases apoptosis.5757 Haidle AM, Myers AG. An enantioselective, modular, and general route to the cytochalasins: synthesis of L-696,474 and cytochalasin B. PNAS. 2004;101:12048-12053. Moreover, cytochalasin D inhibits protein synthesis.5858 Ornelles DA, Fey EG, Penman S. Cytochalasin releases mRNA from the cytoskeletal framework and inhibits protein synthesis. Mol Cell Biol. 1986;6:1650-1662.

The isolated compounds acid piliformic and cytochalasin D we are founded to present promising antifungal activity against C. gloeosporioides (from guarana plant), which causes the anthracnose disease. These compounds were produced by endophytic Xylaria spp. (from Manaus and Maués), in the Amazonia Forest. Thus, it was possible to verify that both Xylaria species presented a biotechnological potential and production of different active compounds. The compounds isolated in the present work are here reported for the first time as antifungal agents against C. gloeosporioides, a relevant plant pathogen worldwide. However, toxicity tests and in vivo bioassays need to be performed.

Acknowledgments

Financial support was provided by FAPESP grant (2014/15760-3) and BIOTA/BIOprospecTA FAPESP grant (2013/50228-8). Scholarships to L. M. Elias. (FAPESP: 2012/02000-5), M. C. Ferreira. (FAPESP: 2014/05940-4), from CNPq (142079/2016-2) and CAPES are also gratefully acknowledged. We thank UFAM, T. E. Bezerra, Dr. R. G. de S. Berlinck, Dr. M. W. Paixão and Dr. N. S. M. Junior.

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Edited by

Associate Editor: Luis Henrique Guimarães

Publication Dates

  • Publication in this collection
    Oct-Dec 2018

History

  • Received
    6 Mar 2017
  • Accepted
    9 Mar 2018
  • Published
    31 Mar 2018
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