b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 840–847 ht tp://www.bjmicrobiol.com.br/ Biotechnology and Industrial Microbiology The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose a a a a Luciana M. Elias , Diana Fortkamp , Sérgio B. Sartori , Marília C. Ferreira , a b c c Luiz H. Gomes , João L. Azevedo , Quimi V. Montoya , André Rodrigues , d a,∗ Antonio G. Ferreira , Simone P. Lira a Universidade de São Paulo, Escola Superior de Agricultura “Luiz de Queiroz”, Departamento de Ciências Exatas, Piracicaba, SP, Brazil b Universidade de São Paulo, Escola Superior de Agricultura “Luiz de Queiroz”, Departamento de Genética, Piracicaba, SP, Brazil c Universidade Estadual Paulista “Júlio de Mesquita Filho”, Instituto de Biociências, Departamento de Bioquímica e Microbiologia, Rio Claro, SP, Brazil d Universidade Federal de São Carlos, Departamento de Química, São Carlos, SP, Brazil a r t i c l e i n f o a b s t r a c t Article history: Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeospo- Received 6 March 2017 rium. These are considered one of the main pathogens, causing significant economic losses, Accepted 9 March 2018 such as in peppers and guarana. The current forms of control include the use of resistant Available online 31 March 2018 cultivars, sanitary pruning and fungicides. However, even with the use of some methods Associate Editor: Luis Henrique of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive Guimarães 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 Keywords: against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The 2-Hexylidene-3-methylbutanedioic compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques acid from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated com- Amazon pounds were identified by spectrometric techniques, as NMR and mass spectrometry. This Anthracnose is the first report that piliformic acid and cytochalasin D have antifungal activity against −1 ␮ Plant pathogen C. gloeosporioides with MIC 2.92 and 2.46 mol mL respectively. Captan and difenocona- −1 zole were included as positive controls (MIC 16.63 and 0.02 ␮mol mL , respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease. © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/). ∗ Corresponding author. E-mail: [email protected] (S.P. Lira). https://doi.org/10.1016/j.bjm.2018.03.003 1517-8382/© 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 840–847 841 High-performance liquid chromatography (HPLC) was Introduction conducted using a Agilent 1100 Series UV/Vis with a qua- ternary pump, coupled to a UV detector MWD (Multiple Anthracnose is one of the most serious plant diseases affect- Wavelength Detector), using a reversed phase column C18 ing different cultures worldwide. This disease is usually (250 mm × 4.60 mm, 5 ␮m; Phenomenex Kinetex), with a caused by filamentous fungi in the genus Colletotrichum or −1 mobile phase H2O/MeOH (80:20, v/v), flow rate 1 mL min Gloeosporium, and these pathogens cause great losses and and = 254 nm. HPLC-grade solvents were utilized. Solid- hence reduction in the quality and quantity of fruits and phase extraction was carried out using silica gel cartridges 1–4 vegetables. Anthracnose is considered the main disease of of different dimensions (Phenomenex). Gel permeation chro- guarana (Paullinia cupana ‘sorbilis’ Mart., Sapindaceae). The matography was performed in a glass column filled with pathogen infects leaves and stems, causing deformation and Sephadex LH-20 (Pharmacia). Silica gel (Macherey-Nagel) 5,6 254 necrotic lesions. The current forms of control include the were used for thin-layer chromatography (TLC). Spots were 7 use of resistant cultivars, sanitary pruning and fungicides. detected under UV light (254 and 365 nm). However, even with the use of some methods of control- ling these cultures, they are not are free of anthracnose. Fungal isolation Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to The endophytic fungi (isolates 249 and 214) were isolated from 8 human health and the environment. leaves of guarana plant (P. cupana) with anthracnose symp- Therefore, the search for natural products with antifungal ◦ toms (necrotic lesions), collected at Manaus-AM (03 22 5), at activity of agronomic relevance is an important alternative for the Experimental Farm of the Federal University of Ama- 9 the control of diseases such as anthracnose. In the search for ◦ zonas (UFAM), and Maués-AM (57 42 0), at Fazenda Santa bioactive compounds, only a small fraction of the estimated Helena (Ambev – American Beverage Company), in Novem- 10 fungal biodiversity has been chemically investigated, and ber 2010. The collected samples were leaves and branches only a small number of these have already been cultured and of a total of 20 adult plants (10 of each locality). Complete 11,12 selected for drug production. Fungi have special interest intact leaves were rinsed with running water and then with because they are known to produce several secondary metabo- distilled water. The cleaned leaves were sterilized by consecu- 13 lites with a wide range of applications. The fungi of the genus tive washes in 75% EtOH (1 min), 3% NaOCl (3 min) and 75% Xylaria stand out in the production of secondary metabolites, EtOH (30 s), and they were rinsed with sterile distilled H2O mainly of the class of cytochalasins, isocumarins, lactones, 16,17 two times. The sterilized material was cut into 8 × 12 mm sesquiterpenos, triterpenos and xanthones, and with the most and 3–4 pieces were deposited on a Petri dish containing diverse biological activities, such as anticancer, antioxidant, TM potato dextrose agar (PDA, Kasvi , Brazil), chloramphenicol antimicrobial, antiinflammatory, antiviral, inhibiting HIV-1 −1 and streptomycin (100 ␮g mL of each) to prevent bacterial 14,15 protease, antibiotic and antitumor, among others. ◦ growth. Plates were incubated at 27 C. Petri dishes were In a previous study, a screening was done with the fractions observed daily, and the individual hypha tips of the emerging of 16 endophytic fungi isolated from Guarana plant from Ama- colonies were re-inoculated in new PDA plates until pure cul- zonia, and two Xylaria spp. were selected because its activity tures were obtained. Pure cultures of the endophytes (isolates against the phytopathogen Colletotrichum gloeosporioides. Thus, 18 249 and 214) were preserved by the Castellani method. this research aimed to evaluate the antifungal potential of sec- ondary metabolites produced by this two endophytic Xylaria Fungal identification spp. isolated from guarana (P. cupana). Pure culture of isolates 249 and 214 were first grown in malt −1 Experimental agar 2% (in g L : 20 malt extract, and 15 agar, Acumedia, TM ◦ Neogen ) at 25 C for seven days. Then, fresh mycelia were General procedures harvested from and used for DNA extraction based on the CTAB method (cetyltrimethyl ammonium bromide) used in 19 Optical rotations were taken on a Polartronic H Schmidt Montoya et al. We amplified the ITS barcoding region using ◦ 1 & Haensch digital polarimeter (1 dm cell) at 23 C. The H ITS4 and ITS5 primers. Amplicons were cleaned up and sub- nuclear magnetic resonance (NMR) was recorded on a Bruker mitted to cycle-sequencing reactions using BigDye Terminator 14.1 Tesla, AVANCE III model spectrometer (operating at v. 3.1 Kit (Thermo Fischer Scientific). Bidirectional sequences TM 600.13 MHz for hydrogen frequency) with crioprobe . The were generated in ABI 3530 (Thermo Fischer Scientific) using chemical shifts are given on a ı (ppm) scale and referenced the same primer pair. to tetramethylsilane (TMS). High-resolution electrospray ion- Forward and reverse sequences were assembled in contigs 20 ization mass spectrometry (HR-ESI MS) was acquired by direct in Bioedit. Then, contigs were compared with homologous infusion on a Q-TOF Maxis Impact in the following conditions: sequences deposited in the NCBI-Genbank. capillary voltage, 4.50 kV; operating in electrospray positive To refine the taxonomic assignment of isolates 249 and 21 mode; The ion source was set to a nebulizer pressure of 0.3 bar, 214, we carried out a phylogenetic analysis in MEGA v.6.0. −1 ◦ a dry gas flow rate of 4 L min , and a dry temperature of 180 C; For this analysis, we retrieved sequences from closet rela- detection range: 100–700 Da with total ion count extracting tive taxa deposited in the database. Sequences were aligned 22 acquisition. Data acquisition was performed using a Bruker in MAFFT, and this dataset was used to infer a phy- Compass Data Analysis 4.2. logenetic tree under the neighbor-joining algorithm and 842 b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 9 (2 0 1 8) 840–847 the Kimura 2-parameters as the nucleotide substitution and placed at one edge of the 60 mm PDA Petri dish plate TM model.