View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Biochemical Systematics and Ecology 39 (2011) 485–490 Contents lists available at ScienceDirect Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco Composition of essential oils from Cupressus lusitanica and a Xylariaceous fungus found on its leaves Florisvaldo C. Santos Filho, Luciana da Silva Amaral, Edson Rodrigues-Filho* Departamento de Química, Universidade Federal de São Carlos, CP 676, 13.565-905, São Carlos, SP, Brazil article info abstract Article history: A Xylariaceous endophytic fungus was isolated from leaves of Cupressus lusitanica. Received 25 November 2010 Essential oil was extracted from these two organisms using the hydrodistillation method Accepted 8 July 2011 and identified by gas chromatography-mass spectrometry and retention index. A variety of Available online 17 August 2011 monoterpenes, sesquiterpenes and diterpenes were identified in the fungus and the plant, and some of them were co-produced by both organisms. The oil extracted from C. lusi- Keywords: tanica was tested against three of the endophytic fungi found in its leaves and showed only Essential oil slightly antifungal activity. Endophytic Ó Cupressus lusitanica 2011 Elsevier Ltd. Open access under the Elsevier OA license. Xylaria Antifungal activity 1. Introduction The “Cypress” plants (Cupressaceae family) are grown in many tropical and subtropical areas for commercial purposes such as ornamentation and source of wood building material (Farjon, 1993; Graniti, 1998). Species of genus Cupressus are frequently attacked by fungi which cause them to develop the disease called “Cypress Canker” (Graniti, 1998; Muthuchelian et al., 2005), which depreciate its commercial value. This disease is most frequently caused by fungi of genera Sphaeropsis and Seiridium, and can spread world-wide pretty fast (Sparapano et al., 2004). C. lusitanica is a good terpenoid producer, including monoterpenes, sesquiterpenes and diterpenes (Adams et al., 1997). Some monoterpenes, such as b-thujaplicin, have being proved to act as phytoalexin in C. lusitanica (Sparapano et al., 2004). Therefore it seems that terpenoid compounds plays important role in interactions between plants and pathogens. Among the endophytic fungi recently isolated from healthy tissues of C. lusitanica leaves, we obtained some strains with macro and micro morphology characteristics of those microorganisms belonging to the genus Xylaria (Rogers and Samuels, 1986), being these the most frequently fungi obtained. We described recently four major sesquiterpenes produced by one of these Xylaria, coded NICL5 (Amaral and Rodrigues-Filho, 2010). Thus, in the present work we report the study of the essential oil produced by C. lusitanica and by a Xylariaceous fungus (NICL5) isolated from this plant, with the aims to compare terpenoid contents and to investigate how the plant oil affect the endophytic fungi growth. The study of essential oils from plants is well established. However, this type of study is not so common for organisms such as endophytic fungi. * Corresponding author. Tel.: þ55 16 3351 8053; fax: þ55 16 3351 8350. E-mail address: [email protected] (E. Rodrigues-Filho). 0305-1978 Ó 2011 Elsevier Ltd. Open access under the Elsevier OA license. doi:10.1016/j.bse.2011.07.001 486 F.C. Santos Filho et al. / Biochemical Systematics and Ecology 39 (2011) 485–490 2. Materials and methods 2.1. Material plant Health leaves of C. lusitanica were collected in São Carlos, São Paulo State, Brazil. A voucher specimen (No. 7281) has been deposited in the Herbarium of the Botanic Department of Universidade Federal de São Carlos, Brazil. 2.2. Fungal material The surface sterilization method employed in this work was similar to that used by Petrini et al. (1992).Afterthe collection, the leaves were washed in abundant water (domestic use grade) and then in distilled water. The leaves were surface sterilization by consecutive immersion in 70% ethanol (2 s), sterile distilled water (2 s), 11% aqueous sodium hypochloride for 1–5 min and 70% ethanol (2 s), and then in sterile distilled water. The material was placed in Petri dishes À containing PDA medium (potato, dextrose and agar) supplemented with 100 mgmL 1 terramycin and incubated at room temperature. Endophytic fungi growing from the plant tissues were picked and recultured on PDA to determine culture purity. The isolated fungi were stored at LaBioMMi – Laboratório de Bioquímica Micromolecular de Microorganismos – in Departamento de Química at Universidade Federal de São Carlos, Brazil. Working stocks were prepared on potato dextrose agar. 2.3. Extraction of essential oil from C. lusitanica The essential oil of C. lusitanica was extracted by hydrodistillation from dried leaves (100 g of each sample in 1000 mL of distilled water) using a Clevenger type apparatus for 3 h. The oil was dried over anhydrous sodium sulfate and stored in sealed glass vials at À22 C until the analysis. 2.4. Extraction of essential oil from endophytic fungus cultivated in liquid and solid medium The fungus was grown under static conditions at room temperature for 14 days in round-bottom flask containing the À1 À1 À1 À1 medium liquid composed of dextrose (26.7 g L ), yeast extract (10.0 g L ), NaNO3 (3.0 g L ), K2HPO4 (1.0 g l ), À1 À1 À1 MgSO4$7H2O (0.5 g L ), KCl (0.5 g L ), FeSO4$7H2O (0.01 g L ). After the period of growth the fungus essential oil was extracted by hydrodistillation using a Clevenger type apparatus for 3 h. The oil was dried over anhydrous sodium sulfate and stored in sealed glass vials at À22 C until the analysis. Hydrodistillation was performed on a flask containing the medium only and the extract analyzed as a blank. In another experiment the fungus was grown at room temperature for 14 days in round-bottom flask containing “Uncle Ben’s” rice as solid medium. After this period 800 mL of distilled water was added to the flask and the essential oil was extracted, dried and stored in the same way used in liquid medium experiment. A flask with the same amount of rice was kept as a control and also hydrodistillated. The oil of this blank experiment was analyzed and the data subtracted from that obtained with the fungus present. 2.5. Analysis of the essentials oils The composition of essential oils was investigated by GC-MS, performed on a chromatographic system CARLO ERBA GC 8000 series equipped with a J&W Scientific DB-1 capillary column (30 m  0.25 mm  0.25 mm). The carrier gas was helium. Oven temperature was programmed (50 C for 5 min), then 50–290 Cat7C/min and subsequently held isothermal for 5 min. Injector port: 180 C, detector: 280 C, split ratio: 1:20. Volume injected: 2 mL diluted in dichloromethane. The GC was coupled to a MICROMASS Platform II mass spectrometer and the data were acquired using 70 eV as ionization voltage; scan time: 1.40 s; mass range: m/z 50–750 Da. The software adopted to handle mass spectra and chromatograms was MassLynx V2.11. The identification of the compounds was based on mass spectra (compared with NIST Library). Further confirmation was achieved from Retention Index (RI) generated from a series of alkanes (C9–C25) on DB-5 column. 2.6. Antifungal activity The essential oil extracted from C. lusitanica leaves was tested against three endophytic fungi, preliminarily identified as Xylaria sp1 (NICL3), Guignardia sp (NICL4) and Xylaria sp2 (NICL5). These are endophytic fungi isolated from C. lusitanica. The test was performed by a modified disc diffusion method. Oil solutions in ethanol at different concentrations were deposited on the plate where the microorganisms were subjected to growth for 8 days. Plates with the addition of ethanol only were used as control. F.C. Santos Filho et al. / Biochemical Systematics and Ecology 39 (2011) 485–490 487 Table 1 Chemical composition of the essential oils from C. lusitanica and the endophytic fungus Xylaria. No Compounds Cupressus lusitanica Xylaria in Solid Xylaria in Liquid Kovats Methods for b Medium Medium Index (lit ) identification Retention Kovats Retention Kovats Retention Kovats Time (min) Indexa Time (min) Indexa Time (min) Indexa þ þ Monoterpenes (M . ¼ 136) and monoterpenes oxygenated (M . ¼ 154) 01 b–Pinene 4.38 978.1 ––––979 MS, KI 02 bÀ(Z)Ocimene 5.63 1038.1 ––––1037 MS, KI 03 p-Mentha-3,8-diene 6.33 1068.8 ––––1073 MS, KI 04 p–Mentha-2,4(8)-diene 6.81 1089.9 ––––1088 MS, KI 05 Terpinolene 6.98 1097.3 ––––1089 MS, KI 06 endo-Fenchol 7.41 1114.0 7.51 1116.0 ––1117 MS, KI 07 neo-allo–Ocimene 8.31 1148.1 ––––1144 MS, KI 08 iso-Isopulegol 8.63 1160.2 ––––1160 MS, KI 09 iso-Verbanol 9.13 1179.1 ––––1180 MS, KI 10 Dihydro Carveol 9.51 1193.5 ––––1194 MS, KI 11 Dihydro Carvone 9.68 1200.0 9.48 1199.2 ––1201 MS, KI 12 trans-Pulegol 9.88 1207.3 ––––1210 MS, KI 13 iso-Dihydro Carveol 10.06 1214.0 ––––1215 MS, KI 14 trans-Carveol 10.28 1222.1 ––––1217 MS, KI 15 cis-Carveol 10.38 1225.8 ––––1229 MS, KI 16 neoiso-Dihydro Carveol 10.58 1233.2 ––––1229 MS, KI 17 Pulegone 10.68 1236.9 ––––1237 MS, KI 18 Geranial 11.61 1271.2 ––––1267 MS, KI 19 Perilla aldehyde 11.81 1278.5 ––––1272 MS, KI 20 a–Terpinen-7-al 11.98 1284.2 ––––1285 MS, KI 21 p-Menth-1-en-9-ol 12.36 1298.8 ––––1295 MS, KI 22 m-Acetanisole 12.53 1305.1 ––––1299 MS, KI 23 dehydro-Elshotzia Ketone 12.76 1313.6 10.13 1210.6 ––1303 MS, KI 24 4-hydroxy-Cryptone 12.81 1315.7 ––––1316 MS, KI 25 Phenyl Ethyl Oxyacetaldehyde 13.06 1324.7 ––––1327 MS, KI 26 (Z)-Hasmigone 13.28 1332.8 ––––1329