Journal of Applied Botany and Food Quality 87, 56 - 61 (2014), DOI:10.5073/JABFQ.2014.087.008 1Laboratoire d’Ecologie et Gestion des Ecosystèmes Naturels, Faculté des Sciences de la nature et de la vie, et des sciences de la terre et l’univers 2 Laboratoire des Substances Naturelles et Bioactives (LASNABIO) Département de Chimie, Faculté des Sciences, Université de Tlemcen, Algérie 3 Université de Corse, UMR CNRS 6134, Laboratoire Chimie des Produits Naturels, Campus Grimaldi, Corte, France Antifungal activity of essential oils of three aromatic plants from western Algeria against five fungal pathogens of tomato (Lycopersicon esculentum Mill) S. Bouayad Alam1, N. Gaouar Benyelles1, M. El Amine Dib2*, N. Djabou2, L. Tabti1, J. Paolini 3, A. Muselli 3, J. Costa 3 (Received December 9, 2013) * Corresponding author Summary The antifungal effect of the essential oils from Thymus capitatus L., Daucus crinitus Desf. and Tetraclinis articulate Vahl., aerial parts was evaluated in vitro against five phytopathogenic fungi of tomato (Fusarium oxysporum, Alternaria solani, Aspergillus niger, Penicillium sp1 and Penicillium sp2). Our results showed that among the three plant species tested, T. capitatus oil was the most potent antifungal against the fungi (inhibition of mycelial growth of 100 % at a concentration of 2 μg mL-1). Furthermore, the essential oil of T. articulata was also effective against F. oxysporum, A. solani, A. niger, Penicillium sp1 and Penicillium sp2 with an inhibition of mycelial growth greater than 57 % at a concentration of 5 μg mL-1. D. crinitus essential oil was less effective. T. capitatus essential oil was dominated by carvacrol (69.6 %) and p-cymene (12.4 %). The isochavicol isobutyrate (44.9 %) and isochavicol 2-methyl- butyrate (9.7 %) were the major compounds in D. crinitus essential oil, while the most abundant compounds in T. articulata were α- pinene (32.0 %), cedrol (11.0 %) and 3-carene (9.6 %). The plant essential oils were found to be an effective antifungal against of mycelial growth and, therefore, can be exploited as an ideal treat- ment against disease rot of tomato or as a new potential source of natural additives for the food and/or pharmaceutical industries. Introduction Tomato (Lycopersicon esculentum) is an important commercial crop in the world. Nutritional values of tomato make it a widely accepted vegetable by consumers. Nevertheless, tomato is a very perishable vegetable with a short shelf-life and high susceptibility to fungal disease. Tomatoes are among the most popular fruits grown in Algeria. They are of an excellent quality and are greatly appreciated for their nutritional value. Furthermore, tomato production repre- sents an important agricultural and economic activity in the country. The growing awareness of consumers concerning the relation be- tween food and health is revolutionizing the food industry. Fungal pathogens are mainly responsible for the decay of fruits and vege- tables during the postharvest period (PATHAK, 1997). Aspergillus, Fusarium and Penicillium are responsible for spoilage of many foods and causes decay on stored fruits damaged by insects, animals, early splits, and mechanical harvesting. Apart from causing diseases in plants, many species of Aspergillus, Penicillium and Alternaria can also synthesize mycotoxins (AGRIOS, 1997; ROJAS et al., 2005). Considerable interest has developed on the preservation of foods by the use of essential oils to effectively retard growth and mycotoxin production. Essential oils and their main components possess a wide spectrum of biological activity, which may be of great importance in several fields, from food chemistry to pharmacology and pharma- ceutics (CRISTANI, 2007). The main aim of this work was to eva- luate the antifungal properties of the essential oils of T. capitatus, D. crinitus and T. articulate against phytopathogens that cause se- vere diseases in tomato, such as F. oxysporum, A. solani, A. niger, Penicillium sp1 and Penicillium sp2. Materials and methods Plant materials and essential oils extraction Aerial parts of D. crinitus were collected in Bensekrane forest area (Tlemcen Province) at the flowering stage, in June 2011. The oil yield was 0.37 % (w/w). T. capitatus aerial parts were collected from Beni snous in Tlemcen city at the flowering stage, during June 2011 and yielded 0.52 % (w/w). T. articulata aerial parts were col- lected from Oujlida region, Tlemcen Province during June 2011 and yielded 0.31 % (w/w). The plant species were stored at -18 °C after harvest. A portion (550-600 g) of material from each plant spe- cies was subjected to a Clevenger-type apparatus according to the European Pharmacopoeia (EUROPEAN PHARMACOPOEIA, 2004). The essential oils were dried over anhydrous sodium sulfate and, after filtration, stored in sterilized amber vials at 4 °C until it was used. Gas chromatography Analyses were carried out using a Perkin Elmer Clarus 600 GC ap- paratus equipped with a dual flame ionization detection system and 2 fused-silica capillary columns (60 m x 0.22 mm I.D., film thick- ness 0.25 μm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax (poly- ethylene glycol). The oven temperature was programmed from 60 °C to 230 °C at 2 °C/min and then held isothermally at 230 °C for 35 min. Injector and detector temperatures were maintained at 280 °C. Essential oils were injected in the split mode (1/50), us- ing helium as the carrier gas (1 mL/min); the injection volume was 0.2 μL. Retention indices (RI) of the compounds were determined from Perkin-Elmer software. Gas chromatography-mass spectrometry Essential oils were analyzed with a Perkin–Elmer TurboMass quadrupole analyzer, coupled to a Perkin–Elmer Autosystem XL, equipped with 2 fused-silica capillary columns and operated with the same GC conditions described above, except for a split of 1/80. Electronic Impact (EI) mass spectra were acquired under the follow- ing conditions: Ion source temperature 150 °C, energy ionization 70 eV, mass range 35-350 Da (scan time: 1 s). Component identification The identification of the components was based on a comparison: (i) between the calculated retention indices on the polar (RI p) and apolar (RI a) columns with those of pure standard authentic com- pounds and literature data (JENNINGS and SHIBAMOTO, 1980; KÖNIG et al., 2001; NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY, Antifungal activity of oils against fungal pathogens of tomato 57 2008); and (ii) of the mass spectra with those of our own library of authentic compounds and with those of a commercial library (MC LAFFERTY and STAUFFER, 1994; MC LAFFERTY and STAUFFER, 1988; NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY, 1999). Component quantification Quantification of the essential oil components was carried out using the methodology reported by COSTA et al. (2008), and modified as follows. The response factor (RF) of 29 standard compounds grouped into 7 chemical groups (monoterpene hydrocarbons, sesquiterpene hydrocarbons, alcohols, ketones, aldehydes, esters, and others) was measured using GC (ZNINI et al., 2011). RFs and cali-bration curves were determined by diluting each standard in hexane at 5 concen- trations, containing tridecane (final concentration = 0.7 g/100 g) as an internal standard. Analysis of each standard was performed in triplicate. For the quantification of the essential oil components, tridecane (0.2 g/100 g) was added as internal standard to the essential oil. The correction factor (average of the response factors from stan- dards) of each chemical group was calculated and used to determine the essential oil component concentration (g/100 g) according to the chemical group. Pathogenic fungi Fusarium oxysporum, Alternaria solani, Aspergillus niger, Peni- cillium sp1 and Penicillium sp2 were isolated from naturally de- cayed tomato after storage of several weeks. These isolates were the most aggressive one in our collection and produced the largest lesions on inoculated fruit. A pure culture of these fungus were main- tained on potato dextrose agar medium (PDA: potato 200, dextrose 20 g and agar 15 gL-1 in distilled water at 25 °C) in the presence of a quantity of lactic acid (25 %) for stop the growth of bacteria. The plates were incubated at 25 ± 2 °C for 8 days and darkness. The developing fungal colonies were purified and identified up to the species level by microscopic examination through the help of the following references (BARNETT and HUNTER, 2006). In vitro antifungal assay The antifungal activity of the three essential oils was tested using radial growth technique (BAJPAI et al., 2007). Appropriate volumes of the stock solutions of the oils in dimethyl sulfoxide (DMSO) were added to PDA medium immediately before it was poured into the Petri dishes (9.0 cm diameter) at 40-45 °C to obtain two concen- trations (2.0 and 5.0 μg mL-1). Each concentration was tested in triplicate. Parallel controls were maintained with DMSO mixed with PDA. The discs of mycelial felt (0.5 cm diameter) of the plant pathogenic fungi, taken from 8-day-old cultures on PDA plates, were transferred aseptically to the centre of Petri dishes. Carbendazim was used as reference fungicide. The treatments were incubated at 27 °C in the dark. Colony growth diameter was measured after the fungal growth in the control treatments had completely covered the Petri dishes. Percentage of mycelial growth inhibition was calculated from the formula: (I%) = [(DC-DT)/DC] x 100 (PANDEY et al., 1982); where DC and DT are average diameters of fungal colony of control and treatment, respectively. Statistical Analysis The inhibitory effect of essential oils on mycelial growth was ex- pressed as mean ± standard error of mean (S.E.M.) and analyzed for ANOVA and post hoc Dunnet’s t-test. The separation of means was done by using the least significant difference test at p <0.05. Analysis of each test was performed in triplicate. Results Essential oils composition A total of 26 components accounting to 99.5 % of the essential oil composition of T. capitatus were identified by comparison of their EI-mass spectra and their retention indices (RI) with those of our own authentic compound library (Tab. 1). The essential oil was high- ly dominated by oxygenated compounds (87.1%) with high amount of aromatic terpenic components (82.6 %). However, hydrocarbons appeared also in appreciable proportion (12.4%) which monoterpene hydrocarbons are well represented (10.7 %). Indeed, the main con- stituents of essential oil were carvacrol (69.6 %), p-cymene (12.4 %) followed by γ-terpinene (4.3 %), myrcene (2.1 %), α-terpinene (1.7 %), linalool (1.7 %) and terpinen-4-ol (1.1 %). These results were in accordance with those previously reported in literature (AMARTI et al., 2008; BOUNATIROU et al., 2007; RUBERTO et al., 2000; TAWAHA and HUDAIB, 2012). Other hand, various chemical profiles of essen- tial oils (thymol, cavacrol or thymol/carvacrol as main components) have been reported according to geographical origins of T. capitatus (KAROUSO et al., 2005; MICELI et al., 2006). The analysis of the es- sential oil from the aerial parts of D. crinitus harvested in the forest of Bensekrane (Tlemcen) identified 30 components, which account- ed for 91.3 % of the total composition. Their retention indices and re- lative percentages are shown in Tab. 1. The main components of the aerial parts oil were phenylpropanoids isochavicol esters, principally the isochavicol isobutyrate (44.9 %). The other major components identified were: isochavicol 2-methylbutyrate (9.7 %), pentadecane (5.1 %) and undecane (4.1 %) (Tab. 1). This result is in according with literature data (LANFRANCHI et al., 2010). A total of 54 components accounting for 95.9 % of the total oil of T. articulata were identified (Tab. 1). The essential oils was highly dominated by the monoterpene hydrocarbons (63.8 %) followed by oxygenated sesquiterpenes (14.7 %) and sesquiterpene hydrocarbons (10.5 %). However, the oxygenated monoterpenes appeared in small proportion (6.4 %). The most abundant compounds were α-pinene (32.0 %), cedrol (11.0 %), 3-carene (9.6 %), limonene (4.3 %), sabi- nene (4.3 %) and (E)-β-caryophyllene (4.0 %). BEN JEMIA et al. (2013) have isolated and identified, by GC-MS, 66 constituents, the major constituents of the oil are: bornyl acetate (31.4 %), α-pinene (24.5 %) and camphor (20.3 %). while TOUMI et al. (2011) have identified, by GC/MS, more 45 compounds, with camphor(23.4- 31,6 %), bornyl acetate (17,1-25,8 %), borneol (6.6-14,3 %), limo- nene (3,70-10,1 %) and α-pinene (6,5-11,3 %) were the major com- ponents of T. articulata essential oil. It was observed that the per- centage of α-pinene (19.8-24.9 %) and bornyl acetate (40.2-59.2 %) for the leaves oils from two different sites in Algeria were the major constituents (CHIKHOUNE et al., 2013). In addition, the percentage of cedrol and 3-carene found in our essential oil was higher than cedrol and 3-carene in previous studies. Generally, the quality and quantity of components available in essential oils may be affected by several factors, such as plant genotype, geographical condition, season, and agronomic condition (GUMUS et al., 2010). Antifungal activity of three essential oils against the develop- ment of fungi of tomato The data presented in Tab. 2 show the antifungal activity of 3 plant species, belonging to 3 botanical families (Lamiaceae, Apiaceae and Cupressaceae), against F. oxysporum, A. solani, A. niger, Penicillium sp1 and Penicillium sp2. The effect of plant essential oils varied ac- cording to plant species. Indeed, 2 plant species out of 3 reduced 58 S. Bouayad Alam, N. Gaouar Benyelles, M. El Amine Dib, N. Djabou, L. Tabti, J. Paolini, A. Muselli, J. Costa Tab. 1: Chemical compositions of aerial parts essential oils of T. capitatus, D. crinitus and T. articulate. No.a Components RIa b RIa c RIpd T. capitatus D. crinitus T. articulata Identification e Nonane 906 902 907 - 0.6 - RI, MS α-Thujene 932 924 1028 0.2 - tr RI, MS α-Pinene 936 931 1028 0.9 0.5 32.0 RI, MS α-Fenchene 941 943 1039 - - 0.6 RI, MS Camphene 950 945 1071 0.2 - 0.3 RI, MS Oct-1-en-3-ol 962 962 1441 0.5 - - RI, MS Sabinene 973 967 1122 - 0.6 4.3 RI, MS β-Pinene 978 972 1113 0.1 0.1 1.4 RI, MS Myrcene 987 982 1160 2.1 0.6 3.3 RI, MS α-Phellandrene 1002 999 1161 0.2 - 1.5 RI, MS 3-Carene 1005 1006 1149 0.1 - 9.6 RI, MS α-Terpinene 1008 1011 1270 1.7 - - RI, MS p-Cymene 1015 1015 1270 12.4 0.2 0.5 RI, MS Limonene 1025 1023 1201 - 0.9 4.3 RI, MS β-Phellandrene 1023 1023 1209 - - 1.4 RI, MS (E)-β-Ocimene 1041 1037 1247 - 0.6 0.7 RI, MS (Z)-β-Ocimene 1029 1022 1234 0.6 - - RI, MS γ-Terpinene 1051 1050 1245 4.3 1.6 0.7 RI, MS (E)-Sabinene hydrate 1051 1054 1445 0.1 - 0.2 RI, MS Nonanal 1076 1074 1403 - 0.1 - RI, MS Terpinolene 1082 1079 1281 0.2 0.4 3.2 RI, MS (Z)-Sabinene hydrate 1087 1084 1537 - - 0.8 RI, MS Linalool 1083 1085 1538 1.7 0.2 0.2 RI, MS Undecane 1100 1098 1101 - 4.1 - RI, MS 3-Octyl acetate 1113 1107 1330 - - 0.2 RI, MS Veratol 1112 1113 1713 - - 0.1 RI, MS Camphor 1123 1124 1506 0.1 - - RI, MS (Z)-Verbenol 1027 1128 1642 - - 0.3 RI, MS Isoborneol 1143 1144 1670 0.5 - - RI, MS Borneol 1148 1150 1688 0.3 - - RI, MS Terpinen-4-ol 1164 1162 1591 1.1 0.1 2.0 RI, MS α-Terpineol 1176 1176 1690 0.1 - 0.1 RI, MS Octyl acetate 1188 1187 1460 - 2.3 - RI, MS Decanal 1188 1187 1483 - 1.4 - RI, MS Linalyl acetate 1239 1239 1552 - - 0.2 RI, MS Decanol 1263 1259 1729 - 0.1 - RI, MS Nonanoic acid 1263 1263 2119 - 0.1 - RI, MS (E)-Anethole 1264 1261 1815 - - 0.1 RI, MS Thymol 1266 1263 2181 0.6 - - RI, MS Bornyl acetate 1269 1269 1562 - - 0.7 RI, MS Carvacrol 1278 1286 2193 69.6 - - RI, MS Eugenol 1330 1329 2164 0,1 - - RI, MS α-Terpinyl acetate 1335 1333 1686 - - 1.8 RI, MS (E)-Myrtanyl acetate 1366 1370 1479 - - 0.1 RI, MS β-Bourbonene 1386 1384 1518 - - 0.1 RI, MS β-Elemene 1389 1386 1584 - - 0.2 RI, MS Dodecanal 1389 1389 1695 - 3.1 - RI, MS Antifungal activity of oils against fungal pathogens of tomato 59 No.a Components RIa b RIa c RIpd T. capitatus D. crinitus T. articulata Identification e β-Funebrene 1419 1411 1591 - - 1.6 RI, MS (E)-β-Caryophyllene 1421 1416 1591 1.6 0.6 4.0 RI, MS Thujopsene 1435 1427 1614 - - 0.2 RI, MS α-Humulene 1455 1448 1668 0.1 - 2.5 RI, MS α-Acoradiene 1444 1455 1616 - - 0.1 RI, MS β-Acoradiene 1458 1459 1642 - - 0.1 RI, MS Alloaromadendrene 1462 1461 1630 - - 0,1 RI, MS γ-Curcumene 1475 1471 1680 - - 0,3 RI, MS Germacrene D 1479 1474 1700 - - 1,3 RI, MS β-Selinene 1482 1480 1703 - - 0,1 RI, MS γ-Humulene 1483 1480 1702 - 0.7 - RI, MS Pentadecane 1500 1497 1502 - 5.1 - RI, MS δ-Cadinene 1520 1511 1760 - 0.1 0.3 RI, MS β-Elemol 1535 1533 2063 - - 0,4 RI, MS Isochavicol isobutyrate 1546 1541 2134 - 44.9 - RI, MS Dodecanoic acid 1554 1560 2474 - 1.1 - RI, MS, ref Caryophyllene oxide 1578 1567 1969 0.1 - 0.4 RI, MS Dodecyl acetate 1585 1580 1882 - 2.5 - RI, MS, ref Globulol 1589 1577 2085 - - 0.9 RI, MS Cedrol 1595 1591 2101 - - 11.0 RI, MS Humulene epoxide II 1602 1599 2044 - - 0.1 RI, MS epi-Cedrol 1613 1614 2141 - - 0.2 RI, MS α-Acorenol 1623 1617 2106 - - 0.3 RI, MS γ-Eudesmol 1619 1624 2198 - - 0.1 RI, MS τ-Cadinol 1633 1632 2146 - - 0.2 RI, MS α-Eudesmol 1632 1636 2211 - - 0.3 RI, MS Isochavicol 2-methyl butyrate 1651 1648 2255 - 9.7 - RI, MS Bulnesol 1665 1664 2198 - - 0.2 RI, MS Heptadecane 1700 1703 1699 - 3.4 - RI, MS Tetradecanoic acid 1761 1756 2649 - 3.1 - RI, MS, ref Cedryl acetate 1764 1750 2160 - - 0.6 RI, MS Neophytadiene 1807 1807 1918 - 0.4 - RI, MS, ref Hexadecanoic acid 1951 1949 2916 - 1.1 - RI, MS Manool 2070 2109 2684 - - 0.3 RI, MS (E)-Phytol 2114 2102 2620 - 1.7 - RI, MS Total identification % 99.5 92.0 96.5 % Hydrocarbon compounds 12.4 20.5 74.5 % Monoterpene hydrocarbons 10.7 5.5 63.8 % Sesquiterpene hydrocarbons 1.7 1.4 10.7 % Non terpenic hydrocarnon compounds - 13.2 - % Diterpenes hydrocarbons - 0.4 - % Oxygenated compounds 87.1 71.5 22.0 % Oxygenated monoterpenes 3.8 0.3 6.5 % Oxygenated sesquiterpenes 0.1 - 14.7 % Non terpenic oxygenated compounds 0.5 14.9 0.2 % Aromatic compounds 82.6 - 0.1 % Phenylpropanoids 0.1 54.6 - % Oxygenated diterpenes - 1.7 0.5 a Order of elution is given on apolar column (Rtx-1), b Retention indices on the apolar Rtx-1 column (RIa), c Retention indices on the polar Rtx-Wax column (RIp), d Retention indices on the polar Rtx-Wax column (RIp), e RI: Retention Indices; MS: Mass Spectrometry in EI mod. 60 S. Bouayad Alam, N. Gaouar Benyelles, M. El Amine Dib, N. Djabou, L. Tabti, J. Paolini, A. Muselli, J. Costa Tab. 2: Percentage of inhibition of mycelial growth at various volumes of essential oils. Incubation F. oxysporum A. solani A. niger Penicillium sp1 Penicillium sp2 25°c ± 2 25°c ± 2 25°c ± 2 25°c ± 2 25°c ± 2 Essential oil (2 μg mL-1) T. capitatus 100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 T. articulata 36.11± 0.08 35.12 ± 0.01 11.11± 0.11 34.56 ± 0.02 45.12± 0.06 D. crinitus - - - - 54.32± 0.21 Essential oil (5 μg mL-1) T. articulata 72.22± 0.06 70.12± 0.20 57.77± 0.11 64.44± 0.12 84.44± 0.08 D. crinitus - - - 5.55± 0.21 77.77± 0.06 mycelial growth of F. oxysporum, A. solani, A. niger, Penicillium sp1 and Penicillium sp2 by more than 50 %. Among these plants T. capitatus, belonging to the families of Lamiaceae, completely in- hibited mycelial growth of tested fungus. T. capitatus essential oil produced the greatest reduction in mycelium growth with these fungi at 2 μg mL-1, with percentage reductions of 100 % (Tab. 2). The sec- ond most effective essential oil with this five fungi was T. articulata essential oil, with percentage of mycelial reduction in F. oxysporum, A. solani, A. niger, penicillium sp1 and penicillium sp2 of 36.11, 35.12, 11.11, 34.56 and 45.12 %, respectively, at the same concen- tration (Tab. 2). However, the data indicate that the percentage in- hibition of mycelial growth increased with increasing concentration of essential oils for all strains tested, suggesting that the essential oil of T. articulata inhibited the growth of all strains in a dose-dependent manner. Essential oil D. crinitus cause no percentage of mycelial re- duction, except against penicillium sp2. This activity was more pro- nounced, where the percentage of inhibition increased to 54.32 % at 2 μg mL-1, reaching a maximum of 77.77 % at 5 μg mL-1, suggesting that this strain was the most sensitive to the essential oil (Tab. 2). Discussion In this study, the antifungal activity of essential oils of three plant species was evaluated against F. oxysporum, A. solani, A. niger, Penicillium sp1 and Penicillium sp2. The mycelial growth of colonies in the presence of the essential oil of T. capitatus and T. articulata showed that it effectively controlled all the fungi tested. The mycelial growth of colonies in the presence of the essential oil of T. capitatus and T. articulata showed that it effectively controlled all the fungi tested. This efficiency can be explained by the presence of active molecules that inhibited the growth of the five phytopathogenic fungi. This activity may be produced by a single major compound or by the synergistic or antagonistic effect of various compounds (DEBA et al., 2008). Several authors have attributed the antifungal capacity of plant essential oils to the presence of components such as phenolic and terpene compounds (BEUCHAT, 1994; DAVIDSON, 1997; NYCHAS, 1995) indicated that mycelial growth inhibition is caused by the monoterpenes present in essential oils. These components would increase the concentration of lipidic peroxides such as hydroxyl, alkoxy and alko peroxyl radicals and so bring about cell death. However, the influence of essential oil or bioactive compounds on flavor and aroma of tomato was not investigated and further work should be conducted to purpose the use efficiency of oil components in real applications such as fumigant. In conclusion, this paper is a part of an overall study that aims to determine the antifungal activities of natural floral resources of Algeria, in order to find new bioactive natural products. The essential oils of these plants studied, exhibited an interesting antifungal activity against mycelial growth. Further work is necessary to explore the efficacy of these essential oils against disease rot of tomato and to exploit these oils as a new potential source of natural additives for the food and/or pharmaceutical industries. Acknowledgements The authors are indebted to the Ministère des Affaires Etrangères et Européennes throughout the research program “Partenariat Hubert Curien Tassili”. References AGRIOS, G., 1997: Plant pathology. 4th Ed., Academic Press, San Diego. 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