Journal of Applied Botany and Food Quality 87, 279 - 285 (2014), DOI:10.5073/JABFQ.2014.087.039 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 2Laboratoire des Substances Naturelles et Bioactives (LASNABIO) Département de Chimie, Faculté des Sciences, Université de Tlemcen, Algérie 3Université de Corse, UMR CNRS 6134, Laboratoire Chimie des Produits Naturels, Campus Grimaldi, Corte, France Control of fungal pathogens of Citrus sinensis L. by essential oil and hydrosol of Thymus capitatus L. Leila Tabti1, Mohammed El Amine Dib2*, Nassim Djabou2, Nassira Gaouar Benyelles1, Julien Paolini3, Jean Costa3, Alain Muselli3 (Received September 11, 2014) * Corresponding author Summary Essential oil, hydrosol extract and hydrosol of Thymus capitatus L. from Algeria were tested for antifungal activity against four phytopathogenic fungi (Aspergillus niger, Aspergillus oryza, Penicillium italicum and Fusarium solani) causing the deterioration of Citrus sinensis fruits. Essential oil and hydrosol extract showed strong in vitro antifungal activity based on the inhibition zone and minimal inhibitory concentration values against the pathogens. Citrus sinensis fruits infected by Penicillium italicum were treated in vivo with essential oil, hydrosol extract and hydrosol. 0.2 μg/mL of Thymus hydrosol was needed for the absence of orange infection and causing 100 % mycelial growth inhibition. This activity can be correlated with chemical composition of extracts which are rich in carvacrol (more than 69 %). Therefore, the preventive and curative effects of T. capitatus essential oil and hydrosol could be exploited as an ideal alternative to synthetic fungicides for using in the treatment of many fungal phytopathogens causing severe destruction to oranges. Introduction Citrus is an important crop with world production estimated at 115 million tons per year. During 2010-2011, 571 thousand tons were producer in Algeria which is the 19th produced in the world and the 3rd in the Arab Maghreb Union (LAGHA-BENAMROUCHE and MADANI, 2013). Citrus are among the most popular fruits grown in Algeria. Furthermore, Citrus production represents an important agricultural and economic activity in the country. Oranges and mandarins are traditionally produced for local consumption and also for export. Citrus is an important crop with world production estimated at 115 million tons per year. During 2010/2011, 571 thousand tons were produced in Algeria which is the 19th producers in the world and the 3rd in the Arab Maghreb Union (FAO, 2012) (LAGHA-BENAMROUCHE and MADANI, 2013). Oranges, lemons, grapefruits and mandarins represent approximately 98 % of industrial cultures. Oranges are most pertinent with about 82 % of total (LAGHA-BENAMROUCHE and MADANI, 2013). Fungal growth on fresh fruits and vegetables is responsible for food spoilage and numerous plant diseases, which lead to significant economic losses. Penicillium and Aspergillus were responsible for spoilage of many foods and causes decay on stored fruits damaged by insects, animals, early splits, and mechanical harvesting (TU et al., 2013; ROJAS et al., 2005). The industries of food products nowadays are using synthetic chemical preservatives to prevent the growth of pathogens, but these chemicals convert certain ingested materials into toxins and carcinogens (FARAG et al., 1989). Alternative control methods are needed because of negative public perceptions about the use of pesticides, development of resistance to fungicides, and high cost for development of new chemicals preservatives (STOJKOVIĆ et al., 2011). Thus, there has been a growing interest on the research of the possible use of plant secondary metabolites for pest and disease control in agriculture (GIKH et al., 2002). The plants have long been recognized to provide a potential source of different class of chemical compounds, known as phytochemicals, such as terpenoids, alkaloids, phenolics, glucosides, etc., which are effective products against pathogenic microorganisms (FENG and ZHENG, 2007). Essential oils are of growing interest both in the industry and scientific research because of their antibacterial and antifungal properties which make them useful as natural additives in foods (PATTNAIK et al., 1997). Such antimicrobial activity is due to the presence of bioactive substances such as monoterpenes, sesquiterpenes and related alcohols, other hydrocarbons and phenols (GRIFFIN et al., 1999; KALEMBA and KUNICKA, 2003). Thyme (Thymus) is a genus containing about 350 species of aromatic perennial herbs and sub-shrubs to 40 cm tall, belong to the family Lamiaceae. This family is distributed throughout the arid, temperate and cold regions including Europe, North Africa and Asia. It is in leaf all year, flowering from July to September (GRUENWALD et al., 2004). Several studies have assessed the ability of the Thymus essential oils and their constituents as fumigants and repellents against a number of insect pests (CLEMENTE et al., 2003; LEE et al., 2001; HORI, 2003, SALAMA et al., 2012). Effective antifungal activity of T. propolis from regions of Algeria was explained by their high content in thymol (49.3 %) and carvacrol (57.7 %) (MELLIOU et al., 2007). Molluscicidal activity of T. capitatus essential oils rich with carvacrol (32.98 %) and thymol (32.82 %) on adult and eggs of Biomphalaria alexandrina as well as on different stages of Culex pipiens was evaluated for their effectiveness on vector control (SALAMA et al., 2012). ASKARNE et al. (2012) showed that aqueous extract of Thymus leptobotrys completely inhibited mycelial growth of Penicillium italicum, a phytopathogenic fungi of Citrus. On the other hand, there is no report on the hydrosol composition from this species. Also, the antifungal activity of this hydrosol was reported for the first time against the development of fungi of Citrus sinensis fruit. Therefore, the present study was made (i) to examine the in vitro antifungal activity of the essential oil and hydrosol extract obtained from T. capitatus against four phytopathogenic fungi (Aspergillus niger, Aspergillus oryza, Penicillium italicum and Fusarium solani) and (ii) to test in vivo the essential oil, hydrosol extract and hydrosol against Penicillium italicum responsible of rotting the oranges. Materials and methods Plant material The aerial parts of T. capitatus were collected from Beni Snous forests near Tlemcen, Algeria in May 2011. Voucher specimens were deposited in the herbarium of the Tlemcen University Botanical Laboratory (Voucher number: UTL 05.11). 280 L. Tabti, M. El Amine Dib, N. Djabou, N. Gaouar Benyelles, J. Paolini, J. Costa, A. Muselli Essential oil, hydrosol and hydrosol extract Essential oil of fresh aerial parts (600 g) was isolated by hydro- distillation (HD) in a Clevenger type apparatus for 5 h, giving clear yellow oil. The yield of the oils was 0.52 %. The obtained essential oil was stored at +4 °C until further tests. The first liter of hydro- distillate is recovered in order to obtain T. capitatus hydrosol. Hydro- sol was submitted to Liquid Liquid Extraction (LLE). Half a liter of hydrosol was extracted three times with 200 mL of diethyl ether at room temperature. The organic layer dried over Na2SO4 and evapo- rated, so giving an oil yellowish, called hydrosol extract, with yield of 0.0016 %. (w/w). The 500 mL of hydrosol remaining were used to study the in vivo activity. Gas chromatography Analyses were carried out using a Perkin Elmer Clarus 600 GC apparatus equipped with a dual flame ionization detection system and 2 fused-silica capillary columns (60 m x 0.22 mm I.D., film thickness 0.25 μm), Rtx-1 (polydimethylsiloxane) and Rtx-Wax (polyethylene 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 oil and hydrosol extract were injected in the split mode (1/50), using 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 oil and hydrosol extract 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 following conditions: Ion source temperature 150 °C, energy ionization 70 eV, mass range 35-350 Da (scan time: 1 s). Component identification and quantification Identification of the components of the essential oil obtained by hydrodistillation (HD) and the hydrosol extract obtained by LLE was based (i) on the comparison of their GC retention indices (RI) on non-polar and polar columns, determined relative to the retention time of a series of n-alkanes with linear interpolation, with those of authentic compounds or literature data (JENNINGS and SHIBAMOTO, 1980; KÖNIG et al., 2001; NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY, 2008) and (ii) on computer matching with commercial mass spectral libraries (MC LAFFERTY and STAUFFER, 1994; MC LAFFERTY and STAUFFER, 1988; NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY, 1999) and comparison of spectra with those of in- house laboratory library. The quantification of essential oil components was carried out using peak normalization including response factors (RFs) with internal standard. The normalized % abundances were calculated, using the methodology reported by (BICCHI et al., 2008) in order to perform the statistical analysis of the oil. Tridecane was introduced in all sample oils at same concentration (0.7 g/100 g) as internal standard. Pathogenic fungi Four fungal isolates causing Citrus rot. Aspergillus niger, Aspergillus oryza, Penicillium italicum and Fusarium solani were isolated directly from rotten C. sinensis fruits harvested from orchards of the El-Fhoul cooperative in Tlemcen (Algeria). All isolated fungal species were transferred to sterilized three replicates 9 cm Petri dishes containing fresh Potato Dextrose agar medium (PDA) in the presence of a quantity of lactic acid (20 %) to 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 T. capitatus essential oil and hydrosol extract was tested using radial growth technique (BAJPAI et al., 2007). Appropriate volumes of the stock solutions of the natural mixtures (essential oil and hydrosol extract) in dimethyl sulfoxide (DMSO) to 10 %, were added to PDA medium immediately before it was poured into the Petri dishes (9.0 cm diameter) at 40-45 °C to obtain a series of concentrations (0.01 to 0.5 μg/mL). 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 7-day-old cultures on PDA plates, were transferred aseptically to the centre of petri’s dishes were incubated. Amphotericin B (5.0 to 126 μg/ml) was used as a positive control for antifungal activity. 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 (PANDEY et al., 1982): (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. The measurements were used to deter-The measurements were used to deter- mine the minimum inhibitory concentration (MIC) (The minimum concentration causing 100 % mycelial growth inhibition). The fun- gistatic-fungicidal nature of essential oil and hydrosol extract was tested by observing resumption of growth of the inhibited mycelial disc following its transfer to non-treated PDA. In order to determine fungistatic or fungicidal activity of volatile vapours of essential oils on mycelial growth, plates were further incubated at 26 °C for 7 days. Fungi resuming mycelial growth were considered to be fungistatic. In vivo antifungal assay For the in vivo antifungal assay, we used method described and developed previously by DIKBAS et al. (2008). The selected orange fruits for the experiments were washed in water, dipped in ethanol (70 %) for 2 min, rinsed twice with double distilled sterile water (10 min each) and air-dried. Surface-sterilized oranges were wounded with a flamesterilized nail to a uniform depth of 3 mm. The fungal inoculums containing 106 spores/mL was prepared by scraping spore material from the surfaces of the colonies with a wet cotton swab and resuspending the material in distilled water containing 0.5 % Tween 80. For testing antifungal in vivo activity against P. italicum, the essential oil and hydrosol extract, were separately mixed vigorously with distilled water to obtain two concentrations 0.1 and 0.2 μg/ mL, respectively. However, the hydrosol was applied directly with the concentration of 20 μg/mL. The essential oil, hydrosol extract and hydrosol of T. capitatus and fungal inoculum were sprayed separately on wounded orange fruits. The antifungal activity was tested on healthy fresh oranges. These experiments were arranged as three different applications. Fruits inoculated with only pathogen were used as positive control for each experiment. Non-inoculated fruits with pathogen were used as negative control. The fruits were sealed in polyethylene-lined plastic boxes to retain 70 % humidity and incubated at 25 °C storage condition. The diameters of decay on fruits were measured at 3, 6, 9, 12 and 15th days after inoculation. Antifungal activity of hydrosol against pathogens of Citrus 281 Tab. 1: Chemical compositions of T. capitatus essential oil (EO) and hydrosol extract (HY). No.a Components lRIa b RIa c RIpd EO HY Identification e 1 α-Thujene 932 924 1028 0.2 - RI, MS 2 α-Pinene 936 931 1028 0.9 - RI, MS 3 Camphene 950 945 1071 0.2 - RI, MS 4 Oct-1-en-3-ol 962 962 1441 0.5 0.2 RI, MS 5 β-Pinene 978 972 1113 0.1 - RI, MS 6 Myrcene 987 982 1160 2.1 - RI, MS 7 3-Octanol 981 982 1366 tr 0.1 RI, MS 8 α-Phellandrene 1002 999 1161 0.2 - RI, MS 9 3-Carene 1005 1006 1149 0.1 - RI, MS 10 α-Terpinene 1008 1011 1270 1.7 - RI, MS 11 p-Cymene 1015 1015 1270 12.4 - RI, MS 12 (Z)-β-Ocimene 1029 1022 1234 0.6 - RI, MS 13 γ-Terpinene 1051 1050 1245 4.3 - RI, MS 14 (E)-Sabinene hydrate 1051 1054 1445 0.1 0.6 RI, MS 15 Terpinolene 1082 1079 1281 0.2 - RI, MS 16 Linalool 1083 1085 1538 1.7 0.5 RI, MS 17 Phenylacetaldehyde 1112 1108 1591 - 0.1 RI, MS 18 Camphor 1123 1124 1506 0.1 - RI, MS 19 Isoborneol 1143 1144 1670 - 0.5 RI, MS 20 Borneol 1148 1150 1688 0.3 - RI, MS 21 Terpinen-4-ol 1164 1162 1591 1.1 - RI, MS 22 α-Terpineol 1176 1176 1690 0.1 0.2 RI, MS 23 trans-dihydro Carvone 1180 1182 1607 tr - RI, MS 24 trans-Myrtanol 1241 1242 1859 tr - RI, MS 25 Thymol 1266 1263 2181 0,6 0.1 RI, MS 26 Carvacrol 1278 1286 2193 69.6 95.1 RI, MS 27 Eugenol 1330 1329 2164 0.1 0.2 RI, MS 28 cis-Carvyl acetate 1343 1345 1858 0.1 0.2 RI, MS 29 (E)-β-Caryophyllene 1421 1416 1591 1.6 - RI, MS 30 (E)-α-Bergamotene 1434 1435 1573 tr - RI, MS 31 α-Humulene 1455 1448 1668 0.1 - RI, MS 32 γ-Humulene 1483 1480 1702 tr - RI, MS 33 β-Bisabolene 1503 1499 1721 0.1 - RI, MS 34 δ-Cadinene 1520 1511 1760 tr - RI, MS 35 (E)-α-Bisabolene 1530 1531 1755 0.1 - RI, MS 36 Spathulenol 1572 1560 2120 - 0,1 RI, MS 37 Caryophyllene oxide 1578 1567 1969 0.1 1.1 RI, MS 38 Humulene epoxide II 1602 1599 2044 - 0.1 RI, MS Total identification % 99.2 99.1 % Hydrocarbon compounds 12.6 - % Monoterpene hydrocarbons 10.7 - % Sesquiterpene hydrocarbons 1.9 - % Oxygenated compounds 86.7 99.1 % Oxygenated monoterpenes 3.5 2.2 % Oxygenated sesquiterpenes 0.1 1.3 % Non terpenic oxygenated compounds 0.5 0.4 % Aromatic terpenes 82.6 95.2 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; Normalized % abundances; MS: Mass Spectrometry in Electronic Impact (EI) mode; EO: essential oil from aerial parts obtained by HD; HY: Extract of hydrosol from aerial parts obtained by LLE. 282 L. Tabti, M. El Amine Dib, N. Djabou, N. Gaouar Benyelles, J. Paolini, J. Costa, A. Muselli Tab. 2: Antifungal activity of essential oil (EO) and hydrosol extract (HY) of T. capitatus against A. niger, A. oryzae, P. italicum and F. solani. Extracts A. niger A. oryzae F. solani P. italicum MIC a EC50 b MIC a EC50 b MIC a EC50 b MIC a EC50 b EO 0.5 ± 0.06 d 0.121 ± 0.80 0.2 ± 0.01 d 0.143 ± 0.80 0.2 ± 0.01 d 0.132 ± 0.22 0.1 ± 0.01 e 0.022 ± 0.06 HY 0.5 ± 0.01 d 0.121 ± 0.81 0.2 ± 0.01 d 0.143 ± 0.56 0.2 ± 0.01 d 0.132 ± 0.32 0.1 ± 0.01 e 0.022 ± 0.06 Am B c 46.2 ± 1.81 15.62 ± 1.06 46.2 ± 1.39 15.62 ± 1.50 126 ± 3.56 62.50 ± 1.79 61.2 ± 2.01 31.25 ± 1.11 a minimum concentration causing 100 % mycelial growth inhibition; b minimum concentration causing 50 % mycelial growth inhibition; c Am B : Amphotericin B (μg/ml) was used as reference Antibiotic; d fungicidal effect; e fungicistatic effect; The results are expressed as mean ± standard deviation. (μg/mL) All treatments consisted of three replicates, and experiments were repeated three times and determined the averages of the repeated experimental results. Taste panel Sensory evaluation of oranges treated with hydrosol was assessed by a group of 20 untrained panellists. Panellists were selected among students and staff of the laboratory of chemistry (LASNABIO). Orange samples were soaked into the hydrosol during 24 h, before the oranges were given to panellists to eat. The panellists were asked to evaluate flavor and odor of the orange samples on a scale from 5 to 1, where 1 = extremely dislike, 2 = dislike, 3 = neither like nor dislike, 4 = like; 5 = extremely like, according to a previous reports (STOJKO- VIĆ et al., 2011). A general taste score was calculated as the average of all grades. Sensory evaluation was accomplished at 1st, 2nd and 3rd day. Results were expressed as average grades given by 20 pa- nellists. Statistical Analysis Statistical analysis of variance (ANOVA) was performed using the SAS software and means were separated using the Least Significant Difference (LSD) test at P ≤ 0.05. Analysis of each test was performed in triplicate. Results Chemical analyses A total of 32 components accounting to 99.2 % of the essential oil (EO) 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 (20 components: 74.4 %) with high amount of aromatic terpenic components (82.6 %). How- ever, hydrocarbons appeared also in appreciable proportion (19 com- ponents: 25.0 %) with monoterpene hydrocarbons are well re- presented (23.1 %). Indeed, the main constituents 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 %). Conversely, the analysis of hydrosol extract (HY) obtained by LLE showed only 14 oxygenated compounds (7 monoterpenes, 3 sesquiterpenes, 3 non-terpenic components and 1 phenylpropanoid), no hydrocarbons were reported. In vitro antifungal activity of T. capitatus extracts against the development of fungi of C. sinensis The results obtained in assays of antifungal activity of T. capitatus essential oil and hydrosol extract by radial growth technique are re- ported in Tab. 2. However, data analysis showed that the antifungal activity of the essential oil and hydrosol extract concentration against the four fungi tested exhibited a significant difference (P < 0.05). The results indicate that the inhibition of the mycelial growth of each strain was significantly influenced by the extract concentrations. Essential oil and hydrosol extract inhibited completely all strains. The potent activity was observed against P. italicum with the EC50 of 0.022 μg/ mL followed by A. niger, F. solani and A. oryzae with the EC50 of 0.121, 0132 and 0.143 μg/mL, respectively. The minimum concentra- tion causing 100 % mycelial growth inhibition against P. italicum, A. oryzae and F. solani phytopathogens were very effective at 0.1, 0.2 and 0.2 μg/mL, respectively. However, the minimum concentra- tion causing 100% mycelial growth inhibition for A. niger strain was 0.5 μg/mL. The essential oil and hydrosol extract tested showed strong activity in comparison to the commercial drug Amphotericin B. The most sensitive species were P. italicum, F. solani and A. oryzae with MIC of 0.1, 0.2 and 0.2 μg/ml, respectively. A. niger (MIC of 0.5 μg/ ml) was the most resistant species to the essential oil and hydrosol ex- tract tested. While, MIC of Amphotericin B ranged to 46.2 to 126 μg/ ml for fungal species isolated. Moreover, it is important to know the fungitoxic nature of the essential oils and hydrosol extract. Indeed, the transfer of a mycelial disk of the plate containing a PDA medium and samples on fresh PDA (without oil and hydrosol) showed that no growth had developed after an incubation period of 7 days, sug- gesting a fungicidal effect of essential oils and hydrosol extract on F. solani, A. oryza, A. niger at 0.2, 0.2 and 0.5 μg/mL, respectively. On the other hand, essential oils and hydrosol extract was fungistatic on P. italicum (Tab. 2). In vivo orange assay The results of in vivo P. italicum Citrus rot treatment with essential oil, hydrosol extract and hydrosol are presented in Tab. 3. According to the increase of concentration, a decrease of disease incidence was recorded. According to the results of lesion diameters on the fruits, both concentrations (0.1 and 0.2 μg/mL) of the essential oil and hydrosol extract showed strong antifungal activity even at the end of 15th day, there was no significant difference in lesion diameters among those treatments in comparison to the negative control. The concentration of 0.2 μg/mL of essential oil and hydrosol extract were needed for the absence of orange infection and low disease incidence. More, the hydrosol showed a complete absence of orange infection and no disease incidence with a concentration of 0.2 μg/mL. The result obtained from the hydrosol was showed on Fig. 1. Taste panel Tab. 4 shows the acceptability scores of orange samples. Results showed that there were no significant differences in the sensory properties between samples treated with hydrosol, essential oil and control (without hydrosol), since the sensory properties of oranges treated with hydrosol (0.2 μg/mL) and essential oil (0.2 μg/mL) Antifungal activity of hydrosol against pathogens of Citrus 283 Tab. 3: Means of decay diameters (mm) measured after 3, 6, 9, 12 and 15 days on orange fruits treated with 0.1 or 0.2 μg/mL of essential oil, hydrosol extract and hydrosol from T. capitatus. Treatments Means ± SD of decay diameters (mm) 3 days 6 days 9 days 12 days 15 days Controls negative 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Controls Positive 1.1 ± 0.2 2.8 ± 0.3 5.7 ± 0.5 8.1 ± 0.8 13.2 ± 0.2 Essential oil 0.1 μg/mL 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.01 1.5 ± 0.2 2.5 ± 0.5 0.2 μg/mL 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.01 1.0 ± 0.2 Hydrosol extract 0.1 μg/mL 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.06 1.5 ± 0.2 0.2 μg/mL 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.02 Hydrosol 0.2 μg/mL 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Fig 1: Decayed orange (left), inoculated with only pathogen P. italicum (positive control), and not decayed orange (right) onto which hydrosol (0.2 μg/mL) was applied 30 min before pathogen inoculation, kept at 25 °C for the period stated below the respective picture. 284 L. Tabti, M. El Amine Dib, N. Djabou, N. Gaouar Benyelles, J. Paolini, J. Costa, A. Muselli were deemed acceptable by the panelists at the supplementation levels. More work on the acceptability of both extracts as oranges preservative will be necessary. Discussion In the present investigation, a total of 14 and 38 compounds comprising 99.1 and 99.2 % of the hydrosol extract and essential oil were identified from T. capitatus respectively, carvacrol being the major component, comprising 95.1 and 69.6 % of the hydrosol and essential oil, respectively. This study agrees with the findings of RUBERTO et al. (1992), AMARTI et al. (2008) and TAWAHA et al. (2012), who reported carvacrol to be the major component of T. capitatus essential oil. In the present investigation, the reduction of the mycelial growth of colonies in presence of essential oil and hydrosol extract of T. capitatus showed that it effectively controlled all strains. This effi- ciency can be explained by the presence of active molecules that inhibited the growth of the phytopathogenic fungi. The antifungal properties of T. capitatus essential oil and hydrosol extract are probably associated with the high amount of phenolic terpenes, especially the main component carvacrol. Indeed, carvacrol is used as a disinfectant, fungicide, and fragrance ingredient in cosmetic formulations. In addition, DAFERERA et al. (2000) reported that the fungitoxic activity of essential oils may have been due to formation of hydrogen bonds between the hydroxyl group of oil phenolics and active sites of target enzymes. Although the extracts or essential oils of T. capitatus have been screened for their antifungal activity under in vitro conditions (MELLIOU et al., 2007; DIKBAS et al., 2008; NYCHAS, 1996), there are no reports on the control of P. italicum under in vivo conditions by using the essential oil and hydrosol of T. capitatus. The essential oil and hydrosol of T. capitatus from Algeria was characterized by high content of carvacrol (69.6 % and 95.1 %, respectively). Little literature exists on the effect of carvacrol on these food pathogenic fungi (MORCIA et al., 2012), although carvacrol was effective on in- hibiting spore germination of Botrytis cinerea when applied to the potato dextrose agar (MARTINEZ-ROMERO et al., 2007). In addition, MARKOVIC et al. (2011) demonstrated that carvacrol has a remarkably antifungal potential against Aspergillus spp. and Penicillium spp. In a study conducted by MULLER-RIEBAU et al. (1995), carvacrol showed a remarkable antifungal activity by inhibition of the mycelial growth of Fusarium spp. BOUDDINE et al. (2012) revealed that Aspergillus niger growth was completely inhibited by carvacrol at concentrations of 0.025 %. Furthermore, this study confirmed the antifungal activ- ity of this component against mycelial growth of P. italicum in vitro and in vivo assays. The World Health Organization (WHO) has stated that carvacrol residues in food are without danger to the consumer as long as they do not exceed 50 mg kg-1 (WHO, 2012). It is thus clear that treatment by hydrosol is an easy to prepare and the cost price of extraction is not expensive. However, the benefits of hydrosol (non- toxic at low doses, biodegradable, and no risk for resistance devel- opment) and disadvantages of chemical fungicides on health and on the environment make hydrolate of T. capitatus more interesting for citrus postharvest treatment. Conclusion In conclusion, 38 compounds of T. capitatus essential oil and hydrosol extract were identified. This oil was characterized by high content of carvacrol. Furthermore, this study confirmed the antifungal activity of the essential oil and hydrolate against mycelial growth and spore production of A. niger, A. oryzae, F. solani and P. italicum from in vitro assay. In vivo inhibitory properties of hydrosol on disease incidence of P. italicum-causal agent of penicillium rot on oranges were recorded. Therefore, the preventive and curative effects of T. capitatus hydrolate could be exploited as an ideal alternative to synthetic fungicides for using in the treatment of many fungal phytopathogens causing severe destruction to oranges. Acknowledgements The authors are indebted to the Ministère des Affaires Etrangères et Européennes through the research program France-Algérie “Partenariat Hubert Curien Tassili”. References AMARTI, F., SATRANI, B., AAFI, A., GHANMI, M., FARAH, A., ABERCHANE, M., EL AJJOURI, M., EL ANTRY, S., CHAOUCH, A., 2008: Composition chimique et activité antimicrobienne des huiles essentielles de Thymus capitatus et de Thymus bleicherianus du Maroc. Phytotherapie 6, 342- 347. ASKARNE, L., TALIBI, I., BOUBAKER, H., BOUDYACH, E.H., MSANDA, F., SAADI, B., SERGHINI, M.A., AIT BEN AOUMAR, A., 2012: In vitro and in vivo antifungal activity of several Moroccan plants against Penicillium italicum, the causal agent of citrus blue mold. Crop Prot. 40, 53-58. BAJPAI, V.K., RAHMAN, A., KANG, S.C., 2007: Chemical composition and anti- fungal properties of the essential oil and crude extracts of Metasequoia glyptostroboides Miki ex Hu, Ind. Crop Prod. 26, 28-35. BARNETT, H.L., Hunter, B.B., 2006: Illustrated genera of imperfect fungi. 4th Ed., The American Phytopatological Society, St. Paul Minnesota. BICCHI, C., LIBERTO, E., MATTEODO, M., SGORBINI, B., MONDELLO, L., ZELLNER, B.A., COSTA, R., RUBIOLO, P., 2008: Quantitative analysis of essential oils: a complex task. Flav. Fragr. J. 23, 382-391. BOUDDINE, L., LOUASTE, B., ACHAHBAR, S., CHAI, N., CHAMRI, F., REMMAL, A., 2012: Comparative study of the antifungal activity of some essential oils and their major phenolic components against Aspergillus niger using three different methods. Afr. J. Biotechnol. 11, 14083-14087. CLEMENTE, S., MAREGGIANI, G., BROUSSALIS, A., MARTINO, V., FERRARO, G., 2003: Insecticidal effects of Lamiaceae species against stored products insects. Bol. San. Veg. Plagas. 29, 1-8. DAFERERA, D.J., ZIOGAS, B.N., POLISSIOU, M.G., 2000: GC-MS analysis of essential oils from some Greek aromatic plants and their fungitoxicity on Penicillium digitatum. J. Agric. Food Chem. 48, 2576-2581. DIKBAS, N., RECEP KOTAN, F., DADASOGLU, F.S., 2008: Control of Asper- gillus flavus with essential oil and methanol extract of Satureja hortensis. Int. J. Food Microbiol. 124, 179-182. FARAG, R.S., DAW, Z.Y., HEWEDI, F.M., EL-BAROTY, G.S.A., 1989: Antimicrobial activity of some egyptian spice essential oils. J. Food. Protect. 52, 665-667. FENG, W., ZHENG, X., 2007: Essential oils to control Alternaria alternata in vitro and in vivo. Food Control 18, 1126-1130. GIKH, M., ABDEL RASSOUL, M.A., ABDELGALEIL, S.A.M., 2012: Compara- tive antifungal activities and biochemical effects of monoterpenes on plant pathogenic fungi. Pestic. Biochem. Phys. 103, 56-61. GRIFFIN, S.G., WYLLIE, S.G., MARKHAM, J.L., LEACH, D.N., 1999: The role of structure and molecular properties of terpenoids in determining their Tab. 4: Effect of essential oil (EO) and hydrosol (HY) on acceptability sensory scores of orange stored at 25 °C. Treatment Days of Storage HY 1 2 3 Acceptabilitya 4.5 4.6 4.2 EO 3.9 4.0 3.8 The results are expressed as the average of all grades. a 1 = extremely dislike, 2 = dislike, 3 = neither like nor dislike, 4 = like; 5 = extremely like. Antifungal activity of hydrosol against pathogens of Citrus 285 antimicrobial activity. Flav. Fragr. J. 14, 322-332. GRUENWALD, J., BRENDLER, T., JAENICKE, C., 2004: PDR for Herbal Medi- cines. 3th Ed. Montvale (NJ), Thompson, PDR, 815-816. HORI, M., 2003: Repellency of essential oils against the cigarette beetle, Lasioderma serricorne (Fabricius) (Coleoptera: Anobiidae). Appl. Entomol. Zool. 38, 467-473. JENNINGS, W., SHIBAMOTO, T., 1980: Qualitative analysis of flavour and fragrance volatiles by glass-capillary gas chromatography. In: Jovanovich, H.B. (ed.), Academic Press, New-York. KALEMBA, D., KUNICKA, A., 2003: Antibacterial and antifungal properties of essential oils. Curr. Med. Chem. 10, 813-829. KÖNIG, W.A., HOCHMUTH, D.H., JOULAIN, D., 2001: Terpenoids and related constituents of essential oils. Library of Mass Finder 2.1. Institute of Organic Chemistry, Hamburg, Germany. LAGHA-BENAMROUCHE, S., MADANI, K., 2013: Phenolic contents and anti- oxidant activity of orange varieties (Citrus sinensis L. and Citrus auran- tium L.) cultivated in Algeria: Peels and leaves. Ind. Crop. Prod. 50, 723- 730. LEE, B.H., CHOI, W.S., LEE, S.E., PARK, B.S., 2001: Fumigant toxicity of essential oils and their constituent compounds towards the rice weevil, Sitophilus oryzae (L.). Crop Prot. 20, 317-320. MARKOVIC, T., CHATZOPOULOU, P., SĬILJEGOVIC, J., NIKOLI, N., GLAMO- CLIJA, J., CÍRIC, A., SOKOVIĆ, M., 2011: Chemical analysis and anti- microbial activities of the essential oils of Satureja thymbra L. and Thym- bra spicata L. and their main components. Arch. Biol. Sci. 63, 457-464. MARTINEZ-ROMERO, D., GUILLEN, F., VALVERDE, J.M., BAILEN, G., ZAPATA, P.J., SERRANO, M., VALERO, D., 2007: Influence of carvacrol on survival of Botrytis cinerea inoculated in table grapes. Int. J. Food Microbiol. 115, 144-148. MC LAFFERTY, F.W., STAUFFER, D.B., 1994: Wiley Registry of Mass Spectral Data, 6th Ed., Mass Spectrometry Library Search System Bench-Top/ PBM version 3.10d. Palisade, Newfield. MC LAFFERTY, F.W., STAUFFER, D.B., 1988: The Wiley/NBS Registry of Mass Spectral Data, 4th Ed.. Wiley-Interscience, New York. MELLIOU, E., STRATIS, E., CHINOU, I., 2007: Volatile constituents of propolis from various regions of Greece ‒ Antimicrobial activity. Food Chem. 103, 375-380. MORCIA, C., MALANATI, M., TERZI, V., 2012: In vitro activity of terpinen- 4-ol, eugenol, carvone, 1,8-cineole (eucalyptol) and thymol against mycotoxigenic plant pathogens. Food Addit. Contam. 29, 415-422. MULLER-RIEBAU, F., BERGER, B., YEGEN, O., 1995: Chemical composition and fungitoxic properties to phytopathogenic fungi of essential oils of selected aromatic plants growing wild in Turkey. J. Agric. Food Chem. 43, 2262-2266. NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY, 2008: Spectral Database for Organic Compounds, NIST WebBook: http://webbook.nist. gov/chemistry. NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY, 1999: PC version 1.7 of the NIST/EPA/NIH Mass Spectral Library Norwalk. Perkin-Elmer Corp., CT, USA. NYCHAS, G.J.E., 1996: Natural antimicrobial from plants. In: Gould, G.W. (ed.), New methods of food preservation, 235-258. CRC Press, London. PANDEY, D.K., TRIPATHI, N.N., TRIPATHI, R.D., 1982: Fungitoxic and phytotoxic properties of essential oil of Hyptis suaveolens. Z. Pfl. Krankh. Pfl. Schutz 89, 344-349. PATTNAIK, S., SUBRAMANYAM, V.R., BAPAJI, M., DIXIT, S.N.Z., 1997: Antibacterial and antifungal activity of aromatic constituents of essential oils. Microbios. 89, 39-46. ROJAS, T.R., SAMPAYO, C.A.F., VÁZQUEZ, B.I., FRANCO, C.M., CEPADA, A., 2005: Study of interferences by several metabolites from Aspergillus spp. in the detection of aflatoxigenic strains in media added with cyclodextrin. Food Control 16, 445-450. RUBERTO, G., BIONDI, D., PIATTELLI, M., 1992: The essential oil of Sicilian Thymus capitatus (L.) Hoffmanns, et Link. J. Essent. Oil Res. 4(4), 417- 418. SALAMA, M.M., TAHER, E.E., EL BAHY, M.M., 2012: Molluscicidal and mosquitocidal activities of the essential oils of Thymus capitatus L. and Marrubium vulgare L. Am. J. Drug Discov. Dev. 2(4), 204-211. STOJKOVIĆ, D., SOKOVIĆ, M., GLAMOČLIJA, J., DŽAMIĆ, A., ĆIRIĆ, A., RISTIĆ, M., GRUBIŠIĆ, D., 2011: Chemical composition and antimicro- bial activity of Vitex agnus-castus L. fruits and leaves essential oils. Food Chem. 128, 1017-1022. TAWAHA, K.A., HUDAIB, M.M., 2012: Chemical composition of the essential oil from flowers, flower buds and leaves of Thymus capitatus Hoffmanns. & Link from Jordan. J. Essent. Oil Bear Pl. 15(6), 988-996. TU, Q., CHENA, J., GUOC, J., 2013: Screening and identification of antagonis- tic bacteria with potential for biological control of Penicillium italicum of citrus fruits. Sci. Hortic. 150, 125-129. WHO, 2012: http://apps.who.int/medicinedocs/en/d/Js2200e/28. html2012 (accessed April). Address of the corresponding author: E-mail: a_dibdz@yahoo.fr