Food Sci. Biotechnol. 22(S): 113-119 (2013) DOI 10.1007/s10068-013-0056-2
RESEARCH ARTICLE
In vitro Antifungal Activity and Chemical Composition of Warionia saharae Essential Oil against 3 Apple Phytopathogenic Fungi Mohamed Znini, Gregory Cristofari, Lhou Majidi, Abdelhay El Harrak, Julien Paolini, and Jean Costa
Received: 8 May 2012 / Revised: 7 September 2012 / Accepted: 6 October 2012 / Published Online: 28 February 2013 © KoSFoST and Springer 2013
Abstract Essential oil of aerial parts of Warionia saharae was obtained by hydrodistillation and analyzed by GC and GC-MS. Thirty-nine compounds were identified, accounting for 93.2% of the total oil. β-Eudesmol (34.9%), nerolidolE (23%), and linalool (15.2%) were the most abundant components. The antifungal activity of the W. saharae oil was tested by poisoned food (PF) technique and the volatile activity (VA) assay against 3 phytopathogenic causing the deterioration for apple. The results indicated that the W. saharae oil inhibited significantly the mycelial growth of all strains tested (p2 µL/mL in PF technique for all strains. Fungal spore production was completely inhibited at 1 µL/mL air for Alternaria sp. and at 2 µL/mL air for Penicillium expansum and Rhizopus stolonifer. Keywords: Warionia saharae, essential oil, GC-MS, antifungal activity, apple
Introduction Phytopathogenic fungi are one of the major economic Mohamed Znini, Lhou Majidi () Universit My Ismail, Laboratoire des Substances Naturelles & Synthèse et Dynamique Moléculaire, Faculté des Sciences et Techniques, BP 509, 52003, Errachidia, Morocco Tel: +212-535574497; Fax: +212-535574485 E-mail:
[email protected] Gregory Cristofari, Julien Paolini, Jean Costa Universit de Corse, CNRS-UMR 6134, Laboratoire de Chimie des Produits Naturels, BP 52, 20250 Corti, France Abdelhay El Harrak Universit My Ismail, Laboratoire de Protection & Amélioration et Ecophysiologie Végétale, Faculté des Sciences et Techniques, BP 509, 52003, Errachidia, Morocco
problems of crop production. Apart from their potentiality to cause yield losses and fruit decay, many of them represent a very serious risk for consumers because of their production of dangerous mycotoxins (1). Many pathogens including Alternaria species (Alternaria rot), Penicillium expansum (blue mold), and Rhizopus stolonifer (Rhizopus rot) reduce the market values and deteriorate the quality of fruits, especially apples, and render them unfit for human consumption and cause undesirable effects on human health (2). For many years, a variety of different synthetic fungicides have been extensively used as antifungal agents to inhibit the growth of phytopathogenic fungi. However, they are not considered as long-term solutions due to the concerns associated with exposure risks health and environmental hazards residue persistence. In addition, development of resistance to commonly used fungicides within populations of postharvest pathogens has also become a significant problem (3). Due to these perceived health risks associated with the use of these compounds, there is an increasing public concern on their use as postharvest treatments and consequently, scientists have been prompted to find alternative treatments. Recently, interests have been generated in the development of safer anti-fungal agents such as plant-based essential oils and extracts to control phytopathogens in food and agriculture industries (3). Essential oils are made up of many different volatile compounds and their production by plants is believed to be predominantly a defense mechanism against pathogens and pests, and they have been shown to possess antimicrobial and antifungal properties (2). They are considered as non-phytotoxic compounds, less environmental effects, and wide public acceptance (2,3). The genus Warionia is a monotypic genus of Asteraceae endemic to the north-western edge of the African Sahara desert. The species W. saharae is a somewhat thistle-like aromatic plant of 1 to 3 m of height, with white latex and
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fleshy, pinnately-partite leaves (4). The thick trunk is covered of a gray peel, structural of very wavy terminal leaf bouquets and of capitulate of yellow flowers. The flowering season has been recorded from April to June, while it may extend to July or August if the spring rains are abundant and well spaced (5). In Morocco, W. saharae grows on slopes of the High Atlas, Anti-Atlas, and Saharian Atlas in the coast of western Morocco and in desert areas on basic and siliceous rocks from 0 to 1,300 m. This plant, known locally by the vernacular name of ‘afessas’, is considered to have medicinal properties (6). Decoction of dried leaves is used as antirheumatic, for gastrointestinal disorders and against epileptic crisis (7). Crude extracts of the plants showed antibacterial and cytotoxic activities against a cancer cell line (KB cells) (8). However, to our knowledge and according to literature survey, only 2 studies have been published concerning the chemical composition of W. saharae essential oil. But there is no report on the antifungal activity of W. saharae essential oil. Therefore, the present study was made to determine the chemical composition of W. saharae essential oil and its antifungal activity against phytopathogenic fungal species causing the deterioration of apple with emphasis on the possible future application of the essential oil as an alternative antifungal agent.
Materials and Methods Plant materials and essential oil isolation The aerial parts of Warionia saharae were harvested in March/April 2009 from Errachidia (south-eastern of Morocco). Identification of the species was confirmed by biology unity and voucher specimens were deposited in the herbarium of Faculty of Sciences and Technology of Errachidia (Marocco). The dried vegetal material (100 g) was water-distillated (3 h) using a Clevenger-type apparatus according to the method recommended in the European Pharmacopoeia (9). The essential oil obtained was dried under anhydrous sodium sulfate and stored at 4oC in the dark before analysis. The average yield of essential oil was about 1.20%. GC analysis GC analysis was carried out using a PerkinElmerAutosystem XL GC apparatus (Waltham, MA, USA) equipped with a dual flame ionization detection (FID) system and the fused-silica capillary columns (60 m×0.22 mm i.d., film thickness 0.25-mm) Rtx-1 (polydimethylsiloxane) and Rtx-wax (polyethyleneglycol). The oven temperature was programmed from 60 to 230oC at 2oC/min and then held isothermally at 230oC for 35 min. Injector and detector temperatures were maintained at 280oC. Samples were injected in the split mode (1/50) using helium as a carrier gas (1 mL/min) and a 0.2 mL injection volume of pure oil.
Znini et al.
Retention indices (RI) of compounds were determined relative to the retention times of a series of n-alkanes (C5C30) (Restek, Lisses, France) with linear interpolation using the Van den Dool and Kratz equation and software from Perkin-Elmer (10). GC-MS analysis Samples were analyzed with a PerkinElmer turbo mass detector (quadrupole) coupled to a Perkin-Elmer autosystem XL equipped with the fusedsilica capillary columns Rtx-1 and Rtx-wax. Carrier gas, helium (1 mL/min); ion source temperature, 150oC; oven temperature programmed from 60 to 230oC at 2oC/min, and then held isothermally at 230oC (35 min); injector temperature, 280oC; energy ionization, 70 eV; electron ionization mass spectra were acquired over the mass range 35-350 Da; split, 1/80; injection volume, 0.2 mL of pure oil. Identification of essential oil constituents 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 compounds and literature data (11,12); and (ii) of the mass spectra with those of our own library of authentic compounds and with those of a commercial library (12,13). Quantification of essential oil constituents Quantification of the essential oil components was carried out using the methodology reported by Costa et al. (14), 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 (2). RFs and calibration curves were determined by diluting each standard in hexane at 5 concentrations, 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 standards) of each chemical group was calculated and used to determine the essential oil component concentration (g/100 g) according to the chemical group. Fungal strains isolation Three fungal isolates causing apples rot, Alternaria sp., Penicillium expansum, and Rhizopus stolonifer were isolated directly from rooted apples collected from different rooms in Midelt station (Morocco). All isolated fungal species were transferred to sterilized 3 replicates 9 cm petri dishes containing fresh potato dextrose agar (PDA) medium in the presence of a quantity of streptomycin to stop the growth of bacteria.
In vitro Antifungal Activity and Chemical Composition of Warionia saharae Essential Oil
The plates were incubated at 25±2oC for 7 days and darkness. The developing fungal colonies were purified and identified up to the species level by microscopic examination through the help of the references (15). The isolates collected were maintained on PDA at 4oC. Antifungal activity assay The antifungal activity of the essential oil of W. saharae against mycelial growth of fungi isolated was assessed using poisoned food (PF) technique (16) and volatile activity (VA) assay (17) with some modifications. PF technique In PF, the essential oil was dispersed as an emulsion in sterile agar suspension (0.2%) and added to PDA immediately before it was emptied into the glass petri dishes (90×20 mm i.d.) at a temperature of 40-45oC. The concentrations tested were 0.125 to 2 µL/mL. The controls received the same quantity of sterile agar suspension (0.2%) mixed with PDA. The tested fungi were inoculated with 6-mm mycelial plugs from 7-day-old cultures cut with a sterile cork and incubated for 3 days for R. stolonifer and 6 days for Alternaria sp. and P. expansum at 25±2oC. VA assay In VA assay, the petri dishes (90×20 mm) were filled with 20 mL of PDA medium and then seeded with a mycelial disc (6-mm diameter), cut from the periphery of 7-day-old mycelium culture of the tested fungi. The petri dishes (90×20 mm, which offer 80 mL air spaces after addition of 20 mL agar media) were inverted and sterile filter paper discs (9-mm i.d.) impregnated with different concentrations of essential oil, 0.125, 0.25, 0.5, 1, and 2 µL/mL air are deposited on the inverted lid and incubated for 3 days for R. stolonifer and 6 days for Alternaria sp. and P. expansum at 25±2oC. For each corresponding control, equal amount of water was poured on the sterilized paper filter. In both types of experiments, 3 replicate plates were inoculated for each treatment (fungi/amount) and the experiment was conducted 3 times and the mycelial growth was measured the diameter following 2 perpendicular lines passing by the center of the dish. Fungitoxicity of essential oil was expressed in terms of percentage of mycelial growth inhibition (I %) and calculated following the formula of Pandey et al. (18). D t – Di I(%) = -------------- × 100 Dt
where, Dt and Di represent mycelial growth diameter in control and treated petri plates, respectively. The measurements were used to determine the minimum inhibitory concentration (MIC) (lowest concentration of the essential oil that inhibits the visible growth of a microorganism after overnight incubation) and the EC50
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values (concentration causing 50% inhibition of mycelial growth on control media). The EC50 value was calculated according to the relationship of essential oil concentrations and percentage inhibition of mycelial growth. The fungistaticfungicidal nature of essential oil was tested by observing revival of growth of the inhibited mycelial disc following its transfer to non-treated PDA. A fungicidal effect was observed where there was no growth, whereas a fungistatic effect was where temporary inhibition of microbial growth occurred. Spore production assay Fungal spore production was tested using the modied method of Tzortzakis and Economakis (19). The spores of the previously exposed colonies by essential oil vapor were collected by adding 5 mL sterile water containing 0.1% Tween-20 to each petri dishes and rubbing the surface 3 times with the sterile Lshaped spreader to free spores. The spore suspensions obtained were filtered through sterilize cheesecloth into a sterile 50-mL glass beaker and homogenized by manual shaking. Spore concentration was estimated using a haemocytometer slide (depth 0.1-mm, 0.0025 mm2). The percentage reduction of spore production was computed by the following equation: Nt – Ni - × 100 I(%) = ------------Nt
where, Nt and Ni represent the number of spore in control and treated petri plates, respectively. Data analysis The inhibitory effect of essential oil on mycelial growth was analyzed by an analysis of variance (ANOVA). Mean and standard error of data were calculated using SAS software (version 9.0; SAS Inc., Cary, SC, USA). The separation of means was done by using the least significant difference (LSD) test at p
