Journal of Experimental Biology and Agricultural Sciences, October - 2014; Volume – 2(5)
Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org
ISSN No. 2320 – 8694
ANTIFUNGAL ACTIVITY OF CLOVE (Syzygium aromaticum L.) ESSENTIAL OIL AGAINST PHYTOPATHOGENIC FUNGI OF TOMATO (Solanum lycopersicum L) IN ALGERIA Hamini-Kadar N*, Hamdane F, Boutoutaou R, Kihal M and Henni J E Laboratory of Applied Microbiology, Department of Biology, Faculty of Nature Sciences and Life, University of Oran, BP 16.Es-Senia, 31100, Oran, Algeria Received – August 20, 2014; Revision – September 08, 2014, Accepted – September 22, 2014 Available Online – ARTICLE IN PRESS
KEYWORDS ABSTRACT Antifungal Activity Essential Oil Syzygium aromaticum Fusarium oxysporum f.sp radicis lycopercisi
Present study was conducted to find the efficiency of Syzygium aromaticum [L.] essential oil in management of three pathogenic fungus viz Fusarium oxysporum f.sp radicis lycopercisi, F. commune and F. redolens. The yield of hydro-distilled essential oil was also computed (7.49%) in study. The results of study revealed moderate to high level antifungal activities against tested microorganisms at all the tested concentration. The highest antifungal activity was recorded at 1µLml-1; on this concentration clove essential oil caused complete growth inhibition of F. oxysporum f.sp radicis lycopercisi and F. redolens while the highest antifungal activity of oil against F. commune was observed at 0.5 µLml-1.
Fusarium commune Fusarium redolens
* Corresponding author E-mail:
[email protected] (Hamini-Kadar N) Peer review under responsibility of Journal of Experimental Biology and Agricultural Sciences.
Production and Hosting by Horizon Publisher (www.my-vision.webs.com/horizon.html). All_________________________________________________________ rights reserved. Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org
448
1 Introduction Higher aromatics plants have traditionally been used in folk medicine; antimicrobial properties of these plants are well documented against bacteria, fungi and yeasts (Karkosh, 2012). Most of the medicinal properties of these plants are directly correlated with the essential oils produced by these plants. Essential oils and extracts of these plants are able to control microorganisms related to skin diseases, dental caries and food spoilage (Chaieb et al., 2007). Essential oils are aromatic volatile oily hydrophobic liquid concentrates that are extracted from plant material, such as flowers, buds, seeds, leaves, twigs, bark, wood, fruits, roots and whole plant. These essential oils are highly complex mixtures of 20 to 60 volatile compounds, albeit some may contain more than 100 different components (Djilani & Dicko, 2012, Ibrahium et al., 2013). These essential oils contain a variety of volatile molecules such as terpenes, terpenoids and phenol derived aromatic and aliphatic compounds, which might have bactericidal, antiviral, and fungicidal consequences (Akthar et al., 2014). Terpenoids are the primary constituents of the essential oils responsible for the aroma and flavor (Nuzhat & Vidyasagar, 2013). Several studies have been published to confirming the effect of essential oil and their major compounds on pathogenic fungi. Kurita et al. (1981) screened 40 plants compounds against seven species of fungi and found that some plant metabolites are highly effective against the fungal pathogens. Similarly, Nosrati et al. (2011) have studies the antifungal properties of spearmint essential oil against F. oxysporum f. sp. radiciscucumerinum and concluded that spearmint essential oil can be used to control the F. oxysporum f. sp. radicis-cucumerinum. Furthermore, Arici et al., (2013) also investigate in vitro antifungal activity of six essential oils viz Cumin, Thymus, Lavandula, Eucalyptus, Rosemary, Nigella and Dill against the F. oxysporum f.sp. radicis-lycopersici and F. oxysporum f.sp. lycopersici. Clove (Syzygium aromaticum L. Merrill and Perry) is one of the most valuable spices that have been used from centuries as food preservative and for many medicinal purposes. Cloves are native of Indonesia but nowadays are cultivated in several parts of the world (Cortés-Rojas et al., 2014). Flower bud have many medicinal proprieties like antiviral, antimicrobial, antifungal general stimulating, hypertensive aphrodisiac, light stomachic, carminative and anesthetic (de Paoli et al., 2007; Politeo et al., 2010; Koba et al., 2011; Machado et al., 2011). Anitifungal properties of clove essential oil exhibited strong inhibitory effects against the mycelial growth of Botrytis cinerea (Sirirat et al., 2009). Similarly, Pinto et al. (2009) investigated antifungal activities of the clove essential oil against clinical isolates and collection strains of Candida, Aspergillus & dermatophyte species and identified its main
_________________________________________________________
Journal of Experimental Biology and Agricultural Sciences http://www.jebas.org
Hamini et al
component as eugenol which is responsible for antifungal properties of essential oil against all the tested strains. Crown and root rot caused by F. oxysporum f.sp radicis lycopersici, F. redolens and F. commune were very common and one of the most destructive soil borne fungal diseases of tomatoes occurring in greenhouse and field crop in Algeria (Hamini-Kadar et al., 2010). Several fungicides have been used for managements of these diseases in tomato. Very limited informations are available about the use of natural product especially essential oils against these destructive pathogens. The purpose of this study was to examine the antifungal activity of clove essential oil extract against these phytopathogenic fungi. 2 Materials and Methods 2.1 Essential oil extraction by hydro-distillation The dried flower buds of Cloves plant used in this study were purchased from local market of Saida, Algeria. The extraction of essential oils was performed by hydro-distillation (Figure 1) in a Clevenger apparatus, 50 g of dry cloves were powdered immersed in a 250 ml flask with distilled water for 3-4 hours. Essential oils recovered in small opaque bottles (Ranjitha & Vijiyalakshmi, 2014). The extraction yield of essential oil was calculated by the method described by Bssaibis et al. (2009) which defined as follows: R (%) =M X 100/M0 Whereas R (%): efficiency M: weigh of extract recovered in g. M0: weigh of dry extract used for the extraction in g 2.2 Preparation of fungi Three species of Fusarium viz F. oxysporum f.sp radicis lycopersici, F. commune and F. redolens were obtained from the Laboratory of Applied Microbiology, Department of Biology, Faculty of Nature and Life Sciences, University of Oran, Algeria. Theses strains were selected by their aggressiveness among tomatoes. All the collected pathogens were molecularly identified by their EF-1α sequences. Pure culture of the fungal isolate was maintained by aseptic transfer to a freshly prepared PDA medium. 2.3. Screening of antifungal activity Effect of clove essential oil was assessed by agar diffusion plate method. Different volumes of crude extracts were incorporated into PDA medium just before pouring in sterilized Petri dishes to obtain different concentrations. Essential oil concentrations were prepared at 0.1, 0.2, 0.4, 0.5, 1, 2 and 5 µl/ml.
Antifungal Activity of Clove (Syzygium aromaticum L.) Essential Oil against phytopathogenic fungi of tomato (Solanum lycopersicum L) in Algeria.
Five millimeter mycelial discs were taken from the margins of 7 days old culture and placed on the middle of a PDA plate; petri plates were sealed with parafilm and incubated at 28±2°C for 7 days. Three replicates of each treatment were arranged in randomized block design (RBD) in incubator. The radial growth of colonies has been measured and on the basis of these values the percentage of inhibition of colony growth has been calculated by formula given by Djordjevic et al. (2013).
Where, gc is the growth of mycelium in control plates, gt is the growth of mycelium in treated plates. 2.4. Statistical Analysis Statistical analyses were carried out using Microsoft Excel data analysis tool; it was used to calculate a significance of correlation (P-value). 3. Results and discussion: 3.1 Performance of essential oil: The essential oil of Cloves was extracted from dry plant material in the equipment of the hydro-distiller (Figure 1) Average yields of essential oils were calculated relative to the dry plant material. The results revealed that the extraction of aromatic components from S. aromaticum is 7.49%, these
result are lower (11.5%) than cited by Guan et al., (2006), with hydro-distillation. 3.2 Antifungal assay This in vitro study proved antifungal activity of clove essential oil against Fusarium sp. Inhibition of mycelium growth of F. oxysporum, F.redolens and F.commune started at the lowest application rates (0.1µL/mL) of essential oil and this inhibition successively increased with the increasing of oil concentration (Table 1). The inhibition in mycelium growth of F. redolens at 0.1µL/mL was lower than the inhibition observed for F.oxysporum and F.commune (7.5%, 21.95%, and 22.95% respectively). A complete inhibition for Fusarium oxysporum f.sp radicis lycopersici and Fusarium redolens, were observed at 1µL/mL of clove oil (Fig.2 and 3), while complete inhibition for F. commune started at 0.5µL/mL, (Fig. 4) but there is no significant difference in inhibition between 0.5µL/mL and 1µL/mL (p
