International Journal of Food Microbiology 143 (2010) 183189

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

In vitro and in vivo antifungal activities of the essential oils of various plants against tomato grey mould disease agent Botrytis cinerea
Emine Mine Soylu, ener Kurt, Soner Soylu
Mustafa Kemal University, Department of Plant Protection, Agriculture Faculty, 31034 Antakya, Hatay, Turkey

a r t i c l e

i n f o

a b s t r a c t
The aim of this study was to nd an alternative to synthetic fungicides currently used in the control of devastating fungal pathogen Botrytis cinerea, the causal agent of grey mould disease of tomato. Antifungal activities of essential oils obtained from aerial parts of aromatic plants, which belong to the Lamiacea family such as origanum (Origanum syriacum L. var. bevanii), lavender (Lavandula stoechas L. var. stoechas) and rosemary (Rosmarinus ofcinalis L.), were investigated against B. cinerea. Contact and volatile phase effects of different concentrations of the essential oils were found to inhibit the growth of B. cinerea in a dosedependent manner. Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. A volatile vapour of origanum oil at 0.2 g/ml air was found to completely inhibit the growth of B. cinerea. Complete growth inhibition of pathogen by essential oil of lavender and rosemary was, however, observed at 1.6 g/ml air concentrations. For the determination of the contact phase effects of the tested essential oils, origanum oil at 12.8 g/ml was found to inhibit the growth of B. cinerea completely. Essential oils of rosemary and lavender were inhibitory at relatively higher concentrations (25.6 g/ml). Spore germination and germ tube elongation were also inhibited by the essential oils tested. Light and scanning electron microscopic (SEM) observations revealed that the essential oils cause considerable morphological degenerations of the fungal hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage and loss of conidiation. In vivo assays with the origanum essential oil, being the most efcient essential oil, under greenhouse conditions using susceptible tomato plants resulted in good protection against grey mould severity especially as a curative treatment. This study has demonstrated that the essential oils are potential and promising antifungal agents which could be used as biofungicide in the protection of tomato against B. cinerea. 2010 Elsevier B.V. All rights reserved.

Article history: Received 10 May 2010 Received in revised form 13 August 2010 Accepted 17 August 2010 Keywords: Antifungal activity Essential oil Botrytis cinerea SEM Tomato

1. Introduction Grey mould, caused by Botrytis cinerea Pers ex.Fr is a severe and constant threat to eld and greenhouses-grown tomatoes (Lycopersicon esculentum) in many countries worldwide (LaMondia and Douglas, 1997), including Turkey. The fungal agent infects leaves, stems, owers and fruits of plants, either by direct penetration or through wounds caused by cultivation practices. Infestation is stimulated by high humidity, particularly if free moisture is present on the plant surface and low temperatures (Shtienberg and Elad, 1997; Williamson et al., 2007). Although B. cinerea is a classical high risk pathogen in the sense of resistance management (Rosslenbroich and Stuebler, 2000; Myresiotis et al., 2007), disease control is generally achieved by the use of synthetic fungicides (Elad et al., 1995). Over the past several decades, various attempts to control plant diseases have been made at eradication or prevention through

Corresponding author. Tel.: + 90 326 245 5845; fax: + 90 326 245 5832. E-mail addresses: soylu@mku.edu.tr, ssoylu69@gmail.com (S. Soylu). 0168-1605/$ see front matter 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.08.015

the development of synthetic fungicides. The control of the disease is based on an integration of several cultural methods with the use of fungicides belonging to several groups. Until the middle of the 1990s, chemical control of grey mould was mainly achieved by site specic fungicides belonging to benzimidazoles, dicarboximides, and Nphenylcarbamate, while multi-specic inhibitors such as chlorothalonil, dichlouanid, iminactodine, and captan were used only in tank mixtures or in rotation with site specic inhibitors (Leroux et al., 2002). Although the synthetic fungicides are effective, their continued or repeated application has disrupted biological control by natural enemies and led to outbreaks in diseases, widespread development of resistance to various types of fungicides (Katan, 1982; Georgopoulos, 1987; Staub, 1991; Elad et al, 1992), toxicity to non-target organisms and environmental problems. Decreasing efcacy and increasing concern over the adverse environmental effects of synthetic fungicides have brought about the need for the development of new types of selective control alternatives and crop protection methods without or with reduced use of conventional fungicides. Essential oil bearing plants may be alternative to currently used disease control agents, since they constitute a rich source of bioactive chemicals (Isman,

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2000; Burt, 2004). These chemicals are often active against a limited number of species, including the specic target species, are biodegradable to nontoxic products and are potentially suitable for integrated use, they could be developed as new classes of possibly safer disease control agents. Therefore, much effort has focused on plant materials for potentially useful products as commercial fungicides or as lead compounds (Balandrin et al., 1985; Miyakado, 1986; Benner, 1993; Hedin et al., 1997). Certain plant-derived materials were found to be highly effective against fungicide-resistant pathogens. For example, natural compounds such as cinnamaldehyde and salicylaldehyde were effective against four strains of thiabendazole-resistant Fusarium sambucinum (Vaughn and Spencer, 1994). Although there are many reports on the antifungal activities of essential oils against plant pathogenic fungi in vitro conditions, to our knowledge the in vivo efcacy of the essential oils against the grey mould of tomato has not been studied. The Eastern Mediterranean region of Turkey has the ora that is rich in indigenous aromatic and medicinal plant species. Origanum (Origanum syriacum var. bevanii L.), lavender (Lavandula stoechas L. subsp. stoechas), and rosemary (Rosmarinus ofcinalis L.), which belong to the Lamiacea family, are widely grown in the Mediterranean basin of Turkey since antiquity and are known for their medicinal and aromatic properties. In the present study, antifungal effects of plant essential oils derived from aerial parts of origanum, lavender, and rosemary have been investigated against fungal disease agent B. cinerea. Both contact and volatile phase effect of the essential oils on hyphal growth, spore germination and germ tube elongation were determined in vitro conditions. Morphological changes in hyphae were investigated by using light and scanning electron microscopes (SEM). Efcacy of the most efcient essential oil, as determined in vitro studies, was evaluated further for control of grey mould diseases in greenhouse conditions (in vivo studies) following their application as a foliar spray with a broader objective of identication of eco-friendly tactics for management of this disease.

2.3. Antifungal activity of essential oils on mycelial growth in vitro conditions The antifungal tests of essential oils were carried out for assessing its contact and volatile phase effects towards mycelial growth of B. cinerea as described previously (Soylu et al., 2006). For determination of contact effects, the essential oils were dispersed as an emulsion in water using ethanol and Tween 20 (0.1% v/v) and added to PDA immediately before it was emptied into the glass Petri dishes (90 20 mm in diameter) at a temperature of 4045 C. The concentrations tested were 0.4 to 25.6 g/ml. The controls received the same quantity of ethanol and Tween 20 mixed with PDA. B. cinerea was inoculated immediately by plating in the centre of each plate with a 7 mm diameter disc of the fungus, cut with a sterile cork borer from the edge of actively growing cultures on PDA plates. The Petri dishes were incubated in the dark at 22 C. For determination of volatile phase effects, glass Petri dishes (90 20 mm, which offer 80 ml air spaces after addition of 20 ml agar media) were used. The Petri dishes inoculated as described above at different concentrations of essential oils were added to sterile lter papers (10 mm diameter, Whatman no.1) and placed on the inner surface of the lid of Petri dishes to obtain nal concentrations of 0.05 to 1.6 m/ml air. The Petri dishes were sealed immediately with paralm to prevent loss of essential oil vapours and incubated at 22 C. The mean radial mycelial growth of the pathogen was determined by measuring the diameter of the colony in two directions at right angels when the plate surface of the control Petri was covered by fungus 7 days after inoculation. The fungistaticfungicidal nature of essential oils was tested by observing revival of growth of the inhibited mycelial disc following its transfer to non-treated PDA. A fungicidal effect was where there was no growth, whereas a fungistatic effect was where temporary inhibition of microbial growth occurred. The agar discs of B. cinerea, which failed to grow were either transferred onto agar media without oils (for contact phase effect of oils) or onto lids of the plate containing ethanol and Tween 20 (0.1% v/v) without oil (for volatile phase effect of oils). Petri plates were incubated for 5 days. Activity of the each concentration of the various oils was considered fungicidal if the pathogen did not grow or fungistatic if the pathogen growth occurred. For each concentration, ve replicate plates were used. The mean growth values were obtained and then converted in to the inhibition percentage of mycelial growth in relation to the control treatment by using the formula, MGI(%) = ((dc dt) / dc) 100, dc and dt represent mycelial growth diameter in control and treated Petri plates, respectively. The experiments were conducted twice. 2.4. Effect of essential oil on conidial germination and germ tube elongation The effects of essential oil upon the spore germination and germ tube elongation of B. cinerea were described in our earlier study (Soylu et al., 2005). Spore suspension (104 spores ml1) of B. cinerea was prepared from actively growing culture (78 days old) in distilled sterile water. Three different 50 l aliquots of the spore suspension drops were spread onto the surface of PDA medium supplemented with different concentrations of essential oil in contact or volatile phases as described before. Sterile distilled water, containing 0.01% Tween 20, was used in place of the essential oils as control. Plates were incubated at 22 C until the germination in the control reached N 80% (1012 h according to the rate of germination of the fungus) Afterward, spore germination was stopped by applying a drop of lactophenol-cotton blue to the inoculation sites on plates. Germination was dened as the point at which the germ tube length exceeded the spore diameter. The percentage of spore germination and the lengths of germ tubes (three replicates were conducted for each treatment, and a minimum of 100 spores were counted in each replicate) were estimated under a microscope (Olympus BX51, Tokyo, Japan), using a micrometer. The percent inhibition was calculated according to Abbott's formula: MGI(%) =

2. Materials and methods 2.1. Plant material and isolation of essential oils For the extraction of essential oils, plants (O. syriacum var. bevanii, L. stoechas var. stoechas, and R. ofcinalis) were collected locally from Samanda (36 16 N; 35 48 E, 38 m) and Alahan (36 19 N; 36 11 E, 141 m) districts of Hatay province situated in the Eastern Mediterranean region of Turkey and were identied by Dr. . Uremis. A voucher specimen has been deposited in the herbarium of the Plant Protection Department, MKU (No. OsbS1, LssA1, and RoS4). Air-dried plant materials (200 g) were placed in a 5 l round-bottom distillation ask and 3 l double distilled water was added. The essential oils were obtained by steam distillation for 3 h using Clevenger-type apparatus (ldam, Ankara). The isolated fractions of plant parts exhibited two distinct layersan upper oily layer and the lower aqueous layer. Both the layers were separated and, after removing water traces with the help of capillary tubes and anhydrous sodium sulphate, the essential oils were stored at 4 C in a clean amber glass bottle until used.

2.2. Isolation of B. cinerea The highly virulent B. cinerea isolate was obtained from a tomato greenhouse by harvesting from diseased leaves and putting on Potato Dextrose Agar (PDA). B. cinerea was grown for 810 days on PDA at 22 C. Stock cultures obtained from single spore were maintained on PDA and kept at 4 C and sub-cultured once a month. The pure culture of the pathogen has been deposited in the culture collection of the Plant Protection Department, MK (No. ToBc09).

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[(Gc Gt) / Gc)] 100, Gc and Gt represent the mean number of germinated conidia in control and treated Petri plates, respectively. The experiments conducted twice. 2.5. Effect of essential oils on hyphal morphology Determination of volatile and contact phase effects of essential oils on hyphal morphology was described in our earlier study (Soylu et al., 2006). For the determination of volatile phase effect of essential oils on hyphal morphology, a mycelial agar disc from a 7-day old culture was rst placed in the centre of PDA plate and incubated at 20 C for 2 days to allow mycelium to grow into the medium. After 2 days of pre-incubation, different concentrations of essential oils used in vitro studies were dropped (onto covers of Petri dishes), sealed by paralm and incubated at 20 C for 3 days. Determination of contact phase effect of essential oils on hyphal morphology was as described in an earlier paper. Thin layers (1 mm) of agar blocks (34 cm2) containing mycelium were removed at one-day intervals for examination by light microscopy. The blocks cut from growing edges were placed in a drop of 50% glycerol on microscope glass slides, covered with glass cover slip and examined using a phase contrast light microscope (Olympus BX51, Tokyo, Japan). For SEM analysis, fungal hypha was processed as described before (Soylu et al., 2006). Mycelial discs (1 cm in diameter) exposed to the most effective concentration of origanum essential oil was xed with 2.5% glutaraldehyde in 0.1 M phosphate-buffer (pH = 7.2) for 2 h at room temperature. They were washed twice, each time for 10 min, in the same buffer. After xation, the samples were dehydrated in a graded ethanol series (70%, 80%, 90%, and three times at 100%) for a period of 30 min in each series. The samples were critical point dried in a drying apparatus (Polaron CPD 7501, UK) up to the critical point with CO2. The xed material was then mounted on stubs using double-sided carbon tape and coated with gold/palladium in a sputter coater system in a high-vacuum chamber (Polaron SC7620, UK) for 150 sec at 9 mA. The samples were examined and digital images captured using a JEOL JSM 5500 SEM at an accelerating voltage of 5 kV. 2.6. Effect of the essential oil on disease development in vivo conditions Origanum essential oil, having the highest effect on the pathogen, was selected for greenhouse trial to study the effect of the essential oil on disease caused by B. cinerea. All experiments were arranged in a completely randomized split-plot design with three replicates of 10 plants per treatment and repeated at least twice. Different concentrations of essential oil (25, 50, 75, and 100 mg/l) were prepared by dissolving the requisite amounts in sterile Tween 20 (0.1%, v/v) solution. Six-week old tomato cv. F-144 plants were sprayed with these emulsions (10 ml for each plant) uniformly with a manually operated glass knapsack sprayer with FanTip 110 spray nozzle at 3.0 bar pressure. The sprayer was held 35 cm away from the plant, yielding a ne mist (approximately 150200 m droplets). Each spray lasted for 510 s. For protective activity of the oil, tomato plants were treated with origanum oil 24 hours before pathogen inoculation. For curative activity of origanum oil, tomato plants were inoculated with suspension of 5 105 conidia per ml of B. cinerea ToBc09 isolate. These plants were incubated for 24 h and then, were treated with different concentrations of origanum oil. The control plants were sprayed uniformly with 10 ml of sterile Tween 20 (0.1%, v/v) or pathogen suspension used as negative or positive control groups of the experiments without using any antimicrobial. Rovral 50 WP (50% active ingredient iprodione; Bayer Crop Science, Turkey) as a commercial fungicide used against grey mould of tomato was also evaluated in both protective and curative assays at the recommended concentration (750 mg/l) for comparison.

Control and essential oil treated plots were assessed 10 days after treatments. The disease severity index of grey mould on the tomato leaves was rated on a scale of 04 (0 = no disease symptom, 1 = 0.1 5%, 2 = 5.120%, 3 = 20.140%, and 4 = 40.1100%) as the percentage of diseased leaf area (Lee et al., 2006). The efcacy of the essential oil was calculated according to Abbott Formula [%effectiveness = (C T) / C 100, where C = mean disease index in the control, and T = mean disease index in the relevant treatment]. 2.7. Statistical analyses SPSS statistic program (Ver.11.5, SPSS Inc., Chicago, IL, USA) was performed for all calculations. Where necessary, arcsine transformation was performed on data before statistical analysis. Analysis of variance was performed at the signicance level of P 0.05. When appropriate, means were separated by using Tukey's test (P 0.05). The data from two independent experiments were analyzed separately but were not signicantly different (P N 0.05). The EC50 value (concentration causing 50% reduction in mycelial growth) was estimated for each essential oil by using Probit analysis (SPSS statistic program, Ver.11.5, SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Antifungal activities of essential oils on mycelial growth The volatile and contact phase effects of different concentrations of essential oils on the mycelial growth of B. cinerea are shown in Fig. 1. All essential oils were found to inhibit the growth of B. cinerea in a dose-dependent manner. Volatile inhibitory effect of essential oils on mycelial growth was greater than contact inhibitory effect (Fig. 1). In the contact phase of essential oils, relatively higher concentrations were required to inhibit mycelial growth as shown in Fig. 1. Among all the essential oils tested, volatile and contact phases of origanum essential oil caused the greatest inhibition of mycelium growth of B. cinerea at low concentrations (0.2 g/ml air and 12.8 g/ ml respectively). Both volatile and contact phases of essential oils of origanum were found to be fungicidal at 0.2 g/ml air and 12.8 g/ml respectively. Volatile and contact phases of essential oils of lavender and rosemary were found to be fungicidal at 1.6 g/ml air and 25.6 g/ ml concentrations respectively. Efcient concentration (EC50) values of each essential oil (volatile and contact phases) were also estimated by using Probit analyses. The lowest EC50 values of volatile and contact phases of oils were recorded for origanum essential oil (0.044 and 2.41 g/ml) was followed by lavender (0.25 and 9.26 g/ml), and rosemary (0.44 and 10.37 g/ml) respectively. 3.2. Effect of essential oil on conidial germination and germ tube elongation Effects of different concentrations of volatile and contact phase of essential oils on the conidial germination and germ tube elongation of B. cinerea were given in Tables 1 and 2. As observed in mycelial growth inhibition experiments, the volatile phase of essential oil was found to be more effective on conidial germination and germ tube elongation than the contact phase (P 0.05). Complete inhibitions of conidial germination and germ tube elongation by origanum, lavender and rosemary oils were observed at 0.4, 1.6 and 1.6 g/ml air concentrations, respectively (Table 1). Complete inhibition of conidial germination of B. cinerea by the contact phase of origanum, rosemary and lavender essential oil was, however, observed at relatively higher concentration (3.2, 25.6 and 51.2 g/ml respectively). 3.3. Effects of essential oils on hyphal morphology Light and scanning electron microscopic (SEM) observations of B. cinerea hyphae exposed to the most effective concentrations of

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A
100,0 90,0
origanum lavander rosemary

c b

3.4. Effects of the essential oil on disease development in vivo conditions Disease development was also controlled in vivo, with the essential oil of origanum which was found to be the most efcient essential oil in vitro studies, reducing infection of tomato leaves signicantly. Most effective control of fungal infection was achieved if the essential oil was applied within 24 h post inoculation. The in vivo fungicidal activity of the different concentrations of origanum oil against B. cinerea is presented in Table 3. As the doses of origanum oil increased, the infection of B. cinerea was suppressed. When we compare the curative and protective activities of the origanum oil on the infection caused by B. cinerea, the greatest effect on the disease severity of the pathogen was observed in curative activity (Table 3). In curative treatments, in vitro effective (25 mg/l) or higher concentrations (50 and 75 mg/l) of origanum essential oil and iprodione at the recommended concentration (375 mg/l) displayed a signicant decrease in disease severity on tomato in comparison to controls (Table 3). The mean disease severity caused by B. cinerea was signicantly (P 0.05) reduced by the highest concentration of origanum oil (75 mg/l) used (Table 3). Protective activity of origanum oil had a very low effect at 12.5, 25 and 50 mg/l doses, in comparison to same concentration used in the curative activities. Only origanum essential oil at the highest concentration (75 mg/l) and iprodione (375 mg/l) signicantly reduced grey mould severity (Fig. 3). No sign of phytotoxicity was found on the tested plants at the highest concentration (75 mg/l) used in the experiments.

Mycelial inhibition (%)

80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0 0.05 b a c

a a a b b b

b a a

a a

0.1

0.15

0.2

0.4

0.8

1.2

1.6

concentration (g/ml air)

B
100,0 90,0
origanum lavander rosemary

Mycelial inhibition (%)

80,0 70,0 60,0 50,0 40,0 30,0 20,0 10,0 0,0 b

c a a c

c b b a a 0.8 b 1.6 a 3.2 a 6.4 a

4. Discussion Food safety is usually ensured by the addition of antimicrobials that prevent or considerably retard microbial spoilage organisms. The widespread use of pesticides has signicant drawbacks including increased cost, handling hazards, concern about pesticide residues on food, and threat to human health and environment. Public awareness of these risks has increased interest in nding safer alternatives protectant to replace currently used synthetic chemical pesticides. One such alternative is the use of natural plant protectants with pesticidal activity such as essential oil and their major components, since they tend to have low mammalian toxicity, less environmental effects and wide public acceptance (Isman, 2000; Kalemba and Kunicka, 2003; Burt, 2004). Natural antimicrobial agents such as essential oils can be used in food industry only if the compounds they release over time and their effects on target plant and pathogen are well-known and understood. Our study indicated that essential oils may possess antifungal activity against grey mould disease agent B. cinerea and can be exploited as an ideal treatment for future plant disease management programs eliminating fungal spread. Suppression of mycelial growth, spore germination and germ tube elongation by essential oil treatments could make a major contribution to limiting the spread of the pathogen by lowering the spore load in the storage atmosphere and on surfaces. Although all plant species are related and belong to the

12.8

25.6

concentration (g/ml)
Fig. 1. The effects of different concentrations of volatile (A) and contact (B) phase of essential oils of origanum, lavender and rosemary on the mycelial growth of B. cinerea. Bars, for each concentration, with the same letters represent values that are not signicantly different according to Tukey's test (P 0.05).

essential oil vapour (volatile phase) or grown on PDA amended with the different concentrations of essential oil (contact phase) showed degenerative changes in the hyphal morphology in comparison to thick, elongated, smooth surfaced hyphae in control plates (Fig. 2A and B). After exposure to the most effective oil concentrations determined in vitro studies, [contact (12.8 g/ml) or volatile phases (0.2 g/ml air)], hyphae appeared degraded (Fig. 2C), large vesicles were visible within the cell walls. Shrivelled hyphal cells had either no cytoplasm or the cytoplasm was depleted of organelles. Under the effect of the oils, the growth of the fungus was suppressed (Fig. 2C and D). Complete absence of conidiation was also observed in oil treated Petri plates.

Table 1 The effects of different concentrations of volatile phases of essential oils on the conidial germination and germ tube elongation. Concentrations (g/ml air) 0 0.2 0.4 0.8 1.6 Conidial germination (%) Origanum 100 c 17 b 0a 0a 0a Lavender 100 e 77 d 57.6 c 19.6 b 0a Rosemary 100 d 90 d 77.3 c 37 b 0a Length of germ tube elongation (m) Origanum 837.5 c 54.6 b 0a 0a 0a Lavender 837.5 d 410.7 c 234.2 b 171.5 b 0a Rosemary 837.5 e 486.4 d 321.7 c 113.2 b 0a

Arcsine transformation was performed prior to statistical analysis. Within the column, mean values followed by the same letter are not signicantly different according to Tukey's test (P 0.05).

E.M. Soylu et al. / International Journal of Food Microbiology 143 (2010) 183189 Table 2 The effects of different concentrations of contact phases of essential oils on the conidial germination and germ tube elongation. Concentrations (g/ml) 0 0.4 0.8 1.6 3.2 6.4 12.8 25.6 51.2 Conidial germination (%) Origanum 100 b 100 b 100 b 100 b 0a 0a 0a 0a 0a Lavender 100 c 100 c 100 c 100 c 100 c 100 c 100 c 59 b 0a Rosemary 100 b 100 b 100 b 100 b 100 b 100 b 100 b 0a 0a Length of germ tube elongation (m) Origanum 825.9 e 502.3 d 307.8 c 112.6 b 0a 0a 0a 0a 0a Lavender 825.9 f 816.6 f 808.6 f 533.3 e 241.2 d 205.3d 115.6 c 34.6 b 0a

187

Rosemary 825.9 d 818.8 d 803.6 d 794.6 d 789.2 d 559.2 c 48.3 b 0a 0a

Arcsine transformation was performed prior to statistical analysis. Within the column, mean values followed by the same letter are not signicantly different according to Tukey's test (P 0.05).

same plant family, essential oil of origanum showed remarkable antifungal effect, whereas lavender and rosemary showed a less inhibitory effect against B. cinerea. This might be attributed to the mode of resistant behaviour of the fungi against various substances present in the various essential oil. We have recently investigated the chemical compositions of the plants used in this study (Soylu et al., 2006). Major compounds found in essential oils of origanum, rosemary and lavender were carvacrol (79.8%), borneol (20.4%), and camphor (20.2%), respectively. The antimicrobial properties of essential oils of origanum, rosemary and lavender and their major constituents, have been shown to be able to suppress several plant pathogenic fungi (Daouk et al., 1995; Paster et al., 1995; Adam et al., 1998; Lambert et al., 2001; Marino et al., 2001; Abou-Jawdah et al., 2002; Bouchra et al., 2003; Daferera et al., 2003; Zambonelli et al.,

2004; Soylu et al., 2006; Bajpai et al., 2007; Martnez-Romeroa et al., 2007; Soylu et al., 2007; Hadian et al., 2008; Kordali et al., 2008; Ozcan and Chalchat, 2008). The majority of the work initiated so far has concentrated on the effect of essential oils on inhibition of fungal mycelial growth in vitro conditions. In previous works, we have shown that the volatile components of the essential oils of taxonomically different medicinal plants possess in vitro activity against a number of tomato pathogens (Soylu et al., 2005; Soylu et al., 2006). Unlike to in vitro studies, very few studies have been conducted in vivo conditions to show fungicidal properties of essential oils against plant pathogenic fungi (Letessier et al., 2001; Oxenham et al., 2005; Soylu et al., 2007). In vivo fungicidal activity of the most efcient essential oil, origanum was also investigated in greenhouse conditions. Essential oil was sprayed on

Fig. 2. Scanning electron microscopy of hyphae exposed to origanum essential oil. (A and B) Healthy hyphae and conidia (arrow) in control Petri plates. (C and D) Effects of essential oils on hyphal morphology. Note alterations in hyphal morphology including hyphal shrivelling, blistering (arrows) in plate (C) and lysis (arrows) in plate (D).

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Table 3 The curative and protective effects of different concentrations of origanum essential oil on the infection caused by B. cinerea in the greenhouse. Concentrationsa Curative effect DIb Negative control Positive controlc 12.5 25.0 50.0 75.0 Iprodione (375.0)
a b c c

Protective effect % protection DI % protection 100 0 8.95 19.47 26.32 33.16 78.95

0a 1.90 1.33 0.70 0.53 0.43 0.37

d c b b b ab

100 0 30.0 63.16 72.11 77.37 80.53

0a 1.90 e 1.73 de 1.50 cd 1.40 cd 1.27 c 0.40 b

Concentration expressed as mg/l. Disease index (DI) according to Lee et al. (2006). Negative control, tomato plants cv. F144 were sprayed sterile distilled water + Tween 20 (0.1%, v/v) only; positive control, tomato plants cv. F144 were only inoculated with B. cinerea. Both controls were sprayed with distilled water. The means in a column followed by the same letter(s) represent values that are not signicantly different according to Tukey's test (P 0.05).

the tomato leaves before (protective activity) or after (curative activity) fungal inoculation in order to reveal whether essential oil has curative or protective activities. Although both treatments were effective in reducing fungal infection, greatest control was consistently achieved when essential oil applications were made 24 h after inoculation (curative activity). This would suggest that the whole oils exerted their greatest effect on early fungal development on the leaf surface, e.g. spore germination, germ tube growth and/or appressorium formation as we also recorded in vitro studies. Similar observations were also reported in different plant-pathogen interactions. Botrytis fabae and the rust fungus Uromyces fabae were also controlled in vivo, with the whole essential oil of basil (Ocimum basilicum), as well as its major compound pure methyl chavicol and linalool, reducing infection of broad bean leaves signicantly (Oxenham et al., 2005). Most effective control of these fungal infections was achieved if the treatments were applied 3 h post inoculation. Letessier et al. (2001) showed that the essential oil of hyssop reduced germination of B. fabae conidia and uredospores of U. fabae. These workers reported differences in the efcacy of hyssop oil when used in vitro and in vivo, with variable and inconclusive results obtained when

D e

100 90 80

Curative Protective C C C CD d

70 60 50 40 30 20 10 0 12.5
ab

c B bc bc

A a

the oil was used to control powdery mildew on barley and apple. Letessier et al. (2001) suggested that if the volatile components of the hyssop oil were responsible for its antifungal activity, the volatiles would be conned within the Petri dish. Inconclusive results obtained when the oil was used to control powdery mildew on barley and apple because of essential oil diffused away from the leaf surface before come into contact with fungal structure. In our case, it could be hypothesized that origanum essential oil presented a better effect as curative rather than protective treatment because of they might poorly be absorbed by fungi and plant cells if they are applied before pathogen inoculation. Although the mechanism underlying the action of essential oil on the vegetative and reproductive phases of fungal development remains to be understood, light and SEM observations of hyphae of B. cinerea exposed to essential oils revealed alterations in the hyphal morphology. Shrivelled hyphal aggregates, reduced hyphal diameters and lysis of hyphal wall were commonly observed in essential oil treated mycelium, compared with thick, elongated, normal mycelial growth in controls. Such modications may be related to the effect of the essential oil as enzymatic reactions regulating wall synthesis (Rasooli et al., 2006). The lipophilic properties of oil components might have also aided in the ability of the oil to penetrate the plasma membrane (Knobloch et al., 1989). The observations made with light and electron microscopy are in accordance with previous studies in which essential oils of aromatic plants caused the morphological alterations on the fungal hyphae (Bianchi et al., 1997; Fiori et al, 2000; Romagnoli et al., 2005; Soylu et al., 2006; Soylu et al., 2007). Similar observations were recently presented by Tripathi et al. (2009) who found that Hyptis suaveolens essential oil caused severe damage and alterations to vegetative hyphae of Fusarium oxysporum f.sp. gladioli leading to complete loss of cytoplasm from the hyphae. The impacts of oils on fungal structures may reect effects of the volatiles emitted by oils on surface mycelial development (and thus the platform to support spore production) and/or the perception/transduction of signals involved in the switch from vegetative to reproductive development. The volatile phases of the essential oils were found to be more toxic than the contact phase to the B. cinerea. Volatile phase of essential oils of different plants were also reported to possess more antimicrobial activity against plant pathogenic fungi and bacteria (Edris and Farrag, 2003; Soylu et al., 2005). Some investigators reported that the antifungal activity resulted from a direct effect of essential oil vapours on fungal mycelium. They further postulated that the lipophilic nature of essential oils render them more absorbable by the fungal mycelia than by agar due to the highly lipophilic nature of the fungal mycelia and the high water content of the agar media (Inouye et al., 2000; Edris and Farrag, 2003). In conclusion, we aimed at the evaluation of antifungal activity of essential oil of medicinal plants such as origanum, lavender and rosemary, in hopes to nd out new natural product(s) to be used as a bio fungicide against B. cinerea. The essential oil of origanum has been shown to reduce growth of B. cinerea on solid media (in vitro) and to control infection of tomato by B. cinerea (in vivo). Since essential oils have low mammalian toxicity, are biodegradable, multifunctional, non-persistent in the environment, and are cheap to produce, the possibility of developing essential oils for use in crop protection may be an attractive venture. However, further studies need to be conducted to evaluate the cost and efcacy of these essential oils on wide range of diseases in commercial greenhouses. 5. Conclusions Considering the reduction in the mycelial growth and germination of conidia in vitro and incidence of disease symptoms on essential oil treated plants, we concluded that essential oils could be used as possible biofungicides alternative to synthetic fungicides against phytopathogenic fungi.

Disease control (%)

25.0

50.0

75.0 iprodione positive negative

Treatments
Fig. 3. Effect of different concentrations of essential oil on the control of the grey mould caused by Botrytis cinerea on tomato plants (in vivo). Iprodione represents the chemical fungicide used at the concentration of 375 mg/l. Positive and negative controls indicate articial inoculation of the fungal pathogen and water treatment, respectively. Bars, for each effect, with the same small or large letters represent values that are not signicantly different according to Tukey's test (P 0.05).

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Acknowledgement This study was supported nancially by The Scientic and Technical Research Council of Turkey. References
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