International Journal of Food Microbiology 143 (2010) 183–189

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International Journal of Food Microbiology

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In vitro and in vivo antifungal activities of the essential oils of various plants againsttomato grey mould disease agent Botrytis cinerea

Emine Mine Soylu, Şener Kurt, Soner Soylu ⁎Mustafa Kemal University, Department of Plant Protection, Agriculture Faculty, 31034 Antakya, Hatay, Turkey

⁎ Corresponding author. Tel.: +90 326 245 5845; faxE-mail addresses: soylu@mku.edu.tr, ssoylu69@gma

0168-1605/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2010.08.015

a b s t r a c t

a r t i c l e i n f o

Article history:Received 10 May 2010Received in revised form 13 August 2010Accepted 17 August 2010

Keywords:Antifungal activityEssential oilBotrytis cinereaSEMTomato

The aim of this study was to find an alternative to synthetic fungicides currently used in the control ofdevastating fungal pathogen Botrytis cinerea, the causal agent of grey mould disease of tomato. Antifungalactivities of essential oils obtained from aerial parts of aromatic plants, which belong to the Lamiacea familysuch as origanum (Origanum syriacum L. var. bevanii), lavender (Lavandula stoechas L. var. stoechas) androsemary (Rosmarinus officinalis L.), were investigated against B. cinerea. Contact and volatile phase effects ofdifferent concentrations of the essential oils were found to inhibit the growth of B. cinerea in a dose-dependent manner. Volatile phase effects of essential oils were consistently found to be more effective onfungal growth than contact phase effect. A volatile vapour of origanum oil at 0.2 μg/ml air was found tocompletely inhibit the growth of B. cinerea. Complete growth inhibition of pathogen by essential oil oflavender and rosemary was, however, observed at 1.6 μg/ml air concentrations. For the determination of thecontact phase effects of the tested essential oils, origanum oil at 12.8 μg/ml was found to inhibit the growthof B. cinerea completely. Essential oils of rosemary and lavender were inhibitory at relatively higherconcentrations (25.6 μg/ml). Spore germination and germ tube elongation were also inhibited by theessential oils tested. Light and scanning electron microscopic (SEM) observations revealed that the essentialoils 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 theoriganum essential oil, being the most efficient essential oil, under greenhouse conditions using susceptibletomato plants resulted in good protection against grey mould severity especially as a curative treatment. Thisstudy has demonstrated that the essential oils are potential and promising antifungal agents which could beused as biofungicide in the protection of tomato against B. cinerea.

: +90 326 245 5832.il.com (S. Soylu).

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© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Grey mould, caused by Botrytis cinerea Pers ex.Fr is a severe andconstant threat to field and greenhouses-grown tomatoes (Lycopersi-con esculentum) in many countries worldwide (LaMondia andDouglas, 1997), including Turkey. The fungal agent infects leaves,stems, flowers and fruits of plants, either by direct penetration orthrough wounds caused by cultivation practices. Infestation isstimulated by high humidity, particularly if free moisture is presenton the plant surface and low temperatures (Shtienberg and Elad,1997; Williamson et al., 2007). Although B. cinerea is a classical ‘highrisk’ pathogen in the sense of resistance management (Rosslenbroichand Stuebler, 2000; Myresiotis et al., 2007), disease control isgenerally achieved by the use of synthetic fungicides (Elad et al.,1995). Over the past several decades, various attempts to controlplant diseases have been made at eradication or prevention through

the development of synthetic fungicides. The control of the disease isbased on an integration of several cultural methods with the use offungicides belonging to several groups. Until the middle of the 1990s,chemical control of grey mould was mainly achieved by site specificfungicides belonging to benzimidazoles, dicarboximides, and N-phenylcarbamate, while multi-specific inhibitors such as chlorotha-lonil, dichlofluanid, iminactodine, and captan were used only in tankmixtures or in rotation with site specific inhibitors (Leroux et al.,2002). Although the synthetic fungicides are effective, their continuedor repeated application has disrupted biological control by naturalenemies and led to outbreaks in diseases, widespread development ofresistance to various types of fungicides (Katan, 1982; Georgopoulos,1987; Staub, 1991; Elad et al, 1992), toxicity to non-target organismsand environmental problems. Decreasing efficacy and increasingconcern over the adverse environmental effects of synthetic fungi-cides have brought about the need for the development of new typesof selective control alternatives and crop protection methods withoutor with reduced use of conventional fungicides. Essential oil bearingplants may be alternative to currently used disease control agents,since they constitute a rich source of bioactive chemicals (Isman,

184 E.M. Soylu et al. / International Journal of Food Microbiology 143 (2010) 183–189

2000; Burt, 2004). These chemicals are often active against a limitednumber of species, including the specific target species, are biode-gradable to nontoxic products and are potentially suitable forintegrated use, they could be developed as new classes of possiblysafer disease control agents. Therefore, much effort has focused onplant materials for potentially useful products as commercialfungicides or as lead compounds (Balandrin et al., 1985; Miyakado,1986; Benner, 1993; Hedin et al., 1997). Certain plant-derivedmaterials were found to be highly effective against fungicide-resistantpathogens. For example, natural compounds such as cinnamaldehydeand salicylaldehyde were effective against four strains of thiabenda-zole-resistant Fusarium sambucinum (Vaughn and Spencer, 1994).Although there are many reports on the antifungal activities ofessential oils against plant pathogenic fungi in vitro conditions, to ourknowledge the in vivo efficacy of the essential oils against the greymould of tomato has not been studied.

The Eastern Mediterranean region of Turkey has the flora that isrich in indigenous aromatic and medicinal plant species. Origanum(Origanum syriacum var. bevanii L.), lavender (Lavandula stoechas L.subsp. stoechas), and rosemary (Rosmarinus officinalis L.), whichbelong to the Lamiacea family, are widely grown in theMediterraneanbasin of Turkey since antiquity and are known for their medicinal andaromatic properties. In the present study, antifungal effects of plantessential oils derived from aerial parts of origanum, lavender, androsemary have been investigated against fungal disease agentB. cinerea. Both contact and volatile phase effect of the essential oilson hyphal growth, spore germination and germ tube elongation weredetermined in vitro conditions. Morphological changes in hyphaewere investigated by using light and scanning electron microscopes(SEM). Efficacy of the most efficient essential oil, as determinedin vitro studies, was evaluated further for control of grey moulddiseases in greenhouse conditions (in vivo studies) following theirapplication as a foliar spray with a broader objective of identificationof eco-friendly tactics for management of this disease.

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. officinalis) were collected locally fromSamandağ (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 EasternMediterranean region of Turkey and were identified by Dr. İ. Uremis.A voucher specimen has been deposited in the herbarium of the PlantProtection Department, MKU (No. OsbS1, LssA1, and RoS4). Air-driedplant materials (200 g) were placed in a 5 l round-bottom distillationflask and 3 l double distilled water was added. The essential oils wereobtained by steam distillation for 3 h using Clevenger-type apparatus(İldam, Ankara). The isolated fractions of plant parts exhibited twodistinct layers—an upper oily layer and the lower aqueous layer. Boththe layers were separated and, after removing water traces with thehelp of capillary tubes and anhydrous sodium sulphate, the essentialoils 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 tomatogreenhouse by harvesting from diseased leaves and putting on PotatoDextrose Agar (PDA). B. cinerea was grown for 8–10 days on PDA at22 °C. Stock cultures obtained from single spore were maintained onPDA and kept at 4 °C and sub-cultured once a month. The pure cultureof the pathogen has been deposited in the culture collection of thePlant Protection Department, MKÜ (No. ToBc09).

2.3. Antifungal activity of essential oils on mycelial growth invitro conditions

The antifungal tests of essential oils were carried out for assessingits contact and volatile phase effects towards mycelial growth ofB. cinerea as described previously (Soylu et al., 2006). For determi-nation of contact effects, the essential oils were dispersed as anemulsion in water using ethanol and Tween 20 (0.1% v/v) and addedto PDA immediately before it was emptied into the glass Petri dishes(90×20 mm in diameter) at a temperature of 40–45 °C. Theconcentrations tested were 0.4 to 25.6 μg/ml. The controls receivedthe same quantity of ethanol and Tween 20mixedwith PDA. B. cinereawas inoculated immediately by plating in the centre of each plate witha 7 mm diameter disc of the fungus, cut with a sterile cork borer fromthe edge of actively growing cultures on PDA plates. The Petri disheswere incubated in the dark at 22 °C. For determination of volatilephase effects, glass Petri dishes (90×20 mm, which offer 80 ml airspaces after addition of 20 ml agar media) were used. The Petri dishesinoculated as described above at different concentrations of essentialoils were added to sterile filter papers (10 mm diameter, Whatmanno.1) and placed on the inner surface of the lid of Petri dishes to obtainfinal concentrations of 0.05 to 1.6 μm/ml air. The Petri dishes weresealed immediately with parafilm to prevent loss of essential oilvapours and incubated at 22 °C. The mean radial mycelial growth ofthe pathogen was determined by measuring the diameter of thecolony in two directions at right angels when the plate surface of thecontrol Petri was covered by fungus 7 days after inoculation. Thefungistatic–fungicidal nature of essential oils was tested by observingrevival of growth of the inhibitedmycelial disc following its transfer tonon-treated PDA. A fungicidal effect was where there was no growth,whereas a fungistatic effect was where temporary inhibition ofmicrobial growth occurred. The agar discs of B. cinerea, which failed togrow were either transferred onto agar media without oils (forcontact phase effect of oils) or onto lids of the plate containing ethanoland Tween 20 (0.1% v/v) without oil (for volatile phase effect of oils).Petri plates were incubated for 5 days. Activity of the eachconcentration of the various oils was considered fungicidal if thepathogen did not grow or fungistatic if the pathogen growth occurred.

For each concentration, five replicate plates were used. The meangrowth values were obtained and then converted in to the inhibitionpercentage of mycelial growth in relation to the control treatment byusing the formula, MGI(%)=((dc−dt)/dc)×100, dc and dt representmycelial 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 andgerm tubeelongation of B. cinereawere described in our earlier study (Soylu et al.,2005). Spore suspension (104 spores ml−1) of B. cinerea was preparedfrom actively growing culture (7–8 days old) in distilled sterile water.Three different 50 μl aliquots of the spore suspension dropswere spreadonto the surface of PDA medium supplemented with differentconcentrations of essential oil in contact or volatile phases as describedbefore. Sterile distilled water, containing 0.01% Tween 20, was used inplace of the essential oils as control. Plateswere incubated at 22 °C untilthe germination in the control reached N80% (10–12 h according to therate of germination of the fungus) Afterward, spore germination wasstoppedbyapplyingadropof lactophenol-cottonblue to the inoculationsites on plates. Germinationwas defined as the point at which the germtube length exceeded the spore diameter. The percentage of sporegermination and the lengths of germ tubes (three replicates wereconducted for each treatment, and a minimum of 100 spores werecounted in each replicate) were estimated under a microscope(Olympus BX51, Tokyo, Japan), using a micrometer. The percentinhibition was calculated according to Abbott's formula: MGI(%)=

185E.M. Soylu et al. / International Journal of Food Microbiology 143 (2010) 183–189

[(Gc−Gt)/Gc)]×100, Gc and Gt represent the mean number ofgerminated 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 oilson hyphal morphology was described in our earlier study (Soylu et al.,2006). For the determination of volatile phase effect of essential oilson hyphal morphology, a mycelial agar disc from a 7-day old culturewas first placed in the centre of PDA plate and incubated at 20 °C for2 days to allow mycelium to grow into the medium. After 2 days ofpre-incubation, different concentrations of essential oils used in vitrostudies were dropped (onto covers of Petri dishes), sealed by parafilmand incubated at 20 °C for 3 days. Determination of contact phaseeffect of essential oils on hyphal morphology was as described in anearlier paper. Thin layers (1 mm) of agar blocks (3–4 cm2) containingmyceliumwere removed at one-day intervals for examination by lightmicroscopy. The blocks cut from growing edges were placed in a dropof 50% glycerol on microscope glass slides, covered with glass coverslip and examined using a phase contrast light microscope (OlympusBX51, Tokyo, Japan).

For SEM analysis, fungal hypha was processed as described before(Soylu et al., 2006). Mycelial discs (1 cm in diameter) exposed to themost effective concentration of origanum essential oil was fixed with2.5% glutaraldehyde in 0.1 M phosphate-buffer (pH=7.2) for 2 h atroom temperature. They were washed twice, each time for 10 min, inthe same buffer. After fixation, the samples were dehydrated in agraded ethanol series (70%, 80%, 90%, and three times at 100%) for aperiod of 30 min in each series. The samples were critical point driedin a drying apparatus (Polaron CPD 7501, UK) up to the critical pointwith CO2. The fixed material was then mounted on stubs usingdouble-sided carbon tape and coated with gold/palladium in a sputtercoater system in a high-vacuum chamber (Polaron SC7620, UK) for150 sec at 9 mA. The samples were examined and digital imagescaptured 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 oilon disease caused by B. cinerea. All experiments were arranged in acompletely randomized split-plot design with three replicates of 10plants per treatment and repeated at least twice. Different concentra-tions of essential oil (25, 50, 75, and 100 mg/l) were prepared bydissolving the requisite amounts in sterile Tween 20 (0.1%, v/v)solution. Six-week old tomato cv. F-144 plants were sprayed withthese emulsions (10 ml for each plant) uniformly with a manuallyoperated glass knapsack sprayer with FanTip 110° spray nozzle at3.0 bar pressure. The sprayer was held 35 cm away from the plant,yielding a finemist (approximately 150–200 μmdroplets). Each spraylasted for 5–10 s.

For protective activity of the oil, tomato plants were treated withoriganum oil 24 hours before pathogen inoculation. For curativeactivity of origanum oil, tomato plants were inoculated withsuspension of 5×105 conidia per ml of B. cinerea ToBc09 isolate.These plants were incubated for 24 h and then, were treated withdifferent concentrations of origanum oil. The control plants weresprayed uniformly with 10 ml of sterile Tween 20 (0.1%, v/v) orpathogen suspension used as negative or positive control groups ofthe experiments without using any antimicrobial. Rovral 50 WP (50%active ingredient iprodione; Bayer Crop Science, Turkey) as acommercial fungicide used against grey mould of tomato was alsoevaluated in both protective and curative assays at the recommendedconcentration (750 mg/l) for comparison.

Control and essential oil treated plots were assessed 10 days aftertreatments. The disease severity index of grey mould on the tomatoleaves was rated on a scale of 0–4 (0=no disease symptom, 1=0.1–5%, 2=5.1–20%, 3=20.1–40%, and 4=40.1–100%) as the percentageof diseased leaf area (Lee et al., 2006). The efficacy of the essential oilwas calculated according to Abbott Formula [%effectiveness=(C−T)/C×100, where C=mean disease index in the control, and T=meandisease index in the relevant treatment].

2.7. Statistical analyses

SPSS statistic program (Ver.11.5, SPSS Inc., Chicago, IL, USA) wasperformed for all calculations. Where necessary, arcsine transforma-tion was performed on data before statistical analysis. Analysis ofvariance was performed at the significance level of P≤0.05. Whenappropriate, means were separated by using Tukey's test (P≤0.05).The data from two independent experiments were analyzed sepa-rately but were not significantly different (PN0.05). The EC50 value(concentration causing 50% reduction in mycelial growth) wasestimated for each essential oil by using Probit analysis (SPSS statisticprogram, 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 ofessential 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 adose-dependent manner. Volatile inhibitory effect of essential oils onmycelial growth was greater than contact inhibitory effect (Fig. 1). Inthe contact phase of essential oils, relatively higher concentrationswere required to inhibit mycelial growth as shown in Fig. 1.

Among all the essential oils tested, volatile and contact phases oforiganum essential oil caused the greatest inhibition of myceliumgrowth of B. cinerea at low concentrations (0.2 μg/ml air and 12.8 μg/ml respectively). Both volatile and contact phases of essential oils oforiganum were found to be fungicidal at 0.2 μg/ml air and 12.8 μg/mlrespectively. Volatile and contact phases of essential oils of lavenderand rosemary were found to be fungicidal at 1.6 μg/ml air and 25.6 μg/ml concentrations respectively.

Efficient concentration (EC50) values of each essential oil (volatile andcontact phases) were also estimated by using Probit analyses. The lowestEC50 values of volatile and contact phases of oils were recorded fororiganum essential oil (0.044 and 2.41 μg/ml) was followed by lavender(0.25 and 9.26 μg/ml), and rosemary (0.44 and 10.37 μg/ml) respectively.

3.2. Effect of essential oil on conidial germination and germ tube elongation

Effects of different concentrations of volatile and contact phase ofessential oils on the conidial germination and germ tube elongation ofB. cinereawere given in Tables 1 and 2. As observed inmycelial growthinhibition experiments, the volatile phase of essential oil was found tobe more effective on conidial germination and germ tube elongationthan the contact phase (P≤0.05). Complete inhibitions of conidialgermination and germ tube elongation by origanum, lavender androsemary oils were observed at 0.4, 1.6 and 1.6 μg/ml air concentra-tions, respectively (Table 1). Complete inhibition of conidial germi-nation of B. cinerea by the contact phase of origanum, rosemary andlavender essential oil was, however, observed at relatively higherconcentration (3.2, 25.6 and 51.2 μg/ml respectively).

3.3. Effects of essential oils on hyphal morphology

Light and scanning electron microscopic (SEM) observations ofB. cinerea hyphae exposed to the most effective concentrations of

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Fig. 1. The effects of different concentrations of volatile (A) and contact (B) phase ofessential 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 notsignificantly different according to Tukey's test (P≤0.05).

186 E.M. Soylu et al. / International Journal of Food Microbiology 143 (2010) 183–189

essential oil vapour (volatile phase) or grown on PDA amended withthe different concentrations of essential oil (contact phase) showeddegenerative changes in the hyphal morphology in comparison tothick, elongated, smooth surfaced hyphae in control plates (Fig. 2Aand B). After exposure to the most effective oil concentrationsdetermined in vitro studies, [contact (12.8 μg/ml) or volatile phases(0.2 μg/ml air)], hyphae appeared degraded (Fig. 2C), large vesicleswere visible within the cell walls. Shrivelled hyphal cells had either nocytoplasm or the cytoplasm was depleted of organelles. Under theeffect of the oils, the growth of the funguswas suppressed (Fig. 2C andD). Complete absence of conidiation was also observed in oil treatedPetri plates.

Table 1The effects of different concentrations of volatile phases of essential oils on the conidial ge

Concentrations(μg/ml air)

Conidial germination (%)

Origanum Lavender Rose

0 100 c 100 e 1000.2 17 b 77 d 900.4 0 a 57.6 c 77.0.8 0 a 19.6 b 371.6 0 a 0 a 0

Arcsine transformation was performed prior to statistical analysis. Within the column, meantest (P≤0.05).

3.4. Effects of the essential oil on disease development in vivo conditions

Disease development was also controlled in vivo, with the essentialoil of origanum which was found to be the most efficient essential oilin vitro studies, reducing infection of tomato leaves significantly. Mosteffective control of fungal infection was achieved if the essential oilwas applied within 24 h post inoculation.

The in vivo fungicidal activity of the different concentrations oforiganum oil against B. cinerea is presented in Table 3. As the doses oforiganum oil increased, the infection of B. cinerea was suppressed.When we compare the curative and protective activities of theoriganum oil on the infection caused by B. cinerea, the greatest effecton the disease severity of the pathogen was observed in curativeactivity (Table 3). In curative treatments, in vitro effective (25 mg/l) orhigher concentrations (50 and 75 mg/l) of origanum essential oil andiprodione at the recommended concentration (375 mg/l) displayed asignificant decrease in disease severity on tomato in comparison tocontrols (Table 3). The mean disease severity caused by B. cinereawassignificantly (P≤0.05) reduced by the highest concentration oforiganum oil (75 mg/l) used (Table 3).

Protective activity of origanum oil had a very low effect at 12.5, 25and 50 mg/l doses, in comparison to same concentration used in thecurative activities. Only origanum essential oil at the highestconcentration (75 mg/l) and iprodione (375 mg/l) significantlyreduced grey mould severity (Fig. 3).

No sign of phytotoxicity was found on the tested plants at thehighest concentration (75 mg/l) used in the experiments.

4. Discussion

Food safety is usually ensured by the addition of antimicrobialsthat prevent or considerably retard microbial spoilage organisms. Thewidespread use of pesticides has significant drawbacks includingincreased cost, handling hazards, concern about pesticide residues onfood, and threat to human health and environment. Public awarenessof these risks has increased interest in finding safer alternativesprotectant to replace currently used synthetic chemical pesticides.One such alternative is the use of natural plant protectants withpesticidal activity such as essential oil and their major components,since they tend to have low mammalian toxicity, less environmentaleffects and wide public acceptance (Isman, 2000; Kalemba andKunicka, 2003; Burt, 2004).

Natural antimicrobial agents such as essential oils can be used infood industry only if the compounds they release over time and theireffects on target plant and pathogen are well-known and understood.Our study indicated that essential oils may possess antifungal activityagainst grey mould disease agent B. cinerea and can be exploited as anideal treatment for future plant disease management programseliminating fungal spread. Suppression of mycelial growth, sporegermination and germ tube elongation by essential oil treatmentscould make a major contribution to limiting the spread of thepathogen by lowering the spore load in the storage atmosphere andon surfaces. Although all plant species are related and belong to the

rmination and germ tube elongation.

Length of germ tube elongation (μm)

mary Origanum Lavender Rosemary

d 837.5 c 837.5 d 837.5 ed 54.6 b 410.7 c 486.4 d3 c 0 a 234.2 b 321.7 cb 0 a 171.5 b 113.2 ba 0 a 0 a 0 a

values followed by the same letter are not significantly different according to Tukey's

Table 2The effects of different concentrations of contact phases of essential oils on the conidial germination and germ tube elongation.

Concentrations(μg/ml)

Conidial germination (%) Length of germ tube elongation (μm)

Origanum Lavender Rosemary Origanum Lavender Rosemary

0 100 b 100 c 100 b 825.9 e 825.9 f 825.9 d0.4 100 b 100 c 100 b 502.3 d 816.6 f 818.8 d0.8 100 b 100 c 100 b 307.8 c 808.6 f 803.6 d1.6 100 b 100 c 100 b 112.6 b 533.3 e 794.6 d3.2 0 a 100 c 100 b 0 a 241.2 d 789.2 d6.4 0 a 100 c 100 b 0 a 205.3d 559.2 c12.8 0 a 100 c 100 b 0 a 115.6 c 48.3 b25.6 0 a 59 b 0 a 0 a 34.6 b 0 a51.2 0 a 0 a 0 a 0 a 0 a 0 a

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

187E.M. Soylu et al. / International Journal of Food Microbiology 143 (2010) 183–189

same plant family, essential oil of origanum showed remarkableantifungal effect, whereas lavender and rosemary showed a lessinhibitory effect against B. cinerea. This might be attributed to themode of resistant behaviour of the fungi against various substancespresent in the various essential oil. We have recently investigated thechemical 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%), andcamphor (20.2%), respectively. The antimicrobial properties ofessential oils of origanum, rosemary and lavender and their majorconstituents, have been shown to be able to suppress several plantpathogenic 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.,

Fig. 2. Scanning electron microscopy of hyphae exposed to origanum essential oil. (A and B) Hoils on hyphal morphology. Note alterations in hyphal morphology including hyphal shrive

2004; Soylu et al., 2006; Bajpai et al., 2007; Martínez-Romeroa et al.,2007; Soylu et al., 2007; Hadian et al., 2008; Kordali et al., 2008; Ozcanand Chalchat, 2008).

The majority of the work initiated so far has concentrated on theeffect of essential oils on inhibition of fungal mycelial growth in vitroconditions. In previous works, we have shown that the volatilecomponents of the essential oils of taxonomically different medicinalplants possess in vitro activity against a number of tomato pathogens(Soylu et al., 2005; Soylu et al., 2006). Unlike to in vitro studies, veryfew studies have been conducted in vivo conditions to show fungicidalproperties of essential oils against plant pathogenic fungi (Letessieret al., 2001; Oxenham et al., 2005; Soylu et al., 2007). In vivo fungicidalactivity of the most efficient essential oil, origanum was alsoinvestigated in greenhouse conditions. Essential oil was sprayed on

ealthy hyphae and conidia (arrow) in control Petri plates. (C and D) Effects of essentiallling, blistering (arrows) in plate (C) and lysis (arrows) in plate (D).

Table 3The curative and protective effects of different concentrations of origanum essential oilon the infection caused by B. cinerea in the greenhouse.

Concentrationsa Curative effect Protective effect

DIb % protection DI % protection

Negative controlc 0 a 100 0 a 100Positive controlc 1.90 d 0 1.90 e 012.5 1.33 c 30.0 1.73 de 8.9525.0 0.70 b 63.16 1.50 cd 19.4750.0 0.53 b 72.11 1.40 cd 26.3275.0 0.43 b 77.37 1.27 c 33.16Iprodione (375.0) 0.37 ab 80.53 0.40 b 78.95

a Concentration expressed as mg/l.b Disease index (DI) according to Lee et al. (2006).c 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 onlyinoculated with B. cinerea. Both controls were sprayed with distilled water. The meansin a column followed by the same letter(s) represent values that are not significantlydifferent according to Tukey's test (P≤0.05).

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the tomato leaves before (protective activity) or after (curativeactivity) fungal inoculation in order to reveal whether essential oil hascurative or protective activities. Although both treatments wereeffective in reducing fungal infection, greatest control was consis-tently achieved when essential oil applications were made 24 h afterinoculation (curative activity). This would suggest that the whole oilsexerted their greatest effect on early fungal development on the leafsurface, e.g. spore germination, germ tube growth and/or appresso-rium formation as we also recorded in vitro studies. Similarobservations were also reported in different plant-pathogen interac-tions. Botrytis fabae and the rust fungus Uromyces fabae were alsocontrolled in vivo, with the whole essential oil of basil (Ocimumbasilicum), as well as its major compound pure methyl chavicol andlinalool, reducing infection of broad bean leaves significantly (Oxenhamet al., 2005). Most effective control of these fungal infections wasachieved if the treatments were applied 3 h post inoculation. Letessieret al. (2001) showed that the essential oil of hyssop reducedgermination of B. fabae conidia and uredospores of U. fabae. Theseworkers reported differences in the efficacy of hyssop oil when usedin vitro and in vivo,with variable and inconclusive results obtainedwhen

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A a

Fig. 3. Effect of different concentrations of essential oil on the control of the grey mouldcaused by Botrytis cinerea on tomato plants (in vivo). Iprodione represents the chemicalfungicide used at the concentration of 375 mg/l. Positive and negative controls indicateartificial inoculation of the fungal pathogen and water treatment, respectively. Bars, foreach effect, with the same small or large letters represent values that are notsignificantly different according to Tukey's test (P≤0.05).

the oil was used to control powdery mildew on barley and apple.Letessier et al. (2001) suggested that if the volatile components of thehyssop oil were responsible for its antifungal activity, the volatileswould be confined within the Petri dish. Inconclusive results obtainedwhen the oil was used to control powdery mildew on barley and applebecause of essential oil diffused away from the leaf surface before comeinto contact with fungal structure. In our case, it could be hypothesizedthat origanum essential oil presented a better effect as curative ratherthan protective treatment because of they might poorly be absorbed byfungi and plant cells if they are applied before pathogen inoculation.

Although the mechanism underlying the action of essential oil onthe vegetative and reproductive phases of fungal developmentremains to be understood, light and SEM observations of hyphae ofB. cinerea exposed to essential oils revealed alterations in the hyphalmorphology. Shrivelled hyphal aggregates, reduced hyphal diametersand lysis of hyphal wall were commonly observed in essential oiltreated mycelium, compared with thick, elongated, normal mycelialgrowth in controls. Such modifications may be related to the effect ofthe essential oil as enzymatic reactions regulating wall synthesis(Rasooli et al., 2006). The lipophilic properties of oil componentsmight have also aided in the ability of the oil to penetrate the plasmamembrane (Knobloch et al., 1989). The observations made with lightand electron microscopy are in accordance with previous studies inwhich essential oils of aromatic plants caused the morphologicalalterations on the fungal hyphae (Bianchi et al., 1997; Fiori et al, 2000;Romagnoli et al., 2005; Soylu et al., 2006; Soylu et al., 2007). Similarobservations were recently presented by Tripathi et al. (2009) whofound that Hyptis suaveolens essential oil caused severe damage andalterations to vegetative hyphae of Fusarium oxysporum f.sp. gladiolileading to complete loss of cytoplasm from the hyphae. The impacts ofoils on fungal structures may reflect effects of the volatiles emitted byoils on surface mycelial development (and thus the ‘platform’ tosupport spore production) and/or the perception/transduction ofsignals involved in the switch from vegetative to reproductivedevelopment.

The volatile phases of the essential oils were found to be moretoxic than the contact phase to the B. cinerea. Volatile phase ofessential oils of different plants were also reported to possess moreantimicrobial activity against plant pathogenic fungi and bacteria(Edris and Farrag, 2003; Soylu et al., 2005). Some investigatorsreported that the antifungal activity resulted from a direct effect ofessential oil vapours on fungal mycelium. They further postulated thatthe lipophilic nature of essential oils render themmore absorbable bythe fungal mycelia than by agar due to the highly lipophilic nature ofthe 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 ofessential oil of medicinal plants such as origanum, lavender androsemary, in hopes to find out new natural product(s) to be used as abio fungicide against B. cinerea. The essential oil of origanum has beenshown to reduce growth of B. cinerea on solid media (in vitro) and tocontrol infection of tomato by B. cinerea (in vivo). Since essential oilshave low mammalian toxicity, are biodegradable, multifunctional,non-persistent in the environment, and are cheap to produce, thepossibility of developing essential oils for use in crop protection maybe an attractive venture. However, further studies need to beconducted to evaluate the cost and efficacy of these essential oils onwide range of diseases in commercial greenhouses.

5. Conclusions

Considering the reduction in the mycelial growth and germinationof conidia in vitro and incidence of disease symptoms on essential oiltreated plants, we concluded that essential oils could be used aspossible biofungicides alternative to synthetic fungicides againstphytopathogenic fungi.

189E.M. Soylu et al. / International Journal of Food Microbiology 143 (2010) 183–189

Acknowledgement

This study was supported financially by The Scientific andTechnical Research Council of Turkey.

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