www.elsevier.com/locate/jbiosc

Journal of Bioscience and BioengineeringVOL. xx No. xx, 1e6, 2016

Antifungal activities of selected essential oils against Fusarium oxysporum f. sp.lycopersici 1322, with emphasis on Syzygium aromaticum essential oil

Abhishek Sharma,1 Sasireka Rajendran,2 Ankit Srivastava,3 Satyawati Sharma,1,* and Bishwajit Kundu3

Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India,1 Kamaraj College of Engineering and Technology,S.P.G.C. Nagar, Madurai Road, Virudhunagar, Tamil Nadu 626001, India,2 and Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas,

New Delhi 110016, India3

Received 19 October 2015; accepted 19 September 2016Available online xxx

* CorrespondE-mail add

1389-1723/$http://dx.doi

Please cite1322, with

The antifungal effects of four essential oils viz., clove (Syzygium aromaticum), lemongrass (Cymbopogon citratus), mint(Mentha 3 piperita) and eucalyptus (Eucalyptus globulus) were evaluated against wilt causing fungus, Fusarium oxy-sporum f. sp. lycopersici 1322. The inhibitory effect of oils showed dose-dependent activity on the tested fungus. Mostactive being the clove oil, exhibiting complete inhibition of mycelial growth and spore germination at 125 ppmwith IC50value of 18.2 and 0.3 ppm, respectively. Essential oils of lemongrass, mint and eucalyptus were inhibitory at relativelyhigher concentrations. The Minimum inhibitory concentration (MIC) of clove oil was 31.25 ppm by broth microdilutionmethod. Thirty one different compounds of clove oil, constituting approximately ‡99% of the oil, were identified by gaschromatographyemass spectroscopy analysis. The major components were eugenol (75.41%), E-caryophyllene (15.11%),a-humulene (3.78%) and caryophyllene oxide (1.13%). Effect of clove oil on surface morphology of F. oxysporum f. sp.lycopersici 1322 was studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM obser-vation revealed shrivelled hyphae while AFM observation showed shrunken and disrupted spores in clove oil treatedsamples. In pots, 5% aqueous emulsion of clove oil controlled F. oxysporum f. sp. lycopersici 1322 infection on tomatoplants. This study demonstrated clove oil as potent antifungal agent that could be used as biofungicide for the control ofF. oxysporum f. sp. lycopersici in both preventive and therapeutic manner.

� 2016, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Fusarium oxysporum f. sp. lycopersici; Essential oils; Minimum inhibitory concentration; Syzygium aromaticum; Microscopy]

Fungal diseases caused by the Fusarium spp. in plants impedeagricultural production throughout the world (1). Besides infectingcrops, the genus Fusarium is known to produce mycotoxins instored grains (2). Fusarium oxysporum is one of the most importantsoil-borne pathogen that causes wilt diseases in wide variety ofcrops (3). The pathogen has the ability to persist for very long pe-riods in soil without a host (4). Chemical strategies involving use ofsynthetic pesticides for the control of Fusariumwilt have now beendiscouraged due to undesirable effects on soil health, humans andnon-targeted organisms in the environment (5). The focus is,therefore, shifted to search potent chemicals of biological origin forthe management of Fusarium wilt diseases.

Phytochemicals are considered to be environmentally safe as theyare biodegradable (6) and have little or nil toxicity to non-targetanimals (7). Plant essential oils are concentrated volatile hydropho-bic liquids extracted from different parts of the aromatic plants (8).Essential oils are known to possess fungicidal (9), antimicrobial (10)and insecticidal activities (11). The remarkable biological activity ofessential oils against phytopathogens has previously been reported(12e14). Moreover, the ability of some essential oils to be used incombination with other essential oil (15) or bio-control agents (16)make them a potential candidate to combat Fusarium wilt diseasesin field conditions. However, there is a dearth of available literature

ing author. Tel.: þ91 11 2659 1116; fax: þ91 11 2658 1121.ress: satyawatis@hotmail.com (S. Sharma).

e see front matter � 2016, The Society for Biotechnology, Japan..org/10.1016/j.jbiosc.2016.09.011

this article in press as: Sharma, A., et al., Antifungal activitiesemphasis on Syzygium aromaticum essential oil, J. Biosci. Bioe

on the application and mechanism of action of essential oils on thevegetative and reproductive phases of F. oxysporum f. sp. lycopersici.

The aim of the present study is to investigate the fungicidalactivity of essential oils of clove (Syzygium aromaticum), lemon-grass (Cymbopogon citratus), mentha (Mentha � piperita) andeucalyptus (Eucalyptus globulus) against F. oxysporum f. sp. lyco-persici 1322. The chemical analysis of most potent clove oil has beenanalysed using gas chromatographyemass spectroscopy (GCeMS).Further, the effect of clove essential oil on hyphal and sporemorphology were studied through scanning electron microscope(SEM) and atomic force microscope (AFM), respectively. In vivobioassays of tomato were performed to evaluate the ability of cloveoil to reduce wilting caused by F. oxysporum f. sp. lycopersici 1322.

MATERIALS AND METHODS

Chemicals and strains The selected essential oils were procured from GogiaChemicals, Okhla, New Delhi (India) and stored at 4�C till further used. Culturemedia, dimethyl sulfoxide (DMSO), Tween-80 and resazurin were purchased fromMerck, India. The resazurin indicator solution was prepared by dissolving a270 mg tablet in 40 ml of sterile distilled water. A vortex mixer was used toensure that it was a well-dissolved and homogenous solution. F. oxysporum f. sp.lycopersici 1322 strain was procured from Indian Type Culture Collection (ITCC),Indian Agricultural Research Institute, PUSA, New Delhi, India. The culture wasmaintained by sub culturing on to potato dextrose agar (PDA) incubated at 28�Cin dark for 7 days and stored at 4�C for long term use.

Antifungal activity assay In vitro antifungal activity of essential oils onmycelia growth of F. oxysporum f. sp. lycopersici 1322 was evaluated by the poisoned

All rights reserved.

of selected essential oils against Fusarium oxysporum f. sp. lycopersicing., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.09.011

2 SHARMA ET AL. J. BIOSCI. BIOENG.,

food technique (17). PDA (20 ml) was poured into sterilized Petri dishes (90 mmdiameter) and measured amount of oil from stock solution in 10% DMSO(100,000 ppm) was added to get the required concentrations 15.62e500 ppm (7).The control sets were prepared using equal amounts of 10% DMSO in place of oil.A fungal disc (5 mm in diameter) of mycelium, cut from the periphery of a five-day-old culture using a cork borer, was inoculated aseptically into the centre ofeach Petri dish. The plates were sealed with polyethylene film and incubated at atemperature of 28 � 2�C until the growth in the control plates reached the edgeof the plates. The plates were used in triplicate for each treatment. Percentageinhibition of the radial growth by different oils compared to control wascalculated using the following formula (18):

Percentage mycelial inhibition ¼ ½ða� bÞ=a� � 100 (1)

where a is the mean colony diameter for the control sets and b is the mean colonydiameter for the treatment sets.

Spore germination assay Six concentrations of oils (15.62e500 ppm) weretested for spore germination of the test fungi. Fungal spores obtained from 10-day-old cultures of the fungi were taken and placed on glass slides in triplicate. Slidescontaining the spores were incubated in a moist chamber at 25 � 2�C for 24 h.Each slide was then fixed in lacto-phenol-cotton blue and observed under themicroscope for spore germination. For each treatment, 100 spores were countedand the number of spores germinated was scored using haemocytometer tocalculate the percentage of spore germination.

Determination of minimum inhibitory concentration and minimumfungicidal concentration of essential oils The determination of minimuminhibitory concentration (MIC) of essential oils was determined by the brothmicrodilution method. The microbial inoculum suspensions were prepared byharvesting spores from 7-day-old slant surface by pouring sterile 0.1% Tween-80 towash off the spores. The suspensions were mixed for 15 s to ensure homogeneityand subsequently diluted to obtain the required working inoculum of5 � 105 CFU/ml. On the day of the test, 100 ml of the 2-fold essential oil dilutionsreceived 80 ml of an inoculum suspension and 20 ml of resazurin solution (the finalvolume in each well was 200 ml). The growth control wells contained 100 ml ofthe corresponding diluted inoculum suspension and 100 ml of the sterile oil-freemedium and DMSO mixture. The negative control wells consisted of 180 ml of themedium and 20 ml of resazurin solution. The contents of the well were mixed andthe plates were incubated at 28�C for 48 h. A colour change from purple to pinkwas indicative of fungal growth. The lowest concentration at which colour changedid not occur was taken as the MIC value. All tests were performed in triplicates.

Now an aliquot of 50 ml from the wells remaining purple were plated onto PDAand incubated for 72 h at 28�C. The least concentration of the essential oil with novisible fungal growth after incubation was taken as minimum fungicidal concen-tration (MFC) (19).

Gas chromatographicemass spectrometry Chemical composition of theessential oil (clove oil) was determined by GCeMS fitted with a flame ionizationdetection (FID) detector having a 60 m � 0.25 mm � 0.25 m WCOT column coatedwith diethylene glycol (AB-Innowax 7031428, Japan). Heliumwas carrier gas used at aflow rate of 3 ml/min at a column pressure of 155 kPa. Both injector and detectortemperatures were maintained at 260�C. Samples (0.2 ml) were injected into thecolumn with a split ratio of 80:1. Component separation was achieved following alinear temperature programme of 60e260�C at 3�C/min and then held at 260�C for10 min, with a total run time of 50 min. The percentage composition was calculatedusing peak normalization method assuming equal detector response. The sampleswere then analysed on same Shimadzu instrument fitted with the same column andfollowing the same temperature programme as above and the MS parameters used

0102030405060708090

100

15.62 31.25 125

Myc

elia

l inh

ibiti

on (%

)

Concent

Clove oil lemongras

FIG. 1. Inhibitory effects of essential oils on the mycelial growth of Fus

Please cite this article in press as: Sharma, A., et al., Antifungal activities1322, with emphasis on Syzygium aromaticum essential oil, J. Biosci. Bioe

were: ionization voltage (EI) 70 eV, peak width 2 s, mass range 40e850 m/z anddetector voltage 1.5 V. Peak identification was carried out by comparison of the massspectra withmass spectra available on database of National Institute of Standards andTechnology (NIST12 or NIST62) and Wiley 229 mass spectrometry libraries.

Scanning electron microscopy and atomic force microscopy For SEM,F. oxysporum f. sp. lycopersiciwere grown on PDA treated with and without essentialoil at 28�C for 5 days. Segments (5 � 10 mm) were cut from cultures growing onpotato dextrose plates and prepared for SEM visualization by primary fixationwith a2.5% glutaraldehyde solution overnight at 4�C. Thereafter, they were washed with0.1 M sodium phosphate buffer solution (pH 7.2) three times for 20 min each.Following which the samples were dehydrated in an ethanol series (30%, 50%, 70%,and 95%), for 20 min in each alcohol dilution and finally with absolute ethanol for45 min. Samples were then critical point dried in liquid carbon dioxide. The sampleswere mounted on silver stub and gold covered by cathodic spraying (Polaron gold).Morphology of fungus was observed on a scanning electron microscope (Zeiss EVO50) operating at 20.00 kv.

AFM images were obtained using the Bioscope Catalyst AFM (Bruker Corpora-tion, Billerica, MA, USA) having a Nanoscope V controller. For sample preparation, aneven layer of 5e8 ml phosphate buffer saline (PBS) suspended spores were poured ona freshly peeledmica surface followed by drying under nitrogen flow. Following this,the morphologies of untreated and treated F. oxysporum f. sp. lycopersici 1322 sporeswere imaged using standard contact mode in air at room temperature. For imaging,silicon nitride cantilevers having a nominal spring constant of 0.03e0.6 N/m wereused. A standard scan rate of 0.5 Hz with 512 samples per line was used for imagingeach sample. Finally, the images were processed using Nanoscope analysis, v.1.4. Asingle third order flattening of height images with a low pass filter was done fol-lowed by section analysis to determine the dimensions in each case.

Effect of the essential oil on disease development in vivo conditions Cloveessential oil, having the highest effect on the pathogen, was selected for polyhousetrial to study the effect of the essential oil on disease caused by F. oxysporum f. sp.lycopersici. The inoculumwas prepared by culturing F. oxysporum f. sp. lycopersici indarkness at 25�C on sterilized wheat grains for 14 days. Inoculated grain wasblended with sterile distilled water into slurry prior to mixing with steam-sterilizedsoil and sand (1:1, v: v) at the rate of 25 g inoculum per kg mix. After 5- dayincubation period, soil mix was treated with different concentrations of cloveessential oil (0.1%, 1%, 5% and 10%) as aqueous emulsions (5.0 ml/150 cm3 soil)and incubated for 48 h. After this period, treated soil mix was transferred to10 cm diameter plastic pots, previously sterilized with 2% sodium hypochlorite.The pots having only fungus infested soil without oil treatment were consideredas control. All experiments were arranged in a completely randomized completeblock design with three replicates of 10 tomato seedlings (6-week-old) pertreatment and repeated twice. Wilt disease started to appear four weeks aftersowing and the wilt development on each tomato plant were determinedaccording to severity scale of 6 (20). They were as follows: 1, no symptom;2, slight infection with plant showed yellowing leaves and wilting 1e20%; 3, plantshowed yellowing leaves and wilting 21e40%; 4, plant showed yellowing leavesand wilting 41e60%; 5, extensive infection with plant showed yellowing leavesand wilting 61e80%; and 6, the whole plant leaves became yellow, 100% of leavesbecame wilting, and the plant was died. The percentage of disease severity index(DSI) and disease reduction were determined using the formula

DSIð%Þ ¼X

ðgrade� number of plants in that gradeÞ�ðmaximum grade� total number of assessed plantsÞ � 100

(2)

62.5 250 500ra on (ppm)

s Mint Eucalyptus

arium oxysporum. Error bars indicate the mean � standard error.

of selected essential oils against Fusarium oxysporum f. sp. lycopersicing., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.09.011

0

10

20

30

40

50

60

15.62 31.25 62.5 125 250 500

Spor

e ge

rmin

atio

n (%

)

Concentration (ppm)

Clove oil lemongrass Mint Eucalyptus

FIG. 2. Effect of different concentration of essential oils on spore germination of Fusarium oxysporum. Error bars indicate the mean � standard error.

VOL. xx, 2016 ANTIFUNGAL ACTIVITIES OF ESSENTIAL OILS 3

% disease reduction ¼ðdisease severity index of control� disease severity index of treatmentÞ=ðdisease severity index of controlÞ � 100

(3)

Statistical analyses All experiments were repeated at least twice. Data wereanalysed by using SPSS (version 10) statistical software. Effect of treatments onF. oxysporum f. sp. lycopersiciwere analysed using one way ANOVA. Duncan multiplerange tests were used to compare the significance of differences among treatmentsat p values of<0.05. The IC50 value (concentration causing 50% reduction in mycelialgrowth and spore germination) was estimated for each essential oil by using probitanalysis (LdP Line).

TABLE 2. Chemical compositions of Syzygium aromaticum essential oil by GCeMS.

Sample no. Constituent RIa Area (%)b

1 3-Ethyl-3-methylheptane 931 Trace2 Ethylene diglycol monoethyl ether 1006 0.233 Benzyl alcohol 1040 Trace4 Cyclohexane, 1,5-dimethyl-2,3-divinyl- 1143 Trace5 Trifluoroacetyl-a-terpineol 1167 Trace6 Chavicol 1252 Trace7 a-Cubebene 1349 Trace8 Eugenol 1357 75.419 Vanillin 1392 Trace10 cis-Nepetalactone 1399 0.1111 g-Caryophyllene 1407 Trace12 E-caryophyllene 1424 15.1113 Methyl acetylsalicylate 1440 Trace14 Isoeugenol (Z) 1455 Trace15 (4-Allyl-2-methoxyphenoxy)

trimethylsilane1470 0.34

16 1-Cycloheptene, 1,4-dimethyl-3-(2-methyl-1-propene-1-yl)-

1480 Trace

17 b-Caryophyllene 1489 0.3718 b-Selinene 1492 Trace19 a-Muurolene 1497 Trace20 a-Farnesene 1504 0.1821 Chamigren 1507 Trace22 d-Cardinene 1518 0.8423 a-Bisabolene (E) 1540 Trace24 a-Calacorene 1544 Trace25 Caryolan-8-ol 1575 0.15

RESULTS

Antifungal activity assay All essential oils inhibited themycelial growth of F. oxysporum f. sp. lycopersici in a dose-dependent manner (Fig. 1). A concentration of 125 ppm of cloveessential oil completely inhibited the mycelial growth of fungus.Essential oils of lemon grass and mint were found to befungicidal at 250 and 500 ppm, respectively. Eucalyptus oilexhibited fungal mycelial growth inhibition percentage rangingfrom 15.93% to 72.5%.

Inhibitory concentration (IC50) values of each essential oil werealso estimated by using Probit analyses. The lowest IC50 values ofoils were recorded for clove essential oil (18.22 ppm) followed bylemongrass (24.25 ppm), mint (60.05 ppm) and eucalyptus(207.86 ppm), respectively.

Spore germination assay Effects of different concentrationof essential oils on the spore germination of F. oxysporum f. sp.lycopersici are shown in Fig. 2. As seen inmycelial growth inhibitionexperiments, there was a remarkable inhibition (p< 0.05) of sporesby all types of essential oils. Clove oil was found to bemost effectivein spore germination inhibition (100% at 125 ppm) with IC50 value0.3 ppm. Complete inhibition of spore germination by lemongrassand mint oil was, however, observed at relatively higherconcentration (250 and 500 ppm). The highest inhibition

TABLE 1. Minimum inhibitory concentration (MIC) and minimum fungicidal con-centration (MFC) of essential oils against F. oxysporum 1322.

Essential oil MIC (ppm) MFC (ppm)

Clove oil 31.25 125Lemon grass oil 62.5 250Mint oil 125 500Eucalyptus oil 500 NF

NF: not found.

Please cite this article in press as: Sharma, A., et al., Antifungal activities1322, with emphasis on Syzygium aromaticum essential oil, J. Biosci. Bioe

recorded by eucalyptus oil was 89.88% at 500 ppm. The IC50values of lemongrass, mint and eucalyptus oil were found to be0.98, 3.2 and 18.26 ppm, respectively. DMSO (10%, v/v), as acontrol, did not inhibit the spore germination of F. oxysporum f.sp. lycopersici (not shown in Fig. 2).

Minimum inhibitory concentration and minimumfungicidal concentration of essential oils MIC and MFCvalues of essential oils against F. oxysporum f. sp. lycopersici ob-tained by colourimetric assay followed by plating out on PDA arepresented in Table 1. The clove oil displayed potent antifungal

26 Spathulenol 1576 Trace27 a-Humulene 1579 3.7828 Caryophyllene oxide 1587 1.1329 Humulene epoxide II 1613 Trace30 allo-Aromadendrene 1644 0.5131 Longifolol 1715 0.3932 Other compound e 0.4

Trace <0.1%.a RI: Retention index literature comparison.b The relative proportions of the essential oil constituents.

of selected essential oils against Fusarium oxysporum f. sp. lycopersicing., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.09.011

4 SHARMA ET AL. J. BIOSCI. BIOENG.,

activity with MIC and MFC values of 31.25 and 125 ppm,respectively. MIC value of lemongrass, mint and eucalyptusessential oil was found within the range of 62.5e500 ppm.Except eucalyptus oil, all essential oils inhibiting fungal growthshowed fungicidal activity. In general, MFC value was higher thantheir respective MIC value. Since clove essential oil showed thelowest MIC and highest inhibition percentage of fungal growth(mycelia and spores), this was selected for further study.

Gas chromatographicemass spectrometry The major con-stituents of clove oil identified by GCeMS are listed in Table 2.GCeMS analysis resulted in identification of 31 constituents(Fig. S1). The most abundant constituent was phenylpropanoidcompound, eugenol (75.41%). The other main components of theoil were E-caryophyllene (15.11%), a-humulene (3.78%),caryophyllene oxide (1.13%), d-cardinene (0.84%) and allo-aromadendrene (0.51%). Rest all constituents were present inamounts less than 0.5%.

Scanning electron microscopy and atomic forcemicroscopy SEM observations of F. oxysporum f. sp. lycopersicigrown on PDA amended with the most effective concentration ofclove essential oil (125 ppm) showed considerable alteration inhyphal morphology in comparison to the control (Fig. S2). Afterexposure to clove oil, the growth of fungus was suppressed withdegraded and shrivelled hyphae. The shrunken hyphae showedlack of cytoplasm with disrupted cell wall. SEM of the controlshowed normal morphology with lengthened, smooth andhomogenous hyphae.

The AFM analysis of untreated samples showed spherical anddisc shaped structures (Fig. 3a). Homogeneity in the curvilinear

FIG. 3. AFM images showing morphology of (a) untreated and (b) clove oil treated F. oxysamplitude error image generated during contact mode AFM imaging. Arrows in amplituderepresenting maximum and minimum heights in each image are also shown. Scale bars in

Please cite this article in press as: Sharma, A., et al., Antifungal activities1322, with emphasis on Syzygium aromaticum essential oil, J. Biosci. Bioe

morphology of F. oxysporum f. sp. lycopersici spores was suggestiveof their physiologically unperturbed state (Fig. 3a, arrows). How-ever, the AFM images of clove oil treated samples showed shrunkenand distorted spores (Fig. 3b). It is evident that the treatment leadsto fragmentation and loss of characteristic curvilinear morphologyof spores (Fig. 3b, arrows).

Further, on analysing the surface morphology, the untreatedsamples showed smooth and consistent topology of spores. Thethree-dimensional map showed that the untreated spores exhibi-ted uniformity in structure (Fig. 4a). The section analysis of un-treated spores showed a homogenous size distribution with anaverage height and width of 20 nm and 32 nm, respectively. On thecontrary, the clove oil treated samples displayed rough, fragmentedand non-uniform surface morphology (Fig. 4b). The section analysisshowed that the clove oil treated samples have a height and widthdistribution of 2e3 nm and 3e4 nm, respectively. This showed thatclove oil treatment had a pestilent effect on F. oxysporum f. sp.lycopersici spores that resulted in more than 10 fold reduction intheir size distribution plausibly due to their rupture and fragmen-tation. Thus, it is evident from these results that clove oil treatmentpotentially severs F. oxysporum f. sp. lycopersici spores and affectstheir morphology, surface attributes and size distribution.

Effect of clove essential oil on F. oxysporum f. sp. lycopersiciinoculum in soil The effect of treatment on incidence, severityof Fusarium wilt and the percentage of disease reduction achievedafter 4 weeks of inoculation are reported in Table 3. Increasingconcentration of the clove oil resulted in significant (p < 0.5)reduction of disease incidence and severity compared to control.The best inhibitory effect was shown by clove oil at 5% providing

porum spores. The left image in each case is the height image and the right image iserror images indicate differences in morphology in both cases. The colour coded barseach case represent 0.5 mm.

of selected essential oils against Fusarium oxysporum f. sp. lycopersicing., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.09.011

FIG. 4. AFM images showing three-dimensional view of (a) untreated and (b) clove oil treated F. oxysporum spores. The zoomed picture in each case depict crucial differences in sizeas well as surface attributes in untreated and treated spores. Each AFM micrograph is accompanied with a height distribution histogram. The height range of untreated spores wasbetween 18 and 20 nm that gets reduced to 2e3 nm after clove oil treatment. The inset in each histogram shows width distribution in both cases. Untreated and clove oil treatedspores show width ranging between 30e35 and 3e5 nm, respectively.

TABLE 3. Effect of clove essential oil on disease severity index and disease reductionof tomato Fusarium wilt.

Treatment (%) Average disease severity grade DSI (%) Disease reduction (%)

0.1 3.71 61.9 � 3.2 35.81 2.21 36.9 � 2.4 61.765 0.78 13.09 � 1.7 86.5Control 5.78 96.42 � 1.2 e

All values within columns are significantly different by ANOVA (p < 0.05). The re-sults are expressed as an average of three replicates of 10 tomato seedlings (6-week-old) per treatment � standard error (SE).

VOL. xx, 2016 ANTIFUNGAL ACTIVITIES OF ESSENTIAL OILS 5

86.5% disease reduction in tomato plants. Treatment of infested soilwith 10% clove oil proved to be phytotoxic and hence not includedin the experiment.

DISCUSSION

The present investigation revealed that all essential oils tested,had moderate to high in vitro antifungal activity againstF. oxysporum f. sp. lycopersici 1322. The varying susceptibility of thepathogen towards essential oils was reflected in trend receivedfrom both qualitative (mycelial growth inhibition) and quantitative(fungal spore germination) bioassays. The efficacy of essentialoils against the pathogen was in the order of clove oil >

lemongrass > mint > eucalyptus. The antifungal activities of theseoils have been shown to suppress several plant pathogenic fungi(8,21,22). The antifungal properties exhibited by these oils could beattributed to the presence of bioactive terpenes, phenolic acids,alcohols, hydrocarbons and aldehydes (23,24). MIC andMFC resultsindicated that clove essential oil was most potent and showedconsistent fungistatic and fungicidal effect. The lower value of MICcompared to MFC value indicated that clove oil is fungistatic atlower concentration and fungicidal at higher concentration. Anti-fungal activities of clove oil have been reported earlier (25,26).

GCeMS analysis of clove essential oil illustrated eugenol (4-allyl-2-methoxyphenol) as dominant compound. On comparing our re-sults with those reported earlier, the variation in the percentage

Please cite this article in press as: Sharma, A., et al., Antifungal activities1322, with emphasis on Syzygium aromaticum essential oil, J. Biosci. Bioe

composition of eugenol (49.7e88.58%) and caryophyllene(1.38e36.94%) were observed (27e30). This notable difference incomposition of clove essential oil might be due to extractionmethods as well as genetic diversity and agronomic treatments(31,32). Although clove oil is a natural product comprising of severalconstituents, it is rational to correlate the antifungal activities ofclove oil in the present study to the presence of high concentrationof eugenol.

It is believed that lipophilic nature of essential oils may facilitatein the penetration of lipid bilayer fungal membrane and causemembrane disruption (33,34). To substantiate this hypothesis SEMand AFM studies were employed. SEM observations of hyphae andAFM observation of spores of F. oxysporum f. sp. lycopersici exposedto clove oil revealed extensive damage and considerable increase incell surface roughness in the hyphae and spores morphology. Our

of selected essential oils against Fusarium oxysporum f. sp. lycopersicing., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.09.011

6 SHARMA ET AL. J. BIOSCI. BIOENG.,

results are in accordance with the previous studies wherein theclove oil or its major constituent, eugenol was shown to disinte-grate the cell membrane causing a major alteration in cell perme-ability (35e37). This could lead to leakage of cell constituents(plasmolysis) and subsequently death of F. oxysporum f. sp. lyco-persici. Spore germination in vascular tissues (xylem) of plants isconsidered as an important mechanism of pathogenicity ofF. oxysporum f. sp. lycopersici (38). Therefore, the appearance ofcollapsed hyphae and spores in clove oil treated samples impliesthat clove oil could be a potential tool to curb the Fusarium wiltdisease in both preventive as well as therapeutic way.

The results obtained during pot bioassay are in accordance to theresults obtained under in vitro conditions. The clove oil showed verystrong inhibitory effect causing significant reduction of wilt diseasein tomato plants. However, the efficacy of clove oil was dosedependent showing highest biocidal activity at 5%. Concentration at10% was phytotoxic perhaps due to high levels of clove oil volatilesstill present in soil at the time of transplantation. In conclusion, thedata obtained provide substantial information in support of essentialoils as natural agents for the control of Fusariumwilt disease. How-ever, further evaluation on the cost and efficacy of these essential oilsas bioformulation in commercial greenhouses/fields is warranted.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jbiosc.2016.09.011.

ACKNOWLEDGEMENTS

This work was supported by Department of Biotechnology,Ministry of Science and Technology, Government of India (grant no.BT/PR7980/AGR/5/617/2013).

References

1. Gullino, L. M., Daughtrey, M. L., Garibaldi, A., and Elmer, W. H.: Fusariumwilts of ornamental crops and their management, Crop Prot., 1e10. http://dx.doi.org/10.1016/j.cropro.2015.01.003 (2015).

2. Masheshwar, P. K., Moharram, S. A., and Janardhana, G. R.: Detection offumonisin producing Fusarium verticillioides in paddy (Oryza sativa. L) usingpolymerase chain reaction (PCR), Braz. J. Microbiol., 40, 134e138 (2009).

3. Hashem, M., Moharama, A. M., Zaiedb, A. A., and Salehb, F. E. M.: Efficacy ofessential oils in the control of cumin root rot disease caused by Fusarium spp.Crop Prot., 29, 1111e1117 (2010).

4. Larena, I., Sabuquillo, P., Melgarejo, P., and De Cal, A.: Biocontrol of Fusariumand Verticillium wilt of tomato by Penicillium oxalicum under greenhouse andfield conditions, J. Phytopathol., 151, 507e512 (2003).

5. De Cal, A., Sztejnberg, A., Sabuquillo, P., and Melgarejo, P.: ManagementFusarium wilt on melon and watermelon by Penicillium oxalicum, Biol. Control.,51, 480e486 (2009).

6. Linde, J. H., Combrinck, S., Regnier, T. J. C., and Virijevic, S.: Chemicalcomposition and antifungal activity of the essential oils of Lippia rehmanniifrom South Africa, S. Afr. J. Bot., 76, 37e42 (2010).

7. Kumar, V., Mathela, C. S., Tewari, A. K., and Bisht, K. S.: In vitro inhibitionactivity of essential oils from some Lamiaceae species against phytopathogenicfungi, Pestic. Biochem. Phys., 114, 67e71 (2014).

8. Pawar, V. C. and Thaker, V. S.: Evaluation of the anti-Fusarium oxysporum f. sp.cicer and anti-Alternaria porri effects of some essential oils, World J. Microbiol.Biotechnol., 23, 1099e1106 (2007).

9. Siddiqui, S. A., Islam, R., Islam, R., Jamal, A. H. M., Parvin, T., and Rahman, A.:Chemical composition and antifungal properties of the essential oil and variousextracts of Mikania scandens (L.) Willd, Arab. J. Chem. http://dx.doi.org/10.1016/j.arabjc.2013.07.050 (2013) (in press).

10. Chutia, M., Bhuyan, P. D., Pathak, M. G., Sarma, T. C., and Boruah, P.: Anti-fungal activity and chemical composition of Citrus reticulata Blanco essential oilagainst phytopathogens from North East India, LWT - Food Sci. Technol., 42,777e780 (2009).

11. Moretti, M. D. L., Sanna-Passino, G., Demontis, S., and Bazzoni, E.: Essentialoil formulations useful as a new tool for insect pest control, AAPS Pharm. Sci.Tech., 3, 64e74 (2002).

12. Arici, S. E., Bozat, G., and Akbulut, I.: Investigation of potential biologicalcontrol of Fusarium oxysporum f. sp. radicis-lycopersici and F. oxysporum f. sp.

Please cite this article in press as: Sharma, A., et al., Antifungal activities1322, with emphasis on Syzygium aromaticum essential oil, J. Biosci. Bioe

lycopersici by essential oils, plant extract and chemical elicitors in vitro, Pak. J.Bot., 45, 2119e2124 (2013).

13. Browers, J. H. and Locke, J. C.: Effect of formulated plant extracts and oils onpopulation density of Phytophthora nicotianae in soil and control of Phytoph-thora blight in the greenhouse, Plant Dis., 88, 11e16 (2004).

14. Browers, J. H. and Locke, J. C.: Effects of botanical extracts on the populationdensity of Fusarium oxysporum in soil and control of Fusarium wilt in thegreenhouse, Plant Dis., 84, 300e305 (2000).

15. Tejeswini, M. S., Sowmya, H. V., Swarnalatha, S. P., and Negi, P. S.: Antifungalactivity of essential oils and their combinations in in vitro and in vivo condi-tions, Arch. Phytopathol. Plant Prot., 47, 564e570 (2014).

16. El-Mougy, N. S., Shaban, A. M. H., and Abdel-Kader, M. M.: Evaluation of seedcoating with some essential oils and bio-agents against root rot disease of fababean, Int. J. Eng. Innov. Technol., 4, 244e248 (2015).

17. Tian, J., Ban, X., Zeng, H., He, J., Huang, B., and Wang, Y.: Chemical compo-sition and antifungal activity of essential oil from Cicuta virosa L. var. latisectaCelak, Int. J. Food Microbiol., 145, 464e470 (2011).

18. Albuquerque, C. C., Camara, T. R., Mariano, R. L. R., Willadino, L.,Marcelino, , Jr., and Ulisses, C.: Antimicrobial action of the essential oil ofLippia gracillis Schauer, Braz. Arch. Biol. Technol., 49, 527e535 (2006).

19. Tyagi, A. K. and Malik, A.: Liquid and vapour-phase antifungal activities ofselected essential oils against Candida albicans: microscopic observations andchemical characterization of Cymbopogon citrates, BMC Complement. Altern.Med., 10, 65 (2010).

20. Silva, J. C. and Bettiol, W.: Potential of non-pathogenic Fusarium oxysporumisolatesor control of Fusariumwilt of tomato, Fitopatol. Bras.,30, 409e412 (2005).

21. Beg, A. Z. and Ahmad, I.: In vitro fungitoxicity of the essential oil of Syzygiumaromaticum, World J. Microbiol. Biotechnol., 18, 313e315 (2002).

22. Ragab, M. M. M., Ashour, A. M. A., Abdel-Kader, M. M., ElMohamady, R., andAbdel-Aziz, A.: In vitro evaluation of some fungicides alternatives againstFusarium oxysporum the causal of wilt disease of pepper (Capsicum annum L.),Int. J. Agric. For., 2, 70e77 (2012).

23. Singh, G., Singh, O. P., and Maurya, S.: Chemical and biocidal investigations onessential oils of some Indian Curcuma species, Prog. Cryst. Growth Charact.Mater., 45, 75e81 (2002).

24. Burt, S.: Essential oils: their antibacterial properties and potential applicationsin foodsda review, Int. J. Food Microbiol., 94, 223e253 (2004).

25. Wang, C., Zhang, J., Chen, H., Fan, Y., and Zhiqi, S.: Antifungal activity ofeugenol against Botrytis cinerea, Trop. Plant Pathol., 35, 137e143 (2010).

26. Deng, J., Li, W., Peng, X. L., and Hao, X. H.: Study on the potential of antifungalactivity of essential oils against fungal pathogens of fruits and vegetable,J. Chem. Pharmac. Res., 5, 443e446 (2013).

27. Srivastava, A. K., Srivastava, S. K., and Syamsundar, K. V.: Bud and leafessential oil composition of Syzygium aromaticum from India and Madagascar,Flavour Fragr. J., 20, 51e53 (2005).

28. Wenqiang, G., Shufen, L., Ruixiang, Y., Shaokun, T., and Can, Q.: Comparison ofessential oils of clove buds extracted with supercritical carbon dioxide and otherthree traditional extraction methods, Food Chem., 101, 1558e1564 (2007).

29. Chaieb, K., Hajlaoui, H., Zmantar, T., Kahla-Nakbi, A. B., Rouabhia, M.,Mahdouani, K., and Bakhrouf, A.: The chemical composition and biologicalactivity of clove essential oil, Eugenia caryophyllata (Syzigium aromaticum L.Myrtaceae): a short review, Phytother. Res., 21, 501e506 (2007).

30. Bhuiyan, M. N. I., Begum, J., Nandi, N. M., and Akter, F.: Constituents of theessential oil from leaves and buds of clove (Syzigium caryophyllatum (L.)Alston), Afr. J. Plant Sci., 4, 451e454 (2010).

31. Mahanta, J. J., Chutia, M., Bordoloi, M., Pathak, M. G., Adhikary, R. K., andSarma, T. C.: Cymbopogon citratus L. essential oil as a potential antifungal agentagainst key weed moulds of Pleurotus spp. Spawns, Flavour Fragr. J., 22,525e530 (2007).

32. Kumar, P., Mishra, S., Malik, A., and Satya, S.: Compositional analysis andinsecticidal activity of Eucalyptus globulus (family: Myrtaceae) essential oilagainst housefly (Musca domestica), Acta Trop., 122, 212e218 (2012).

33. Lambert, R. J. W., Skandamis, P. N., Coote, P. J., and Nychas, G. J. E.: A study ofthe minimum inhibitory concentration and mode of action of oregano essentialoil, thymol and carvacrol, J. Appl. Microbiol., 91, 453e462 (2001).

34. Ultee, A., Slump, R. A., Steging, G., and Smid, E. J.: Antimicrobial activity ofcarvacrol toward Bacillus cereus on rice, J. Food Prot., 63, 620e624 (2000).

35. Gill, A. O. and Holley, R. A.: Inhibition of membrane bound ATPase ofEscherichia coli and Listeria monocytogenes by plant oil aromatics, Int. J. FoodMicrobiol., 111, 170e174 (2006).

36. Pinto, E., Vale-Silva, L., Cavaleiro, C., and Salgueiro, L.: Antifungal activity ofthe clove essential oil from Syzygium aromaticum on Candida, Aspergillus anddermatophyte species, J. Med. Microbiol., 58, 1454e1462 (2009).

37. Devi, K. P., Nisha, S. A., Sakthivel, R., and Pandian, S. K.: Eugenol (an essentialoil of clove) acts as an antibacterial agent against Salmonella typhi by disruptingthe cellular membrane, J. Ethnopharmacol., 130, 107e115 (2010).

38. Elarosi, H. M.: Sporulation of fungi inside the plant host cell, Nature, 177,665e666 (1956).

of selected essential oils against Fusarium oxysporum f. sp. lycopersicing., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.09.011