Industrial Crops and Products 55 (2014) 116–122

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Antifungal activity of selected essential oils against fungi isolated

from medicinal plant
Tatjana Stević a , Tanja Berić b , Katarina Šavikin a , Marina Soković c , Dejan God̄evac d ,
Ivica Dimkić b , Slaviša Stanković b,∗
a
Institute for Medicinal Plants Research, Tadeuša Košćuška 1, 11000 Belgrade, Serbia
b
Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia
c
Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia
d
Institute for Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12, 11000 Belgrade, Serbia

a r t i c l e i n f o a b s t r a c t

Article history: The development of protective products with natural origin as alternatives to synthetic fungicides is
Received 8 October 2013 currently in the spotlight. Qualitative and quantitative chemical analysis of 16 selected essential oils was
Received in revised form 3 February 2014 determined. Then, antifungal properties determined by in vitro microdilution method against 21 fungi
Accepted 7 February 2014
isolated from herbal drugs were evaluated. All tested oils showed some antifungal activity against all
fungi used. Savory, thyme and oregano oils, characterized by the presence of phenol such as carvacrol
Keywords:
and thymol, and rose oil containing mainly monoterpene alcohols (citronellol and geraniol) proved to be
Antifungal activity
the most effective inhibitor of all fungi tested. Also, combination of particular oils showed reduction of
Essential oils
MIC
the MIC values when combined, commendatory mixtures for potential application in practice. Moreover,
In situ the reduction of the total number of fungi, in situ, using selected essential oils was determined.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Interest in traditional medicine and, in particular, herbal
medicines, has increased substantially in both developed and
developing countries over the past two decades. Global and
national markets for medicinal herbs have been growing rapidly
and significant economic gains are being realized. As a conse-
quence, the safety and quality of herbal medicines have become
increasingly important concerns for health authorities and the pub-
lic alike. The safety and quality of raw medicinal plant materials
and finished products depend on factors that may be classified as
intrinsic (genetic) or extrinsic (environment, collection methods,
cultivation, harvest, post-harvest processing, transport and stor-
age practices). Inadvertent contamination by microbial or chemical
agents during any of the production stages can also lead to deteri-
oration in safety and quality. In addition to the crop loses, presence
of the fungi in medicinal plants reduces their quality and usefulness
(Essono et al., 2007). Moreover, under certain conditions, some of
∗ Corresponding author. Tel.: +381 11 2637364; fax: +381 11 2638 500.
E-mail addresses: tstevic@mocbilja.rs (T. Stević), tanjab@bio.bg.ac.rs
(T. Berić), ksavikin@mocbilja.rs (K. Šavikin), mris@ibiss.bg.ac.rs (M. Soković),
dgodjevac@chem.bg.ac.rs (D. God̄evac), ivicad@bio.bg.ac.rs (I. Dimkić),
slavisas@bio.bg.ac.rs (S. Stanković).
the ubiquitous fungal contaminants could secrete the toxic metabo-
lites, mycotoxins, with powerful mutagenic and carcinogenic effect.
Mycotoxins are thermo stable and cannot be destroyed by cooking.
They have cumulative ability and are eliminated hard from the
organism (Hashem and Alamri, 2010).
The usual antimicrobial chemicals used in agriculture for plant
disease control (benzimidazoles, aromatic hydrocarbons and sterol
biosynthesis inhibitors) are associated with series of problems. Cur-
rently, there is a strong debate about the safety aspects of chemical
preservatives since they are considered responsible for many car-
cinogenic and teratogenic attributes as well as residual toxicity
(Skandamis et al., 2001). For these reasons, consumers tend to be
suspicious of chemical additives and thus the demand for natural
and socially more acceptable preservatives has been intensified.
The increase of fungal resistance to classical drugs, the treatment
costs, and the fact that most available antifungal drugs have only
fungistatic activity, justify the search for new strategies (Rapp,
2004). The exploration of naturally occurring antimicrobials for
food preservation receives increasing attention due to awareness
of natural food products and a growing concern of microbial resis-
tance towards conventional preservatives (Schuenzel and Harrison,
2002).
In the past decade, due to concerns regarding safety of the
synthetic antimicrobial agents, the particular interest has been
focused on the potential applications of essential oils as alternative

http://dx.doi.org/10.1016/j.indcrop.2014.02.011
0926-6690/© 2014 Elsevier B.V. All rights reserved.
 T. Stević et al. / Industrial Crops and Products 55 (2014) 116–122 117

Table 1 Trichotechium roseum, Phomopsis sp., Phoma sp. and Myrothechium

Essential oils.
verrucaria. As a source of isolation we used herbal drugs that
Essential oil Plant origin Specification showed the highest level of contamination with phytopathogenic
Orange Citrus aurantium amara P0104578 and saprophytic fungi: mint (Mentha piperita L. folium et herb),
Bergamot Citrus aurantium bergamia P0112136 nettle (Urtica dioica L. folium), marigold (Calendula officinalis L.
Lemon Citrus limon P0110499 flowers), horsetail (Equisetum arvense L. herb) and corn silk (Zea
Coriander Coriandrum sativum P0112145 mays L. stigma) (Stević et al., 2012).
Eucalyptus Eucalyptus globulus P0112145
Anise Illicium verum S0100154
Lavender Lavandula angustifolia P0123527
Chamomile (blue) Matricaria recutita P0115610
2.4. Antifungal assay in vitro
Tea tree Melaleuca alternifolia P0123084
Basil Ocimum basilicum P0118460 To investigate the antifungal activity of essential oil, a modi-
Geranium Pelargonium graveolens P0114231 fied version of the microdilution technique was used (Hanel and
Rose Rosa damascena P0100578
Raether, 1988; Daouk et al., 1995). Fungal spores were washed from
Savory Satureja hortensis P0118884
Thyme Thymus vulgaris P0123774 the surface of malt agar (MA) plates (in g/L: malt extract 50 and
Viola Viola odorata P0105637 agar 15) with sterile 0,85% saline containing 0.1% Tween 80 (v/v).
Oregano Origanum heracleoticum na The spore suspension was adjusted with sterile saline to a concen-
tration of approximately 1.0 × 105 in a final volume of 100 ␮L per
well. The inocula were stored at 4 ◦ C for further use. Dilutions of the
chemical control measures. They have a broad spectrum of anti- inocula were cultured on solid MA to verify the absence of contam-
fungal properties (Kalemba and Kunicka, 2003; Soković and van ination and to check the validity of the inoculum. Determination of
Griensven, 2006; Carmo et al., 2008; Koul et al., 2008) and they MIC values was performed by a microdilution technique using 96-
are environmentally friendly (biodegradable, do not leave toxic well microtiter plates. Tryptic Bile Soy Broth (TBS) (in g/L: casein 20,
residues or by-products to contaminate the environment) (Abdel- bile salts 1.5, X-B-D glucuronic acid 0.075 and dimethyl sulfoxide 3)
Kader et al., 2011). was used as the basis in the well to which 0.01% Tween 80, different
This study was undertaken to investigate the in vitro inhibitory volumes of the investigated essential oils and fungal inoculum were
effects of a number of essential oils, differing in chemical composi- added. The microplates were incubated for 72 h at 28 ◦ C. The lowest
tion, against 21 phytopathogenic and saprophytic, pre-harvest and concentrations without visible growth were defined as the minimal
post-harvest, fungi isolated from medicinal herbs, antifungal activ- concentrations which completely inhibited fungal growth (MIC).
ity of combinations of two oils in vitro as well as the in situ effect of The minimal fungicidal concentrations (MFC) were determined by
selected oils. serial subcultivation of a 2 ␮L volume on microtiter plates contain-
ing 100 ␮L of broth per well and further incubation for 72 h at 28 ◦ C.
2. Materials and methods The lowest concentration with no visible growth was defined as the
MFC, indicating 99.5% killing of the original inoculum compared to
2.1. Essential oils fluconazole, used as a positive control.

Essential oils used in this study are listed in Table 1. The oils were
purchased from a Frey + Lau GmbH, Henstedt-Ulzburg, Germany,
except for the essential oil of oregano, which was derived from the
company Herba, Serbia.
2.2. Gas chromatography
Gas chromatography-flame ionization detector (GC-FID) and
Gas chromatography–mass spectrometry (GC-MS) analysis was
performed on an Agilent 7890A GC equipped with 5975 C inert XL
EI/CI MSD and FID detector connected by capillary flow technol-
ogy 2-way splitter with make-up. An HP-5MSI capillary column
(30m × 0.25 mm × 0.25 ␮m) was used. The temperature for GC
oven was programmed from 60 ◦ C to 300 ◦ C at 3 ◦ C/min and hold
for 10 min. Helium was used as carrier gas at 16.255 psi (constant
pressure mode). The sample was analyzed in the splitless mode. The
injection volume was 1 ␮L. GC detector temperature was 300 ◦ C. MS
data was acquired in EI mode, with scan range 30–550 m/z, source
temperature was 230 ◦ C and quadrupole temperature was 150 ◦ C.
Solvent delay was 3 min.
2.3. Fungi
Antifungal activity was tested using the twenty one fungi
isolated from medicinal drugs and identified: Aspergillus flavus,
Aspergillus niger, Alternaria alternata, Fusarium solani, Fusarium
tricinctum, Fusarium sporotrichioides, Fusarium verticillioides, Fusa-
rium oxysporum, Fusarium semitectum, Fusarium subglutinans,
Fusarium equiseti, Penicillium sp., Chaetomium sp., Gliocladium
roseum, Curvularia lunata, Verticillium dahliae, Trichoderma viride,
2.5. Screening antifungal activity of combinations of two oils
in vitro
The synergism, indifference, and antagonism of the combina-
tions of two essential oils were screened on the selected eight
fungal strains. MICs were transformed into fractional inhibitory
concentration (FIC) to determine the interaction of two essential
oils, in the following manner:
MIC of A oil in presence of B oil
FIC of A oil =
MIC of A oil
MIC of B oil in presence of A oil
FIC of B oil =
MIC of B oil
Fractional inhibitory concentration index (FIC index) for each oil
was then calculated from FIC values as follows:
FIC index = FICA + FICB
where A was first oil and B was second oil in combinations.
The FIC index (FICi) was interpreted as: synergistic effect when
FICi ≤ 0.5; indifferent effect when FICi was 0.5–2 and antagonistic
effect when FICi ≥ 2 (da Silva et al., 2011).
Criteria for choosing oils for this assay was to combine two oils of
different antifungal potential (high/low) and chemical composition
and, on the other hand, two oils with similar antifungal potential
(both high) and chemical composition. Also, target fungi for inves-
tigating combine activity of two oils were ones potential producers
of mycotoxins and most frequently isolated from herbal drugs in
our previous investigation (Stević et al., 2012).
118 T. Stević et al. / Industrial Crops and Products 55 (2014) 116–122
2.6. Antifungal activity in situ
A modified method of evaporation of the essential oils from the
filter paper in a sealed bag with the investigated herbal drug, called
“pad delivery system” or “soaking pad system” was used (Plaza
et al., 2004; Arrebola et al., 2010). Sterile Whatman filter discs,
cut 1 cm in diameter, were instilled with certain volumes of essen-
tial oils. Used volumes of the essential oils were from 10 ␮L/filter
disc till 100 ␮L/filter disc. Filter papers with administered essen-
tial oil were placed in sterile tea bags with thread, and those in
paper packaging with drugs that are used for packing tea. Packag-
ing was detained and placed in a sterile plastic bag with sample
drugs. After 5 and 7 days at 25 ◦ C the total number of fungi in the
samples, expressed as the number of yeasts and molds in one gram
of herbal drug was determine on DG 18 agar (Dichloran 18% glyc-
erol agar, in g/L: casein enzymatic digest 5, d-glucose 10, potassium
dihydrogenphosphate 1, magnesium sulphate 0.5, dichloran 0.002,
glycerol anhydrous 220, agar 15, chloramphenicol 0.1).
2.7. Statistical analysis
Means separation of minimal inhibitory concentrations (MIC)
was accomplished by Duncan’s multiple range tests. Analysis of
variance was performed on data of MIC of sixteen oils on 21
pathogenic fungi. Significance was evaluated at p < 0.05 for all tests.
Statistical analyses were conducted by the general procedures of
STATISTICA v.7 (StatSoft, Inc.) and IBM SPSS Statistics v.20 (SPSS,
Inc.).
3. Results and discussion
Most of the oils selected for this study predominantly contained
one or two active compounds and therefore they can be divided
into specific chemo-types (Table 2). The idea was to use oils that
can be easily purchased and to exclude oils with small share of
many constituents, because they are more susceptive to variation
in composition and therefore in activity. The percent share of the
main components very well reflects the biological characteristics of
the oil. In the most oils used in this study monoterpenes were the
main components, while sesquiterpenes and phenylpropans were
under-represented. Also, there are some similarities between par-
ticular oils in sharing of the dominant components. Results from the
GC-MS and GC-FID analyses of the commercial essential oils used
in this study are listed in Table 2. The components of the oil were
identified by comparison of their mass spectra to those from Adams,
Wiley 7, and NIST05 libraries. The identification was confirmed by
retention time lock (RTL) method and RTL Adams Database. For
quantification purpose, area percent data obtained by FID were
used. The essential oils of summer savory, oregano and thyme were
characterized by phenol compounds carvacrol and thymol which
was in agreement with the results of previous studies of these oils
(Lee et al., 2007; Dikbas et al., 2008). Oregano oil contained a sub-
stantially higher concentration of carvacrol (75,8%) than savory oil
(50%). Thymol was predominant component of thyme oil (43,7%).
Also, these three oils contained a significant proportion of biologi-
cal precursors of phenolic components, ␥-terpinene and p-cymene.
Rose and pelargonium oil were found to be characterized by two
major compounds, monoterpene alcohols, citronellol and geraniol,
as well as in other studies (Yeon-Suk et al., 2008). Both compo-
nents are proportionally over-represented in the rose oil, 51,3%
of citronellol compared to 39% in pelargonium oil, while geran-
iol was more than twofold present in rose oil (25,8% compared
to 11,3%). The essential oils of lavender, coriander and bergamot
are characterized by monoterpene components: linalool (monoter-
pene alcohol), limonene (monoterpene hydrocarbons) and linalool
acetate (monoterpene acetate). The essential oils of anise and basil
are dominated by one component, phenylpropan, E-anethole with
91,8% in anise oil and methyl chavicol with 89% in basil oil. Basil
oil is known to be variable in composition and often contains high
concentration of linalool (Marotti et al., 1996). Our results were in
agreement with the results of Tiziana-Barrata et al. (1998) accord-
ing to which the methyl chavicol was the main component in basil
oil. Lemon, orange and eucalyptus essential oils were the most
similar in the chemical composition, with high concentration of
monoterpene alcohol, limonene. Limonene content was as high as
90% in orange and eucalyptus oil and up to 65,9% in lemon oil. Tea
tree oil was characterized by 40,7% of terpinen-4-ol (monoterpene
alcohol) and high level of ␥-terpinene (17,8%). Similarly, viola oil
was dominated with triacetin (triester) with 53,7% and relatively
high level of ␣-ionone as a representative carbonyl compound
(9,4%). Chamomile oil is also known to be variable in composition
and often contains hamazulen as dominant component (Gosztola
et al., 2010). In chamomile oil we used, three sesquiterpene com-
pounds are present in high amounts, ␣-bisabolol oxide A (38%) as
dominant, and also bisabolol (21,1%) and farnesene (18,5%).
In the investigation of antifungal potential of essential oils, we
used fungi isolated and identified from plant drugs: mint, net-
tle, marigold, horsetail and corn silk, which in previous studies
showed the highest level of contamination with phytopathogenic
and saprophytic fungi (Stević et al., 2012). Results of antifun-
gal activity of the essential oils are shown in Table 3. Among all
oils tested, savory, oregano and thyme oils proved to be the best
inhibitor of all pathogens tested. For the same three oils has been
found to control Botrytis cinerea and Monilinia laxa growth on stone
fruit (Lopez-Reyes et al., 2013). These oils in our investigation uni-
formly inhibited the growth of most fungi by concentrations of
0,14 mg/mL. Inhibitory concentration of oregano oil for F. oxyspo-
rum and Phomopsis sp. as well as savory oil for A. niger was as low
as 0,07 mg/mL. The highest concentrations of these three potent
oils were required for inhibition of F. subglutinans (about 1 mg/mL
of savory and oregano oils and 2,07 mg/mL of thyme oil). The effi-
cacy of these oils can be attributed to activity of phenol compounds,
carvacrol and thymol. Results from different studies (Griffin et al.,
1999; Kalemba and Kunicka, 2003; Carmo et al., 2008; Gallucci et al.,
2014) mark phenol compounds as agents with the highest anti-
fungal potential among all terpene components of essential oils.
According to Dikbas et al. (2008) inhibition of phytopathogenic
fungi by carvacrol and thymol were similar or superior to complete
oil. Also, it was found that the synergistic activity between thy-
mol and carvacrol and their mutual interaction plays an important
role in the overall activity of the essential oils. Moreover, it can be
assumed that the activity of these oils was significantly affected by
the presence of biological precursors of phenol compounds, namely
␥-terpinene and p-cymene. When consider chemical composition
of these three oils through sum of phenol compounds and precursor
of the phenol compounds, very similar content was observed (73%
for thyme, 78% for savory and 84% for oregano oils). These findings
could explain very similar and strong antifungal activity of savory,
oregano and thyme oils. Rose oil with slightly lower inhibitory con-
centration compared to savory, oregano and thyme oils, however
below 1 mg/mL for all fungi tested, assign it for very potent antifun-
gal agent. High antifungal potential of rose oil can be explained by
the dominance of monoterpene alcohols, citronellol and geraniol,
highly active due to its good solubility in water and the presence
of functional alcohol group. Pelargonium graveolenses essential oil
with high presence of citronellol (35%) and geraniol (28,8%) was
found to be highly active against Rhizoctonia solani (Bouzenna and
Krichen, 2013). It is considered that monoterpene alcohols increase
the permeability of the plasma membrane and inhibit process of
respiration on mitochondrial membrane of fungi (Cox et al., 2000;
Deba et al., 2008; Imelouane et al., 2009). Lower effect of geranium
Table 2
Quantitative composition of seven most abundant compounds identified in 16 essential oils by GC-FID and GC-MS.

Essential oils Seven most abundant compounds (GC-FID)> 1%

Compound 1 Compound 2 Compund 3 Compound 4 Compound 5 Compound 6 Compound 7

Carvacrol-rich
Savory Carvacrol (50,0%) p-Cymene (12,2%) Thymol (9,6%) ␥-Terpinene (6,5%) E-caryophyllene (5,2%) Borneol (2,41%) Linalool (1,6%)
␥-Terpinene (2,61%)

T. Stević et al. / Industrial Crops and Products 55 (2014) 116–122

Oregano Carvacrol (75,8%) Thymol (3,7%) E-Caryophyllene (2,76%) Linalool (2,54%) p-Cymene (2,11%) Borneol (1,2%)

Timol-rich
Thyme Thymol (43,7%) p-Cymene (23,2%) ␥-Terpinene (6,9%) Linalool (4,9%) 1,8-Cineole (3,7%) E-Caryophyllene (3,1%) Carvacrol (2,9%)

Citronellol + geraniol-rich
Rose Citronellol (51,3%) Geraniol (25,8%) Phenethyl alcohol (6,0%) Citronellyl formate (1,3%) 6,9-Guaiadiene (1,4%) Linalool (1,2%) Eugenol (1,8%)
Geranium Citronellol (39,0%) Geraniol (11,3%) Phenethyl alcohol (7,5%) Citronellyl formate (5,1%) 6,9-Guaiadiene (4,1%) Linalool (1,7%) Geranyl butanoate (4,8%)

Linalool + Lin. acetate-rich

Lavander Linalool (35%) Linalool acetate (37%) ␣-Pinene (3,2%) E-Caryophyllene (3,2%) Borneol (2,6%) Neryl acetate (2,4%) Terpinen-4-ol (2,1%)
Coriander Linalool (71,2%) ␣-Pinene (7,8%) ␥-Terpinene (4,2%) Camphor (4,3%) Geranyl acetate (3,2%) Limonene (2,8%) Geraniol (1,1%)
Bergamot Limonene (37%) Linalool acetate (37%) Linalool (13%) ␥-Terpinene (3,0%) ␤-Pinene (3,0%) - -

Limonen-rich
Lemon Limonene (65,9%) ␤-Pinene (13,9%) ␥-Terpinene (6,2%) Geranial (2,3%) Sabinene (2,2%) ␣-Pinene (2,1%) p-Cymene (1,4%)
Orange Limonene (90,1%) Linalool acetate (1,6%) Myrcene (1,4%) ␥-Terpinene (1,0%) - - -
Eucalyptus Limonene (89,9%) ␥-Terpinene (2,5%) p-Cymene (2,2%) ␣-Pinene (2,1%) - - -

Anethole-rich
Anise E-Anethole (91,8%) Methyl chavicol (3,6%) Foeniculin (1,20%) Linalool (1,0%) - - -

Terpinen-4-ol-rich
Tea tree Terpinen-4-ol (40,7%) ␥-Terpinene (17,8%) ␣-Terpinene (7,1%) ␣-Terpineol (4,5%) Terpinolene (4,2%) Limonene (3,9%) ␣-Pinene (3,5%)

Triacetin-rich
Violet Triacetin (53,7%) ␣-Ionone (9,4%) Phenyl ethyl propionate (6,8%) ␤-Ionone (4,8%) Linalool (3,9%) Linalool acetate (3,8%) Citronellol (1,5%)

Methyl chavicol-rich
Basil Methyl chavicol (89%) 1,8-Cineole (3,0%) Linalool (1,3%) ␥-Terpinene (1,1%) - - -

Bisabolol + bisabolol oxide-rich

Chamomile ␣-Bisabolol oxide A (38%) Bisabolol (21,1%) Farnesene (18,5%) Bisabolol oxide II (5,1%) Z-Spiroether (4,7%) Hamazulene (2,2%) Germacrene D (1,2%)

119
120 T. Stević et al. / Industrial Crops and Products 55 (2014) 116–122

Mean value of MIC (n = 3) for each oil was shown, * Values of MIC, followed by the same letter are not significantly different (p < 0,05), according to Duncan‘s multiple range test, The values in bold are the lowest concentrations
oil compared to rose oil, with its qualitative composition similar to
Chamomila
rose oil, could be attributed to quantitative differences in represen-

16,00 gh
23,40 cd

25,20 bc
28,70 a*

14,80 hi
14,80 hi
14,80 hi
17,30 fg
12,00 jk
11,50 kl

10,60 kl

11,00 kl

13,40 ij

13,40 ij

13,40 ij
23,50 d

26,20 b
21,25 e

21,25 e
18,70 f

10,30 l
tation of citronellol and geraniol (Table 2). Tea tree oil and anise oil
exhibited also excellent activities against most of the tested fungi.
Tea tree oil, with fairly uniform concentrations inhibited the growth
6,80 abc of most fungi; MIC values were about 1 mg/mL. The most resistant

6,80 abc

6,80 abc

6,80 abc
5,95 cd

5,95 cd

5,95 cd

5,95 cd
5,95 cd
5,95 cd
bc

5,10 d

5,10 d

5,10 d
7,65 a
7,65 a
7,65 a

7,65 a

7,65 a

7,65 a
Basil

to this oil were F. subglutinans, F. equiseti, T. viride and A. flavus. It

6,12

6,12
is considered that high antifungal potential of tea tree essential oil
is consequence of the high content of terpinen-4-ol, monoterpene
bcd

3,12 bcd

3,12 bcd

2,91 cde
2,08 fgh

2,08 fgh

2,08 fgh
2,60 def

2,50 def

2,28 efg
1,66 gh
3,53 bc

4,88 a*

alcohol, which in previous studies showed good antifungal activity

1,56 h

1,56 h
1,56 h

1,45 h
3,64 b

3,64 b
3,64 b
4,60 a
Viola

3,12

against Aspergillus and Penicillium (Hammer et al., 2003). Anise oil

inhibited the growth of most fungi with concentrations between
0,7 mg/mL and 2,2 mg/mL, which could be considered as high anti-
Tea tree

1,05 de

1,05 de
1,14 cd

1,14 cd

1,14 cd
a*

0,91 ef

0,96 ef

0,91 ef

0,91 ef

0,91 ef
1,40 b

1,40 b

1,40 b
1,40 b
1,00 e
1,82 a
1,82 a

1,82 a
1,18 c

0,86 f

fungal activity. Investigation of Huang et al. (2010) suggested that

1,82

the high activity of anis oil was liable of trans-anethole which is

equally active in inhibiting the growth of bacteria and fungi. Slightly
1,83 abc

1,83 abc
1,92 abc
1,22 de

1,22 de
1,20 de

1,20 de
less potency in inhibiting the growth of most fungi exhibited violet
ab

1,57 cd

1,60 cd
1,70 bc
1,60 bc
2,18 a*
0,93 ef

0,96 ef
1,00 ef

1,00 ef

1,00 ef
2,18 a

2,18 a
Anise

0,70 f
2,00

oil. F. semitectum and F. solani were the most resistant to this oil,
while T. roseum and Phomopsis sp. were the most susceptible. MIC
values for all the rest fungi tested was between 2 and 3 mg/mL.
Good antifungal potential of violet oil mainly against all tested
Eucalip.

10,10 bc
10,50 bc

10,50 bc
11,60 a*
10,70 b

10,70 b

7,40 gh
8,90 de

8,90 de
9,70 cd

7,90 fg
ef

8,70 ef

6,80 h

6,60 h

5,20 ij

5,40 ij
7,76 g

pathogenic fungi can probably be attributed to the high proportion

4,85 j

4,85 j

4,85 j
8,70

of triacetin, but also to under-represented components: ␣-ionone,

phenyl ethyl propionate and ␤-ionone for which there are data of
14,00 cd
14,60 bc
14,60 bc

the antifungal activity (Lalko et al., 2007). Similar uniformity in

16,40 a*
Orange

15,20 b

15,20 b
12,15 e
12,10 e

12,10 e

12,20 e
12,10 e
12,00 e
a

16,20 a

14,20 c

10,40 f
8,70 g

8,10 g
16,20

9,70 f
9,70 f

9,70 f

MICs values for essential oils of coriander, lavender and bergamot

for most fungi tested was detected (Table 3). Due to the dom-
ination of linalool, monoterpene alcohol with moderate to good
10,40 de

11,13 cd
15,60 a*

6,09 ghi
13,40 b
12,70 b

13,40 b
b

13,00 b

13,00 b
11,50 c
Lemon

6,61 gh

6,26 gh

antifungal activity, coriander oil exhibit relatively high efficiency.

5,74 hi
6,94 fg

6,90 fg
6,90 fg
13,00
7,65 f

8,00 f

8,00 f

5,20 i

Limonene and linalool acetate were predominant components in

lavender and bergamot oils. Despite the fact that limonene and
linalool acetate are component with low antifungal activity, good
Bergam.

1,91 ghi

1,91 ghi
2,10 ghi
2,00 ghi
2,18 gh

4,60 ab

4,50 ab
4,35 bc

4,80 a*
1,83 hi

2,80 ef
3,74 d

3,65 d
d

2,26 g

3,04 e

4,20 c
1,17 j

1,74 i

1,09 l
1,04 l

antifungal potential of bergamot and lavender oils was probably

3,50

a result of the contributory activities of other compounds present

in oils (Lis-Balchin et al., 1998; Hammer et al., 2003). Low activ-
Antifungal activity of the essential oils is expressed through the minimal inhibitory concentration (mg/mL).

Corian.

2,98 gh

3,65 de

ity in inhibition of mycelia growth of all tested fungi was recorded

5,10 a*

3,23 fg
ef

2,72 h
2,13 ij

2,13 ij

2,21 ij
3,74 d
4,76 b

1,76 k
4,25 c
4,08 c

1,23 l

2,38 i

2,38 i
0,97 l

1,10 l

1,10 l

1,06 l
3,40

for basil oil. MIC values were between 5 and 7 mg/mL. Interest-
ingly, F. subglutinans, the utmost resistant fungus to most tested
oils, was among the most sensitive to this oil. Weaker antifun-
gal potential of the basil oil can be attributed to the dominance
3,04 cdef

3,12 cde

3,12 cde
2,57 fgh
2,85 def

2,76 efg

2,30 gh
3,31 cd
Laven.
a*

2,11 hi
1,38 jk
1,33 jk
1,77 ij

4,78 b
4,60 b

4,60 b
1,24 k

1,19 k
1,15 k
1,19 k

of methyl chavicol that in earlier trials showed variable activity

3,40 c
6,80

depending on the test organism. Tadtong et al. (2009) established a

moderate to weak antimicrobial activity of methyl chavicol. Weak
1,91 bcd

2,20 bcd

antifungal activity of essential oils of lemon, orange and eucalyp-

2,09 cde

2,11 def
def

1,37 gh
Geran.

2,28 bc

2,28 bc

2,28 bc
4,40 a*

1,18 hi

1,91 ef
1,90 ef

2,37 b
2,37 b
1,55 g
1,82 f

1,82 f
0,66 j

0,62 j
1,00 j
2,00

tus can be explained by the dominance of limonene which was,

together with monoterpene acetates, the weakest inhibitor of fun-
gal growth among the pure monoterpene compounds. Our results
0,64 a*
0,29 b

0,29 b

0,29 b

0,29 b
0,29 b
0,31 b
0,31 b
0,35 b
0,31 b
0,31 b

0,31 b
0,30 b
a

0,14 c

0,14 c

0,14 c

0,18 c
0,19 c
0,17 c
0,14 c
Rose

are consistent with previous studies where all three oils showed
0,62

required to affect 100% of inhibition for a particular pathogen.

low antimicrobial potential (Combrinck et al., 2011). The highest

MIC values were recorded for chamomile oil in which sesquiter-
Thyme

0,16 de
0,19 de

0,19 de

0,28 de
0,34 de

0,34 de
0,17 de

0,34 de

0,21 de

0,17 de
0,41 cd
a*

1,18 b
0,98 b
0,14 e

0,14 e

0,14 e

0,14 e

0,14 e

penes are dominant compounds. For A. alternata it was needed as

0,61 c

0,14 c
2,07

much as 30 mg/mL for total inhibition of growth. Soković and van

Griensven (2006) showed that the essential oil of chamomile had
Oregano

weak antifungal potential which was in agreement with our results.

a*

0,07 d

0,07 d
0,28 b

0,28 b
0,28 b

0,28 b
0,28 b

0,28 b

0,28 b
0,14 c
0,14 c

0,14 c

0,14 c
0,14 c

0,14 c
0,14 c

0,14 c

0,14 c

0,14 c
0,14 c

Parallel with obtaining of MIC, MFC were determined for all oils
1,16

against all fungi tested (data not shown). With just a few excep-
tions, MFC values were exactly the same or very near the value of
Savory
a*

0,14 d
0,14 d

0,14 d
0,14 d
0,14 d

0,14 d
0,14 d
0,14 d
0,14 d
0,14 d
0,14 d
0,14 d
0,14 d
0,14 d
0,14 d
0,14 d
0,62 b

MIC, suggesting fungicidal activity of the tested oils.

0,07 e
0,27 c

0,27 c
0,95

In this paper, we also examined the antifungal activity of three

mixtures of two essential oils: rose and lavender, oregano and
F. sporotrichioides

lavender, and thyme and oregano. Inhibitory activity of a combina-

Chaetomium sp.
F. verticillioides
F. subglutinans

Penicillium sp.

tion of these oils was tested on the growth of the following selected
Phomopsis sp.
F. semitectum

M. verrucaria
F. oxysporum
F. tricinctum

A. alternata

fungi: A. flavus, Fusarium equiseti, F. subglutinans, F. sporotrichioides,

Phoma sp.
G. roseum
F. equiseti

V. dahliae

T. roseum
C. lunata
A. flavus
F. solani

T. viride
A. niger

F. solani and F. semitectum, Penicillium sp. and A. alternata. Results

Table 3

are shown in Table 4. The combination of the essential oils of thyme

and oregano exhibited excelling antifungal activity than when
a
 T. Stević et al. / Industrial Crops and Products 55 (2014) 116–122 121

Fig. 1. Reduction of total number of fungi in samples of herbal drugs treated with essential oils in situ. Change in log CFU/g of fungi with standard errors in herbal drug samples
treated with different essential oils was shown. Volume (and actual concentration) of the pure oil administered to filter disc was as follows: 30 ␮L for: thyme (0,81 mg/mL),
oregano (0,84 mg/mL), rose (0,87 mg/mL) and savory oils (0,81 mg/mL): 40 ␮L for tea tree (1,08 mg/mL) and anis (1,04 mg/mL) oil; 50 ␮L for coriander (1,25 mg/mL) and
lavender (1,35 mg/mL) oils; and 80 ␮L for geranium oil (2,16 mg/mL). Each point represents mean of three independent experiments.

Table 4
Antifungal activity of mixture of essential oils.

Combination of oils A. flavus A. alternata F. subglutinans F. equiseti F. sporotrichioides F. solani F. semitectum Penicillium sp.

FIC of rose oil 0,0625 – 0,0625 0,0625 0,0625 – – 0,0625

FIC of lavender oil 1,0000 – 0,2500 0,0625 0,0063 – – 2,0000
FICi 1,0625 – 0,3125 0,125 0,0685 – – 2,0625
Interaction I – S S S – – A

FIC of thyme oil 0,0625 – 2,0000 – – 0,0625 0,1250 0,0625

FIC of oregano oil 0,0625 – 4,0000 – – 0,0625 0,2500 0,0625
FICi 0,1250 – 6,0000 – – 0,1250 0,3750 0,1250
Interaction S – A – – S S S

FIC of oregano oil 1,0000 0,0625 0,0625 – – 1,0000 0,0625 –

FIC of lavender oil 0,0625 2,0000 0,0625 – – 0,0625 0,2500 –
FICi 1,0625 2,0625 0,1250 – – 1,0625 0,3125 –
Interaction I A S – – I S –

FIC, fractional inhibitory concentration; FICi, fractional inhibitory concentration index; S, synergistic effect; I, indifferent effect; A, antagonistic effect.

tested individually against all fungi tested, except for F. subgluti-
nans. Synergistic activity was observed for the combination of rose
and lavender essential oils, against Fusarium species that showed
the greatest resistance to both oils when tested individually while
against A. flavus effect was indifferent and for Penicillium sp. even
antagonistic. The combination of oregano and lavender essential
oils was found to have synergistic effect in respect to F. subglutinans
and F. semitectum while indifferent effect was observed for A. flavus
and F. solani. Antagonistic effect of these two oils was recorded for
A. alternata. Generally, reduction of the MIC values for the most
oils (FIC in the Table 4) when combined in comparison to the MIC
values when tested individually (Table 3) was recorded. Increased
fungistatic potential of mixture of essential oils may be due to the
combined activities of two or more components of the essential oils
(Goñi et al., 2009).
As a pilot investigation, we tried to determine effectiveness
of selected essential oils, which showed good antifungal activity
in vitro, in the reduction of the total number of fungi, in situ. A
modified method of evaporation of the essential oils from the fil-
ter paper in a sealed bag with the investigated herbal drug has
been used. Among all essential oils tested for antifungal activity
in situ, the highest efficacy in reducing the total number of fungi in
samples of herbal drugs exhibited essential oils of thyme, oregano
and rose. Slightly lower inhibitory activity was detected for savory,
coriander, lavender, anise and tee tree oils (Fig. 1). Geranium oil
showed the weakest inhibitory activity. When tested in situ, thyme,
oregano and rose oils (concentrations about 0,8 mg/mL (30 ␮L of
pure oil) per filter disc) reduced the total number of fungi over 50%
(Fig. 1). These results are in agreement with results of the activities
of individual oils obtained in MIC test, except for savory oil.
4. Conclusions
According to obtained results, essential oils of summer savory,
oregano, thyme and rose represent a good basis for the formula-
tion of products with potential efficacy in the control of fungi. The
results indicate that all tested fungi were relatively uniformly sus-
ceptible to all four oils. Also, combination of particular oils showed
reduction of the MIC values when combined, commendatory mix-
tures for potential application in practice. This synergism would
have the advantage in pre- and post-harvest protection because
pathogens cannot easily acquire resistance to multiple components
122 T. Stević et al. / Industrial Crops and Products 55 (2014) 116–122
of the two essential oils. Additionally, using smaller amount of oils,
when projecting on the application in large scales, could have con-
siderable economic effects.
Acknowledgements
This work was supported by the Ministry of Education, Science
and Technological Development, Republic of Serbia (Grants No.
46013 and 173026).
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