Bioresource Technology 99 (2008) 8788–8795

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Antifungal, phytotoxic and insecticidal properties of essential oil isolated from

Turkish Origanum acutidens and its three components, carvacrol, thymol
and p-cymene
Saban Kordali a, Ahmet Cakir b,*, Hakan Ozer c, Ramazan Cakmakci c, Memis Kesdek a, Ebru Mete d
a
Atatürk University, Faculty of Agriculture, Department of Plant Protection, 25240 Erzurum, Turkey
b
Atatürk University, Kazım Karabekir Education Faculty, Department of Chemistry, 25240 Erzurum, Turkey
c
Department of Field Crops, Faculty of Agriculture, Ataturk University, 25240 Erzurum, Turkey
d
Atatürk University, Faculty of Arts and Sciences, Department of Chemistry, 25240 Erzurum, Turkey

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

Article history: The chemical composition of essential oil isolated by hydrodistillation from the aerial parts of Origanum
Received 17 December 2007 acutidens was analyzed by GC–MS. Carvacrol (87.0%), p-cymene (2.0%), linalool acetate (1.7%), borneol
Received in revised form 14 April 2008 (1.6%) and b-caryophyllene (1.3%) were found to be as main constituents. Antifungal, phytotoxic and
Accepted 15 April 2008
insecticidal activities of the oil and its aromatic monoterpene constituents, carvacrol, p-cymene and thy-
Available online 29 May 2008
mol were also determined. The antifungal assays showed that O. acutidens oil, carvacrol and thymol com-
pletely inhibited mycelial growth of 17 phytopathogenic fungi and their antifungal effects were higher
Keywords:
than commercial fungicide, benomyl. However, p-cymene possessed lower antifungal activity. The oil,
Origanum acutidens
Carvacrol
carvacrol and thymol completely inhibited the seed germination and seedling growth of Amaranthus ret-
Thymol roflexus, Chenopodium album and Rumex crispus and also showed a potent phytotoxic effect against these
Antifungal activity plants. However, p-cymene did not show any phytotoxic effect. Furthermore, O. acutidens oil showed
Phytotoxicity 68.3% and 36.7% mortality against Sitophilus granarius and Tribolium confusum adults, respectively. The
findings of the present study suggest that antifungal and herbicidal properties of the oil can be attributed
to its major component, carvacrol, and these agents have a potential to be used as fungicide, herbicide as
well as insecticide.
Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction result in soil and groundwater contamination, and development

In recent years, scientists have focused on the increase of food
production needed for the fast expansion of world population.
Unfortunately, substantial yield losses occur due to insects and
plant diseases caused by fungi, bacteria and viruses (Fletcher
et al., 2006). Fungi and bacteria have also unfavorable effects on
quality, safety and preservation of food. Synthetic chemicals are
widely used in the control of plant diseases. However, these chem-
icals may cause toxic residues in treated products (Barnard et al.,
1997; Isman, 2000). Synthetic pesticides can also cause environ-
mental pollution owing to their slow biodegradation (Barnard
et al., 1997; Misra and Pavlostathis, 1997). In addition, the risk of
developing the resistance by microorganisms and the high cost–
benefit ratio are other disadvantages of synthetic pesticide usage
(Brent and Hollomon, 1998).
Weeds are another major problem in world agriculture because
they cause losses in crop yield. Therefore, farmers have increased
herbicide use. However, intensive use of synthetic herbicides can
* Corresponding author. Tel.: +90 442 2314012; fax: +90 442 2360955.
E-mail address: cakira@atauni.edu.tr (A. Cakir).
of weed resistance (Duke et al., 2000). Herbicides at high concen-
trations can also increase the risk of toxic residues in agricultural
products. Therefore, researchers have focused on new potential
bio-herbicides, having different and selective herbicidal mecha-
nisms in comparison to their synthetic herbicides (Dudai et al.,
1999; Duke et al., 2000; Kordali et al., 2007b).
Insect pests often cause extensive loss of the stored food grains
and their products in tropical and semitropical environments
(Isman, 2000). Therefore, there is a need for environmentally safe
insecticides or repellents for use in food grain. Plant-derived sec-
ondary metabolites play an important role in plant resistance to in-
sects. Therefore, screening plant essential oils and plant extracts
for insecticidal properties could lead to discovery of new agents
for pest control (Isman, 2000; Yildirim et al., 2005; Kordali et al.,
2006; Caglar et al., 2007).
The genus Origanum (oregano) is an important genus of the
Lamiaceae family and comprises about 900 species, widespread
throughout the world. There are about 20 species of Origanum
genus in Turkish flora (Davis, 1982; Baytop, 1999). Origanum
species have traditionally been used as a spicy additive for food in-
stead of thyme. This genus is rich in essential oils and bitter

0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2008.04.048
 S. Kordali et al. / Bioresource Technology 99 (2008) 8788–8795
substances (Baytop, 1999; Esen et al., 2007). The species of Origa-
num genus are known in Anatolia as ‘‘Yalancı kekik”, ‘‘Kekik”,
_
‘‘Istanbul kekiği” and ‘‘Keklik otu”. Origanum species are tradition-
ally used as sedative, diuretic, degasifier, sweater and antiseptic,
and also in the treatment of gastrointestinal diseases and constipa-
tion (Baytop, 1999). There are numerous reports on the chemical
composition and their various biological activities of Origanum
species (Daouk et al., 1995; Muller-Riebau et al., 1995; Sokovic
et al., 2002; Bouchra et al., 2003; Yildirim et al., 2005; Kizil and
Uyar, 2006; Caglar et al., 2007; Esen et al., 2007; Lee et al., 2007;
Soylu et al., 2007). Origanum acutidens is an endemic species grow-
ing in northeastern Turkey. The essential oil composition of this
species has been previously reported (Baser et al., 1997; Figueredo
et al., 2006). However, there is only one study on its biological
properties, which reported the antimicrobial and antioxidant re-
sults of the essential oil and extract (Sokmen et al., 2004).
This study was carried out to determine antifungal, herbicidal,
phytotoxic and insecticidal properties of the essential oil isolated
from the aerial parts of O. acutidens and its aromatic monoterpene
constituents, carvacrol, p-cymene and thymol.
2. Methods
2.1. Plant material and isolation of essential oil
Origanum acutidens (Hand.-Mazz) letswaart was collected at the
flowering stage in August 2006 in Ispir, Erzurum, Turkey. The taxo-
nomic identification of plant materials was confirmed by a senior
plant taxonomist Dr. Ali Kandemir, in Department of Biology, Erzin-
can University, Erzincan, Turkey. The voucher specimen has been
deposited at the Herbarium of the Department of Science, Erzincan
University, Education Faculty, Erzincan, Turkey. Collected plant
materials were dried in shadow and ground in a grinder. The dried
plant samples (500 g) were subjected to hydrodistillation (plant
material in boiling water) using a Clevenger-type apparatus for 4
hours. Hydrodistillation of O. acutidens yielded 0.6% (w/w) of essen-
tial oil. The yields were based on dry materials of plant samples.
2.2. GC–MS analysis
The analysis of the essential oil was performed with a Thermo-
finnigan Trace GC/Trace DSQ/A1300, (E.I Quadrapole) equipped
with a SGE-BPX5 MS fused silica capillary column
(30 m  0.25 mm i.d., film thickness 0.25 lm). For GC–MS detec-
tion, an electron ionization system with ionization energy of
70 eV was used. Carrier gas was helium at a flow rate of 1 ml/
min. Injector and MS transfer line temperatures were set at
220 °C and 290 °C, respectively. The oven temperature was pro-
grammed from 50 °C to 150 °C at 3 °C/min, then held isothermal
for 10 min and finally raised to 250 °C at 10 °C/min. Diluted sam-
ples (1/100, v/v, in methylene chloride) of 1.0 ll were injected
manually in the splitless mode. The relative percentage of the oil
constituents was expressed as percentages by FID peak area
normalization.
The identification of individual compounds was based on com-
parison of their relative retention times with those of authentic
samples on SGE-BPX5 capillary column, and by matching of their
mass spectra of peaks with those obtained from authentic samples
and/or the Wiley 7N and TRLIB libraries spectra and published data
(Adams, 2007).
2.3. Antifungal activity assays
Seventeen agricultural pathogenic fungi were obtained from the
culture collection at Atatürk University (Faculty of Agriculture,
8789
Department of Plant Protection, Erzurum). Cultures of each of the
fungi were maintained on potato dextrose agar (PDA) and were
stored at +4 °C. The fungal species used in the experiments were
shown in Table 2.
Antifungal activity was studied by using a contact assay
(in vitro), which produces hyphal growth inhibition (Kordali
et al., 2007a). Briefly, potato dextrose agar (PDA) plates were
prepared using 9 cm diameter glass Petri dishes. The essential oil,
carvacrol (Fluka; 96% purity), thymol (Fluka; >99% purity) and p-
cymene (Aldrich; 99% purity) were dissolved in dimethyl sulfoxide
(DMSO) (Merck)-water solution (10% v/v) and required amounts of
the solutions (25 and 10 mg/Petri dish (1500 and 500 mg/l concen-
tration)) were added to each of the PDA plates containing 20 ml of
agar at 50 °C. A disc (5 mm diameter) of the fungal species was cut
from one week-old cultures on PDA plates and then the mycelia
surface of the disc was placed upside down on the center of a dish
with fungal species in contact with growth medium on the dish.
Then, the plates were incubated in the dark at 22 ± 2 °C. The exten-
sion diameter (mm) of hyphae from centers to the sides of dishes
was measured at 24 h intervals for 6 days. Mean of growth mea-
surements were calculated from four replicates of each of the fun-
gal species. PDA plates containing DMSO–water solution (10% v/v),
without essential oil and the compound solutions were used as
negative control. In addition, PDA plates treated with benomyl
(12.0 mg/Petri dish or 600 mg/l concentration) were used as posi-
tive control.
The percentage of growth inhibition by treatment was calcu-
lated using the following equation:
CT
% Inhibition ¼  100
C
Where, C is the mean of four replicates of hyphal extension (mm) of
controls and T is the mean of four replicates of hyphal extension
(mm) of plates treated with essential oil and the compound
solutions.
2.4. Seed germination and seedling growth experiments
The seeds of Amaranthus retroflexus, Chenopodium album and
Rumex crispus were collected in Erzurum region (Turkey) in Octo-
ber 2007. Empty and undeveloped seeds were discarded by float-
ing in tap water. To avoid possible inhibition caused by toxins
from fungi or bacteria, the seeds were surface-sterilized with 15%
sodium hypochlorite for 20 min (Kordali et al., 2007b) and then
rinsed with abundant distilled water. Two layers of filter paper
were placed on the bottom of each Petri dish (9 cm diameter)
and then 50 seeds of A. retroflexus, C. album and R. crispus were
placed on the filter paper (Kordali et al., 2007b). Then, 10 ml of dis-
tilled water was added to each Petri dish. Lids of Petri dishes were
closed with Whatman No. 1 filter paper wrapping up tightly with a
transparent ribbon.
To determine the herbicidal effects of vapors of the oil and its
constituents, the assays were applied at 10 ll/Petri dish contents
of p-cymene, carvacrol and the oil (corresponding to 8.6, 9.8 and
10.0 mg/Petri dish, respectively), and 10 mg/Petri dish content of
thymol. Seeds and filter papers were moistened with 10 ml of dis-
tilled water. Ten microliters of the essential oil, p-cymene and car-
vacrol were dripped on the paper placed on the lid using a micro
pipette. Thymol was dissolved in DMSO (1:1; w/v) and appropriate
amount of the solution (10 lg/Petri dishes) were impregnated to
Whatman No. 1 filter paper placed on the lid of Petri dish. Petri
dishes were closed with an adhesive tape to prevent escaping of
volatile compounds and were kept at 23 ± 2 °C on a growth cham-
ber supply with 12 h of fluorescent light and humidity of 80% (Du-
dai et al., 1999). After 10 days, the number of germinated seeds and
seedling lengths were measured. Germination was measured as
8790 S. Kordali et al. / Bioresource Technology 99 (2008) 8788–8795
the percentage of seeds from which a radicle emerges. The treat-
ments were arranged in a completely randomized design with
three replications including controls.
To determine the contact herbicidal effect of the oil and its con-
stituents, the oil and the compounds were dissolved in DMSO–
water solution (1%; v/v). The final concentration of the treatments
was 1.0 mg/ml. The emulsions (10 ml) were transferred to Petri
dish (9 cm diameter) placed on the bottom two layers of filter pa-
per. Afterward, 50 seeds of A. retroflexus, C. album and R. crispus
were placed on the filter paper (Kordali et al., 2007b). Petri dishes
were closed with an adhesive tape to prevent escaping of volatile
compounds and were kept at 23 ± 2 °C on a growth chamber sup-
ply with 12 h of fluorescent light and humidity of 80% (Dudai et al.,
1999). After 10 days, the number of germinated seeds was deter-
mined and seedling lengths measured. Germination was measured
as the percentage of seeds from which a radicle emerges. In addi-
tion, 2,4-D, isooctyl ester (10 ll/Petri dishes) was used as positive
control. The treatments were arranged in a completely randomized
design with three replications including controls.
2.5. Phytotoxicity assays
Phytotoxic effects of the oil and its constituents (carvacrol, thy-
mol and p-cymene) on greenhouse-grown herbs were determined
by means of a bioassay. To avoid possible contaminants caused by
fungi or bacteria, weeds of the plant samples were surface-steril-
ized with 15% sodium hypochlorite for 20 min and then rinsed
with abundant distilled water (Kordali et al., 2007b). Pots (10 x
10 cm) were filled with 550 g soil (organic material ratio: 2.02%;
cation change capacity: 43.34 me/100 g; pH 7.5) sterilized in auto-
clave. Then, 50 seeds of the plant samples were sown into the pots
and kept under photoperiod conditions (23 ± 2 °C, 80 ± 5 relative
humidity and 12 h light and 12 h dark photoperiod) in a growth
chamber to allow germination and growth of the plant samples.
The pots were irrigated with tap water when necessary. The num-
ber of germinated seeds of the respective plant sample in each pot
was counted. Afterwards, the oil, carvacrol, thymol and p-cymene
were dissolved in 10 ml of DMSO–water solution (1% v/v). The final
concentration of the treatments was 1.0 mg/ml. These emulsions
(10 ml for each pot) were sprayed uniformly with a glass atomizer
on the surface of whole plants in each pot in the stage of 3–4 real
leaves. The plants in each pot, sprayed uniformly with 10 ml of
DMSO–water solution (1%), were used as negative control groups.
In addition, the plants sprayed with 2,4-D, isooctyl ester (10 mg for
each pot) were used as positive control. Killed plants was counted
and recorded at 24th and 48th hour after sample applications. The
treatments were arranged in a completely randomized design with
three replications including controls. The phytotoxicity of the
treatments was expressed as percent mean of killed plants.
2.6. Insects and bioassays
Sitophilus granarius adults were collected from an Eastern Ana-
tolia storage house (Pasinler town), and Tribolium confusum adults
were collected from Ankara storage house and also from Plant Pro-
tection Central Research Institute in Ankara. The pest adults were
maintained in the Plant Protection Department, Faculty of Agricul-
ture, Atatürk University and feed on wheat (Triticum vulgare) at
25 ± 1 °C, 64 ± 5% relative humidity and 12 h:12 h (L:D). The insect
adults were also controlled once a week. In order to test the toxic-
ity of the oil against to pests adults (4–6 days old), 20 adults of the
insects with 20 grains of wheat were placed in Petri dishes (9 cm).
Thirty microliters of the oil were applied with an automatic pipette
on Whatman No. 1 filter papers (2 cm  2 cm) attached to the up-
side of the Petri dish, corresponding to 375 ll/l air concentrations.
After exposure, mortality of the adults was determined at 12, 24,
48 and 96 h. Petri dish applied with only sterile water was used
as control group. Three replicates were used for each dose and
exposure time combination and insecticidal activity of the oil
was expressed as % mean mortality of the adults.
2.7. Statistical analysis
In order to determine whether there is a statistically significant
difference among the obtained results for antifungal, herbicidal
and insecticidal activity assays, variance analyses were carried
out using SPSS 10.0 software package. Differences between means
were tested through Duncan and LSD tests and values with p < 0.05
were considered significantly different.
3. Results and discussion
3.1. Chemical composition of the oil
GC–MS analyses of hydro-distillated essential oil of O. acutidens
allowed the identification of 28 different components in the oil,
representing 99.0% of the compounds in the oil (Table 1). Accord-
ing to the analysis results, carvacrol was the most abundant com-
ponent of the oil (87.0%). Other main components of the oil were
found to be p-cymene (2.0%), linalool acetate (1.7%), borneol
(1.6%) and b-caryophyllene (1.3%). The essential oil of O. acutidens
was previously investigated (Baser et al., 1997; Sokmen et al.,
2004; Figueredo et al., 2006), and it was shown that carvacrol
(66.0–72.0%) and p-cymene (7.5–14.0%) were the major compo-
nents of the oil. As seen in Table 1, our findings are in agreement
with those previously reported. As it has been reported previously,
the essential oils of the genus Origanum are rich in carvacrol, thy-
mol, c-terpinene and p-cymene (Daouk et al., 1995; Muller-Riebau
et al., 1995; Sokovic et al., 2002; Bouchra et al., 2003; Daferera
et al., 2003; Soylu et al., 2006; Esen et al., 2007). However, thymol
has a higher percentage than carvacrol in some Origanum oils
belonging to different genotypes (Daferera et al., 2003; Esen
et al., 2007; Bendahou et al., 2008). For instance, the essential oil
of O. vulgare growing in Greece was found to be rich in thymol
(63.7%), p-cymene (13.0%) and carvacrol (8.6%; Daferera et al.,
2003). However, we determined in the present study that O. acuti-
dens oil contained thymol in trace amounts (Table 1).
3.2. Antifungal activity of the oil and its constituents
The inhibitory effects of the essential oil isolated from O. acuti-
dens on the mycelial growth of 17 phytopathogenic fungal species
are shown in Table 2. The results of the antifungal tests revealed
that the oil was inhibitory against all of the tested fungi, and the
mycelial growth of all tested fungi was completely inhibited at
25 mg/Petri dish dose of the oil (Table 2). Inhibitory effects of the
oil against the pathogenic fungi were also higher than commercial
benomyl (Table 2). Our results was further supported by Sokmen
et al. (2004) findings that the essential oil of O. acutidens showed
remarkable antifungal activity with inhibitory effect against 12 of
the 18 fungi. In general, major components are responsible for
activity of essential oils. Essential oils of Origanum and Thymus spe-
cies contain mainly aromatic monoterpenes, carvacrol, thymol and
p-cymene and their activity are often attributed to these com-
pounds (Daouk et al., 1995; Muller-Riebau et al., 1995; Sokovic
et al., 2002; Bouchra et al., 2003; Daferera et al., 2003; Sokmen
et al., 2004; Soylu et al., 2006; Esen et al., 2007; Bendahou et al.,
2008). Therefore, in the present study, pure commercial carvacrol,
thymol and p-cymene were also tested for antifungal activity
against plant pathogenic fungi. As can be seen in Table 2, carvacrol
and thymol completely inhibited the mycelial growth of all tested
 S. Kordali et al. / Bioresource Technology 99 (2008) 8788–8795 8791

Table 1
Chemical composition of Origanum acutidens essential oil

Number Rt RIa Component % Identification methods

1 12.96 994 Myrcene 0.2 GC, MS, RI
2 14.29 1023 a-Terpinene 0.1 GC, MS, RI
3 14.78 1034 p-Cymene 2.0 GC, MS, RI
4 16.35 1067 c-Terpinene 0.7 GC, MS, RI
5 17.17 1079 cis-Sabinene hydrate 0.3 MS, RI
6 18.66 1117 trans-Sabinene hydrate 0.6 MS, RI
7 22.27 1172 Borneol 1.6 GC, MS, RI
8 23.44 1190 a-Terpineol 0.2 GC, MS, RI
9 23.91 1200 c-Terpineol 0.3 MS, RI
10 26.10 1255 Linalool acetate 1.7 GC, MS, RI
11 26.62 1264 Geranial 0.2 MS, RI
12 26.74 1267 Thymoquinone tr GC, MS, RI
13 27.95 1289 Thymol tr GC, MS, RI
14 28.68 1296 Carvacrol 87.0 GC, MS, RI
15 30.67 1356 Nerol acetate 0.1 GC, MS, RI
16 31.53 1377 Geraniol acetate 0.2 GC, MS, RI
17 33.08 1419 b-Caryophyllene 1.3 GC, MS, RI
18 33.87 1433 b-Gurjunene 0.2 GC, MS, RI
19 34.64 1460 a-Humulene 0.1 GC, MS, RI
20 35.42 1478 c-Muurolene 0.1 MS, RI
21 35.68 1486 Germacrene D 0.2 MS, RI
22 36.05 1494 Viridiflorene 0.2 GC, MS, RI
23 37.01 1512 c-Cadinene 0.1 MS, RI
24 37.17 1517 d-Cadinene 0.3 MS, RI
25 40.00 1574 Spathulenol 0.7 MS, RI
26 40.24 1579 Caryophyllene oxide 0.5 GC, MS, RI
27 47.39 1694 Eudesma-4(15),7-dien-1b-ol 0.1 MS, RI
Grouped components (%)
Aromatic monoterpenes 89.0
Monoterpene hydrocarbons 1.0
Oxygenated monoterpenes 5.2
Sesquiterpene hydrocarbons 2.5
Oxygenated sesquiterpenes 1.3
Total identified 99.0

tr: traces (less than 0.1%).

a
Retention index relative to n-alkanes on SGE-BPX5 capillary column; GC: identification based on retention times of authentic compounds on SGE-BPX5 capillary column;
MS, RI: tentatively identified based on computer matching of the mass spectra of peaks with Wiley 7N and TRLIB libraries and published data (Adams, 2007), and comparison
of retention index of the compounds compared with published data (Adams, 2007).

Table 2
Antifungal activities of essential oil and its components

Fungal species O. acutidens oil Carvacrol Thymol p-Cymene Benomyl Control

(25 mg/Petri dish) (10 mg/Petri dish) (10 mg/Petri dish) (10 mg/Petri dish) (10 mg/Petri dish) (12 mg /Petri dish)
Growtha (mm) Growtha (mm) Growtha (mm) Growtha (mm) Growtha (mm) Growtha (mm) Growtha (mm)
Alternaria alternata 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 35.8 ± 4.0 a 14.7 ± 1.2 b 35.9 ± 3.9 a
Alternaria solani 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 25.2 ± 2.9 a, b 12.9 ± 1.2 b 21.1 ± 2.7 a
Botrytis sp. 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 62.2 ± 6.7 a 5.0 ± 0.0 b 68.7 ± 7.6 a
Fusarium acuminatum 5.0 ± 0.0 c 6.0 ± 0.3 c 5.0 ± 0.0 c 5.0 ± 0.0 c 19.6 ± 3.0 b 5.0 ± 0.0 c 29.2 ± 3.2 a
Fusarium culmorum 5.0 ± 0.0 c 6.1 ± 0.3 c 5.0 ± 0.0 c 5.0 ± 0.0 c 44.6 ± 4.9 a 5.0 ± 0.0 c 26.5 ± 2.7 b
Fusarium equiseti 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 35.5 ± 4.2 a 5.0 ± 0.0 c 16.8 ± 2.1 b
Fusarium nivale 5.0 ± 0.0 c 5.8 ± 0.3 c 5.0 ± 0.0 c 5.0 ± 0.0 c 45.5 ± 4.8 a 5.0 ± 0.0 c 30.9 ± 4.1 b
Fusarium oxysporum 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 35.8 ± 4.4 a 5.0 ± 0.0 c 27.7 ± 3.7 b
Fusarium sambucinum 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 46.1 ± 5.9 a 5.0 ± 0.0 b 46.2 ± 6.1 a
Fusarium semitectum 5.0 ± 0.0 c 5.4 ± 0.1 c 5.3 ± 0.1 c 5.2 ± 0.1 c 29.9 ± 2.7 b 5.0 ± 0.0 c 39.1 ± 5.3 a
Fusarium solani 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 24.4 ± 2.9 a 5.0 ± 0.0 b 23.3 ± 2.7 a
Monilinia sp. 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 29.3 ± 5.0 a 5.0 ± 0.0 b 26.1 ± 3.0 a
Pythium ultimum 5.0 ± 0.0 d 5.0 ± 0.0 d 5.0 ± 0.0 d 5.0 ± 0.0 d 53.2 ± 5.8 b 19.7 ± 3.1 c 76.4 ± 5.8 a
Phytophthora capsici 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 28.8 ± 3.8 a 5.0 ± 0.0 b 27.5 ± 3.1 a
Rhizoctonia solani 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 65.8 ± 6.8 a 5.0 ± 0.0 c 56.9 ± 7.4 b
Sclerotinia minor 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 5.0 ± 0.0 c 61.6 ± 8.0 a 5.0 ± 0.0 c 37.6 ± 8.6 b
Verticillium dahliae 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 5.0 ± 0.0 b 14.6 ± 1.5 a 5.0 ± 0.0 b 13.2 ± 1.4 a
a
Means in the same column by the same letter are not significantly different to the test of Duncan (a = 0.05).

fungi. p-Cymene showed weak antifungal activity against Fusarium
acuminatum and P. ultimum (Table 2), whereas it significantly in-
creased the mycelial growth of F. culmorum, F. equiseti, F. nivale,
F. oxysporum and Sclerotinia minor. The essential oils of O. acutidens
contained carvacrol (87.0%), p-cymene (2.0%), linalool acetate
(1.7%) and borneol (1.6%) as major components, and thymol was
detected in trace amount (Table 1). These results show that potent
antifungal activity of O. acutidens oil can be attributed to its major
component, carvacrol. Our findings were in agreement with previ-
ous reports about antifungal activity of carvacrol, thymol and
essential oil of Origanum species, which are rich in carvacrol and
thymol and exhibited the highest antifungal activity against vari-
8792 S. Kordali et al. / Bioresource Technology 99 (2008) 8788–8795

ous phytopathogenic fungi (Daouk et al., 1995; Muller-Riebau
et al., 1995; Tsao and Zhou, 2000; Sokovic et al., 2002; Bouchra
et al., 2003; Daferera et al., 2003; Sokmen et al., 2004; Kizil and
Uyar, 2006; Soylu et al., 2006; Esen et al., 2007; Lee et al., 2007;
Bendahou et al., 2008). For instance, as similar to our results,
Sokmen et al. (2004) reported that the essential oil of Turkish O.
acutidens, which contains carvacrol (72.0%), p-cymene (7.5%) and
c-terpinene (5.3%) as major components have fungitoxic effects
against F. oxysporum, F. solani, Monilinia fructicola and Rhizoctonia
solani. Likewise, the essential oil of O. compactum, which contains
carvacrol (58.1%), p-cymene (11.4%) and thymol (9.0%) as major
components, carvacrol and thymol were more toxic against Botrytis
cinerea (Bouchra et al., 2003). Potent fungitoxic properties of carva-
crol and thymol against various plant pathogens were also previ-
ously documented (Muller-Riebau et al., 1995; Tsao and Zhou,
2000; Sokovic et al., 2002).
On the basis of the above results, it is clear that thymol and car-
vacrol, containing free hydroxyl group in p-cymene skeleton were
more fungitoxic than p-cymene (Table 2). These results showed
that hydroxyl group in aromatic ring is important in antifungal
activity of the compounds, contain p-cymene skeleton. As to the
mechanism of action of carvacrol, thymol and oregano essential
oils, it has been found that these agents cause alterations in the hy-
phal morphology and hyphal aggregates, resulting reduced hyphal
diameters and lyses of hyphal wall interacting with the cell mem-
brane of the pathogen (Thompson, 1996; Soylu et al., 2006, 2007).
On the other hand, Veldhuizen et al. (2006) reported the structural
requirements for the activity of carvacrol and found that the hy-
droxyl group of carvacrol itself is not essential for the antimicrobial
activity and aliphatic ring substituents of carvacrol also affect its
antimicrobial effect. Furthermore, it was documented that volatile
aromatic components in plant kingdoms were more fungitoxic
than those of non-aromatic volatile components of essential oils
(Cheng et al., 2006; Wang et al., 2005; Lee, 2007).
3.3. Herbicidal and phytotoxic effects of the oil and its constituents
Herbicidal effects of the essential oil of O. acutidens, and its aro-
matic monoterpenes, carvacrol, p-cymene and thymol were evalu-
ated for assessing their volatile and contact phase effects on the
germination of A. retroflexus, C. album and R. crispus, which are
important weeds in cultivated area. In our study, their herbicidal
effects were compared with a commercial herbicide, 2,4-D, iso-
octylester (Tables 3 and 4). The results obtained from volatile
and contact phase effect assays showed that the oil and its pheno-
lic constituents, carvacrol and thymol completely inhibited the
germination and seedling growth of all tested weeds (Tables 3
and 4). Their suppressing effects on the germination and seedling
growth of the weeds were also higher than that of commercial her-
bicide, 2,4-D, isooctyl ester (Tables 3 and 4). As shown in Table 1,
the oil of O. acutidens contained carvacrol (87.0%) as major compo-
nent, which showed potent herbicidal effects in the present study.
Therefore, the herbicidal effects of the oil can be attributed to its
major component, carvacrol. It has been documented that essential
oils isolated from some Origanum species and its phenolic com-
pounds, carvacrol and thymol possess potent herbicidal effect on
weed germination and seedling growth of various plant species
(Dudai et al., 1999; Angelini et al., 2003). This is compatible with
our results. Although the herbicidal mechanism of the oil and its
constituents was not investigated in the present study, it is well
known that monoterpenes in the essential oils have phytotoxic ef-
fects that may cause anatomical and physiological changes in plant
seedlings leading to accumulation of lipid globules in the cyto-
plasm, reduction in some organelles such as mitochondria, possi-
bly due to inhibition of DNA synthesis or disruption of
membranes surrounding mitochondria and nuclei (Koitabashi
et al., 1997; Zunino and Zygadlo, 2004; Nishida et al., 2005). The
herbicidal effects of the oil and its constituents in the present study
may be due to one or more of these factors.
On the other hand, this is a first report on the herbicidal effect of
p-cymene, other aromatic monoterpene constituent of O. acutidens
oil. This compound was found to be no inhibitory effect against
weeds of A. retroflexus and R. crispus at contact phase assays. Fur-
thermore, this compound increased the germination of C. album,
but did not affect the seedling growth of the weeds in contact
phase assays (Table 3). In contrast to its contact phase effect, va-
pors of p-cymene inhibited the weed germination of C. album
and R. crispus. However, the vapors of p-cymene weakly sup-
pressed the germination of A. retroflexus weeds. It is interesting
to find that the vapors of p-cymene exhibited a different behavior

Table 3
Contact inhibitory effects of the essential oil and its components on seed germination and seedling growth of A. retroflexus, C. album and R. crispus

Samples Dose (mg/Petri dishes) Germination (%) Seedling Growth (mm)

Root Aerial part
A. retroflexus
Essential oil 10.0 0.0 ± 0.0 c 0.0 ± 0.0 d 0.0 ± 0.0 c
Carvacrol 9.8 0.0 ± 0.0 c 0.0 ± 0.0 d 0.0 ± 0.0 c
Thymol 10.0 0.0 ± 0.0 c 0.0 ± 0.0 d 0.0 ± 0.0 c
p-Cymene 8.6 52.7 ± 4.4 a 23.3 ± 1.2 a 11.5 ± 0.6 a
2,4-D 10.0 5.3 ± 1.8 b 3.3 ± 0.3 c,d 2.9 ± 0.4 c
Control – 47.3 ± 2.9 a 18.2 ± 1.1 a,b 10.4 ± 0.7 a,b
C. album
Essential oil 10.0 0.0 ± 0.0 d 0.0 ± 0.0 c 0.0 ± 0.0 c
Carvacrol 9.8 0.0 ± 0.0 d 0.0 ± 0.0 c 0.0 ± 0.0 c
Thymol 10.0 0.0 ± 0.0 d 0.0 ± 0.0 c 0.0 ± 0.0 c
p-Cymene 8.6 50.0 ± 2.0 a 15.0 ± 1.0 a 6.3 ± 0.2 a
2,4-D 10.0 17.3 ± 3.5 c 2.2 ± 0.1 b,c 2.5 ± 0.2 b,c
Control – 39.3 ± 2.4 b 15.8 ± 1.2 a 6.7 ± 0.4 a
R. crispus
Essential oil 10.0 0.0 ± 0.0 c 0.0 ± 0.0 b 0.0 ± 0.0 d
Carvacrol 9.8 0.0 ± 0.0 c 0.0 ± 0.0 b 0.0 ± 0.0 d
Thymol 10.0 0.0 ± 0.0 c 0.0 ± 0.0 b 0.0 ± 0.0 d
p-Cymene 8.6 89.3 ± 1.8 a 30.8 ± 1.9 a 7.0 ± 0.2 a
2,4-D 10.0 42.0 ± 8.1 b 2.8 ± 0.1 b 2.2 ± 0.1 c,d
Control – 90.0 ± 1.2 a 30.3 ± 1.5 a 6.3 ± 0.2 a,b

Means in the same column by the same letter are not significantly different to the test of Duncan (a = 0.05).
 S. Kordali et al. / Bioresource Technology 99 (2008) 8788–8795 8793

Table 4
Inhibitory effects of vapors of essential oil and its components on seed germination and seedling growth of A. retroflexus, C. album and R. crispus

Samples Dose (mg/Petri dishes) Germination (%) Seedling Growth (mm)

Root Aerial part
A. retroflexus
Essential oil 10.0 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 b
p-Cymene 8.6 21.3 ± 3.5 b 19.9 ± 1.5 b 17.2 ± 1.3 a
Carvacrol 9.8 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 b
Thymol 10.0 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 b
Control – 38.0 ± 3.5 a 32.8 ± 1.4 a 16.8 ± 0.8 a
C. album
Essential oil 10.0 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 c
p-Cymene 8.6 47.3 ± 4.4 a,b 20.6 ± 1.2 b 10.5 ± 0.6 a,b
Carvacrol 9.8 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 c
Thymol 10.0 0.0 ± 0.0 c 0.0 ± 0.0 c 0.0 ± 0.0 c
Control – 50.7 ± 0.7 a 36.3 ± 1.3 a 13.8 ± 0.6 a
R. crispus
Essential oil 10.0 0.0 ± 0.0 b 0.0 ± 0.0 c 0.0 ± 0.0 c
p-Cymene 8.6 72.7 ± 5.3 a 42.7 ± 2.2 a 12.6 ± 0.2 a,b
Carvacrol 9.8 0.0 ± 0.0 b 0.0 ± 0.0 c 0.0 ± 0.0 c
Thymol 10.0 0.0 ± 0.0 b 0.0 ± 0.0 c 0.0 ± 0.0 c
Control – 72.0 ± 7.0 a 24.3 ± 1.4 b 14.3 ± 0.4 a

Means in the same column by the same letter are not significantly different to the test of Duncan (a = 0.05).

Table 5
Phytotoxic effects of O. acutidens essential oil and its components against A. retroflexus, C. album and R. crispus

Samples Dose (mg/pot) Hour Phytotoxicity (% mean death)

A. retroflexus C. album R. crispus
The oil 10 24 73.2 ± 5.0 b,c 89.6 ± 2.7 a 100.0 ± 0.0 a
10 48 86.7 ± 4.4 a,b 100.0 ± 0.0 b 100.0 ± 0.0 a
p-Cymene 10 24 5.0 ± 3.4 d 6.5 ± 2.9 f 0.0 ± 0.0 f
10 48 7.5 ± 3.3 d 11.1 ± 1.6 f 1.5 ± 1.5 f
Carvacrol 10 24 89.2 ± 4.0 a,b 69.4 ± 2.4 d 65.9 ± 9.9 c
10 48 96.3 ± 2.5 a 88.6 ± 3.5 b 80.1 ± 5.9 b
Thymol 10 24 73.1 ± 4.5 b,c 59.3 ± 9.3 e 18.7 ± 3.2 e
10 48 89.9 ± 4.3 a,b 77.8 ± 5.2 c,d 48.0 ± 5.9 d
2,4-D 10 24 58.1 ± 9.7 c 82.1 ± 3.8 b,c 81.1 ± 2.1 b
10 48 75.8 ± 9.7 b 100.0 ± 0.0 a 100.0 ± 0.0 a
Control – 24 0.0 ± 0.0 d 1.9 ± 1.3 f 0.7 ± 0.5 f
48 0.7 ± 0.7 d 1.9 ± 1.3 f 0.7 ± 0.5 f

Means in the same column by the same letter are not significantly different to the test of Duncan (a = 0.05).

on the seedling growth of the weeds (Table 4). It reduced the root
growths of A. retroflexus and C. album, whereas, the root grow of R.
crispus was increased by the vapors of p-cymene (Table 4). On the
other hand, as shown in Table 4, its vapors did not affect the
growths of aerial parts of A. retroflexus and C. album.
Phytotoxic effects of the oil and its aromatic monoterpene con-
stituents (carvacrol, p-cymene and thymol) against A. retroflexus, C.
album and R. crispus in greenhouse conditions at 1.0 mg/ml con-
centration were determined for the first time in the present study.
Their phytotoxic effects also were compared with 2,4-D, commer-
cial herbicide. The oil, carvacrol, thymol and commercial herbicide,
2,4-D, isooctyl ester showed potent phytotoxic effects against all
plant species tested (Table 5). After 48 h the application, 10 mg/
pot concentration of the oil and 2,4-D, isooctyl ester completely
killed exposed R. crispus and C. album (Table 5). Phytotoxicity re-
sults also showed that carvacrol was more phytotoxic than thymol.
However, p-cymene, other component of the oil did not show any
significant phytotoxic effect against the tested plant samples. Thus,

Fig. 1. Percent mortality of S. granarius and T. confusum adults after treatment with the oil of O. acutidens according to treatment times. , Statistically significant at p < 0.05;
, statistically significant at p < 0.01; , statistically significant at p < 0.001.
8794 S. Kordali et al. / Bioresource Technology 99 (2008) 8788–8795
the potent phytotoxic effect of the oil can be attributed to its major
component, carvacrol (Table 1). However, essential oils consisting
of numerous components and other major and/or minor com-
pound(s) in oils may affect phytotoxic activity.
3.4. Insecticidal activity of the oil
In the current study, the toxicity of the oil on two stored prod-
uct pests, S. granarius and T. confusum, were also determined. The
mortality increased with increasing in the exposure times
(Fig. 1). The oil was found to be more toxic against S. granarius as
compared with its toxicity against T. confusum. O. acutidens oil
caused 68.3% and 36.7% mortality of S. granarius and T. confusum
adults, respectively, after 96 h of exposure. It has been previously
shown that the essential oil isolated from O. acutidens contain
mainly carvacrol (72.0%) and p-cymene (7.5%) possessed potent
toxic effects against S. granarius (Sokmen et al., 2004; Yildirim
et al., 2005; Caglar et al., 2007). In the present study, the oil of O.
acutidens containing carvacrol (87.0%) and p-cymene (2.0%) as ma-
jor components was found to be toxic against two stored pests (S.
granarius and T. confusum). Our findings are agreement with above
reports. Furthermore, in support of our results, Knio et al. (2008),
who studied the larvicidal activity of carvacrol and thymol, docu-
mented that these compounds possessed more toxic effects against
Ochlerotatus caspius larvae.
4. Conclusion
The development of natural herbicides and pesticides would
help to decrease the negative impact of synthetic agents, such as
residues, resistance and environmental pollution. In this respect,
natural pesticides and herbicides may be effective, selective, biode-
gradable, and less toxic to environment. O. acutidens oil and its aro-
matic monoterpene components showed potent antifungal activity
against plant pathogenic fungi and phytotoxic effects against A. ret-
roflexus, C. album and R. crispus. Based on the present results, the oil
and its phenolic components, carvacrol and thymol could be sug-
gested as alternative pesticides, herbicides as well as insecticides.
However, further studies are required to determine the cost, appli-
cability, safety and phytotoxicity against the cultured plants of
these agents as potential herbicide and fungicides.
Acknowledgement
The authors thank Dr. Ali Kandemir, Department of Biology,
Erzincan University, Erzincan, Turkey, for the taxonomic identifica-
tion of plant material.
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