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International Journal of AgriScience Vol. 2(3): 228-236, March 2012
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ISSN: 2228-6322© International Academic Journals
International Journal of AgriScience Vol. 2(3): 228-236, March 2012 228
Study on Thymus vulgaris, Lavandula officinalis and Eucalyptus camaldulensis
extracts on the two-spotted spider mite
Modarres Najafabadi S.S.*1,Taji M.2,Hajihassani A.2
1Department of Entomology, Agriculture and Natural Resources Research Center, Arak, Iran. *Author for
correspondence (email: s_modarres_705@yahoo.com)
2 Young Researchers Club, Arak Branch, Islamic Azad University, Arak, Iran.
Received December 2011; accepted in revised form January 2012
ABSTRACT
The Two-Spotted Spider Mite (TSSM), Tetranychus urticae Koch, is a significant pest to bean
plants in Markazi Province (Iran). This study was done to evaluate the acaricidal activity of
extracts from three essential oils; thyme, lavender and eucalyptus on the pest T. urticae. The
essential oils were applied in five different concentrations (0.5%, 1.0%, 2.0%, 3.0% and 4.0%)
and the experiment was done in 2009-2011. The results showed that lavender (Lavandula
officinalis) had the most potent efficient as an acaricidal agent against Tetranychus, this was
followed by thyme (Thymus vulgaris) and then eucalyptus (Eucalyptus camaldulensis). The LC50
values of lavender, thyme and eucalyptus for adult mites were 0.65, 1.84 and 2.18, respectively.
The findings of this research revealed that extracts of these three essential oils (lavender, thyme
and eucalyptus) all had an active acaricidal effect on the two-spotted spider mite on bean plants.
Keywords: Tetranychus urticae, plant essential oils, lavender, thyme, eucalyptus
INTRODUCTION
The two-spotted spider mite, Tetranychus
urticae is one of the most important pests
responsible for yield losses in horticulture
on both ornamental and agronomic crops. A
major problem in the struggle to control T.
urticae is that the pest develops resistance to
many acaricides due to an approximate 5-
fold increase in the mixed function of
oxidase activity (Puinean et al. 2010).
Chemical control of these mites has been
extensively practiced for several years
(Soliman 2007, Sanil and Shetty 2010).
In Egypt, different extracts from Syzygium
cumini L. against T. urticae have been used
to control mite populations and it was
determined that the most potent extract to
effect acaricidal activity was ethanol (Afify
et al. 2011). Because of the problem of
resistance and high residual levels from
pesticides, the use of botanical insecticides
has long been touted as an attractive
alternative to synthetic chemical insecticides
for pest management as they reputedly pose
little threat to the environment or to human
health (Murray 2006).
There are methods used to detect and
determine multiresidues in food products by
LC-MS-MS tandem spectroscopy that may
hinder their marketability (Afify et al. 2010).
Two-spotted spider mite females were
repelled by spinosad and they mostly
oviposited and fed on areas that had not
been treated with spinosad. Spinosad did not
affect the behavior of Panonychus ulmi
females. When T. urticae females were
released on potted bean plants (at the two-
229 International Journal of AgriScience Vol. 2(3): 228-236, March 2012
leaf stage) in which leaves received a
sprayed application of spinosad on the
adaxial or abaxial leaf surfaces, or complete
spinosad coverage on one or two of the
leaves, the mite population increase lagged
significantly behind those released on
control plants. These results indicate that S.
cumini and spinosad had significant
acaricidal effect on T. urticae but not on P.
ulmi (Afify et al. 2010).
Use of essential oil of plant extracts in pest
management programs has recently attracted
the attention of many scientists. Pesticides
from plant origin generally remain on a
plant for a relatively short duration and are
therefore deemed more suitable than
chemicals for pest control (Murray 2006,
Raina et al. 2009). However, products from
plant extracts need to be carefully selected
and strictly evaluated for their effects on
different species of natural enemies, as
effects can be either deleterious or positive
(Khan et al. 2007, Jourdie et al. 2010).
There is an enzyme present in insects,
glutathione-S-transferase that is of interest
as it functions in the detoxification
mechanism due to its involvement in
tolerance to acaricides (Afify et al. 2011,
Gui et al. 2009). It has been reported that
most xenobiotics are subject to enzymatic
modification after penetration through
protein binding and transportation in a
biological interaction such as that of
acaricide on insects. It has been clearly
demonstrated that several enzymatic systems
such as esterase (α and β), and phosphatase
(alkaline and acid) can play a vital role in
the detoxification of xenobiotics to nontoxic
materials (Afify 2010).
This study aimed to evaluate the acaricidal
activity of extracts of essential oils of
lavender, thyme and eucalyptus against T.
urticae. The study evaluated the biochemical
changes to T. urticae from treatments of
LC50 of the tested oils.
MATERIAL AND METHODS
Mite colony: Adult TSSM were originally
collected from common bean plants (P.
vulgaris) growing in the fields of the
Khomein region, Iran in May 2009. These
mites were reared on bean plants that were
cultivated in plastic pots (20cm diameter ×
25 cm height) in a growth chamber (27±2ºC,
70±5% RH and a photoperiod of 16 L:8 D
h.) for at least two months (several
generations) prior to the experiment. All
experiments were performed in growth
chambers in the same conditions as herewith
mentioned. The cultivars required for the
experiments were obtained from the Bean
Research Institute of Khomein, Iran. The
seeds were sown in plastic pots (20cm
diameter × 25 cm height) filled with
fertilized field soil. The bean cultivars were
planted in 20 replications and maintained in
a greenhouse. After four weeks, leaves were
detached from the bean plants and used for
leaf disc preparation. All plants were
irrigated at the same time during the
experiment and no fertilizers or pesticides
were used.
Leaf discs: The experiments employed the
leaf disc method (Pedigo and Buntin 1994,
Naher et al. 2006). Each leaf disc was a
4cm2 section from the central area of a leaf
separated by plastic padding 2cm×2cm.
Each leaf disc was placed on a plastic Petri
dish (8cm diameter × 1.5cm height with a
hole at its center). Thereafter, one fully
expanded young leaf (third leaf below the
apical meristem of one month-old plants)
was randomly collected and used for
preparation of the leaf disc. Leaves of bean
plants were selected from all replications
and leaf discs of (2cm×2cm) were cut and
then placed on water-saturated cotton in the
Petri dish with the underside facing upward.
During the experiments, bean seeds were
periodically planted in the greenhouse
(every 10 days), and to reduce the effects of
International Journal of AgriScience Vol. 2(3): 228-236, March 2012 230
plant age on mite development and
fecundity, the new leaf discs were prepared
from their leaves and the mites were
transferred on to them.
Plant materials: Common thyme (Thymus
vulgaris) (Lamiaceae), Common lavender
(Lavandula officinalis) (Lamiaceae) and
River Red Gum, (Eucalyptus camaldulensis)
(Myrtaceae) were collected from the
Medicine Plants Research Institute of Arak,
Iran.
Preparation of essential oils: The whole
plants (herbs) of thyme and lavender and the
leaves of eucalyptus were dried for a week
at room temperature, and then crushed
according to the method of Calmasur et al.
(2006). Essential oils were extracted by
hydro distillation (deionized water for 4 h)
in a vacuum according to the method of
Aroiee et al. (2005). Essential oils and
components were kept under freezing
conditions until use. Series of aqueous
concentrations of each essential oil were
prepared with Triton X-100 as surfactant at
a rate of 0.1%.
Treatment of eggs: Leaf discs (4 cm2) cut
from leaves of bean plants were used as a
substrate for the ovipositor. Four leaf discs
were used for each treatment and five mite
females were transferred to each disc and
left for 24 h to lay eggs, then females were
removed. Thereafter, forty eggs, on four
discs, were treated with one of the five
concentrations (0.5%, 1.0%, 2.0%, 3.0% and
4.0%). Eggs were sprayed by a glass
atomizer, with a series of concentrations for
each essential oil. 1 mL/200 cm of the
solution was used. Eggs were then incubated
at (27±2ºC, 70±5% RH) for seven days until
hatching. Records were taken for numbers
of eggs to hatch and numbers of eggs that
failed to hatch.
Treatment of adult females: T. urticae
females, 3 days old, were obtained by
placing 100 nymphs onto the culture, and
wet cotton pads in Petri dishes were placed
on to the excised bean leaves. Emerged
females and males were transferred to new
bean leaves for 2-3 days and allowed to
mate. Afterwards, forty females were
transferred equally to four discs (4 cm2), and
then treated with one of the previous
treatments. The control treatment was
operated by Triton X-100 at a rate of 0.1%.
Mortality was estimated for adult females 24
h after spraying and was estimated by
Abbot’s formula (1925) and LC50, LC90;
the slope values were estimated according to
Finney (1971).
Statistical analysis: Data were statistically
analyzed using Costa software (cohort
software, Berkeley). Significance of results
was obtained by randomized one-way
ANOVA, and the means were separated
using the Duncan’s multiple range test
(Duncan 1955) at P<0.01.
RESULTS
Essential oils of plant extract are a
promising natural alternative for the control
of T. urticae. These extracts facilitate easy
handling and application, as well as a low
cost option in place of chemical pest control.
Data presented in Table 1 demonstrates that
essential oil extract of lavender was the most
potent acaricidal agent against T. urticae,
which enhanced the highest adult female
mortality and the lowest egg hatchability.
Adult mortality percentages after 24 h were
42.50%, 75.00%, 90.00%, 95.00% and
100.00% for lavender by spraying
concentrations of 0.5%, 1.0%, 2.0%, 3.0%
and 4.0%, respectively. The percentages of
corresponding mortalities for thyme were
20.00%, 30.00%, 42.50%, 72.50%, and
85.00%, while 17.50%, 27.50%, 40.00%,
70.00%, and 80.00% for eucalyptus,
respectively. Hatchability percentages after
six days were 75.00%, 55.00%, 30.00%,
16.00% and 10.00% for lavender; 95.00%,
87.50%, 80.00%, 72.50%, 57.50% for thyme
231 International Journal of AgriScience Vol. 2(3): 228-236, March 2012
and 95.00%, 92.50%, 82.50%, 77.50% and
67.50% for eucalyptus, respectively, for
control treatment (Triton X-100 at a rate of
0.1%), adult mortality was 10.00% and egg
hatchability was 95.00%. Table 2
demonstrated that essential oil extract of
lavender demonstrated the most potent
acaricidal activity followed by thyme and
then eucalyptus. The LC50 values after 24 h
for adults were 0.65%, 1.84% and 2.18%,
respectively, while for eggs percentages of
1.17%, 6.26% and 7.33% were recorded
after seven days. The slope values of the
regression line were 2.41, 2.53 and 2.49 for
adults and 2.28, 1.89 and 2.15 for eggs,
respectively. LC90 values were 2.27%,
5.91% and 7.13% for adults and 4.34%,
9.81% and 28.95% for eggs, respectively.
Table 1. Effect of three essential oil plant against T. urticae egg hatchability and adult mortality (mean±SD)(%).
Concentration (%)
Lavandula officinalis
Thymus vulgaris
Eucalyptus camaldulensis
Adult mortality
Egg hatchability
Adult mortality
Egg hatchability
Adult mortality
Egg hatchability
control
10.00±1.29
95.00±0.58
10.00±1.14
95.00±0.58
10.00±1.29
95.00±0.58
0.5
42.50±1.71
75.00±1.29
20.00±1.29
95.00±0.58
17.50±0.96
95.00±0.58
1.0
75.00±1.70
55.00±2.38
30.00±0.82
87.50±0.50
27.50±1.71
92.50±0.96
2.0
90.00±0.82
30.00±0.58
45.50±2.06
80.00±1.15
40.00±1.41
82.50±1.26
3.0
95.00±0.58
16.00±0.10
75.50±1.50
72.50±1.50
70.00±2.24
77.50±1.71
4.0
100.00±0.00
10.00±1.41
85.00±0.82
57.50±2.36
80.00±0.82
67.50±0.58
Table2. Effect of three essential oil plant against T. urticae egg hatchability and adult mortality (mean±SD)(%).
Concentration (%)
Lavandula officinalis
Thymus vulgaris
Eucalyptus camaldulensis
Adults
Eggs
Adults
Eggs
Adults
Eggs
LC50 (%)
0.65
1.17
1.84
6.26
2.18
7.33
Lower limit
0.46
0.94
1.53
4.18
1.82
4.74
Upper limit
0.82
1.45
2.21
25.40
2.67
39.05
Index
100.00
100.00
35.44
19.11
29.82
16.31
Slope
2.41
2.28
2.53
1.89
2.49
2.15
LC99 (%)
2.27
4.34
5.91
9.81
7.13
28.95
DISCUSSION
The results of this experiment showed that
spider mites demonstrated no resistance to
essential oils and that evidently there were
differences in potency according to the type
of essential oil. These highly effective
essential oils were used at an early stage to
control mite populations at isolated loci, to
preserve the pest’s natural enemies and to
maximize their role in natural pest control.
The rotation of different, highly effective
International Journal of AgriScience Vol. 2(3): 228-236, March 2012 232
extracts for control by means of acaricide
was effective.
The results herewith presented for treatment
with lavender extract are in agreement with
those documented by Ma et al. (2008)
reporting that the highest effect of terpinene-
4-ol on esterase activity was noted during
the recovery stage of the adult housefly
(Musca domestica). The activities of both
acid phosphatase and alkaline phosphatase
in insects were induced by terpinen-4-ol.
The activities of glutathione-S-transferase
were inhibited at exciting, convulsing and
paralysis stages, but gradually recovered at
the recovery stage. The activities of
glutathione-S-transferase probably had
relations with the toxicity of terpinen-4-ol
against the larvae of the Mythimna separate
(Ma et al. 2008). This point will be taken in
to consideration in the near future to clarify
the effect of inhibition from multiple
phosphatases, or individual ones. The
activity of glutathione-S-transferase was
inhibited in exciting, convulsing and
paralysis stages of the 5th star larvae of
Mythimna separata, but it gradually
recovered during the recovery stage. This
affected the metabolism and activity of
phosphatase and esterase enzymes.
However, inhibited insect glutathione-S-
transferase inhibited normal metabolism.
The activity of glutathione-S-transferase at
LC50 of the essential oil indicated that this
enzyme activity was recovered and was able
to act in defense of free radicals and it was
more active when it could be detected at
specific LC50 of essential oil extract in
recovered mites.
Acaricidal activities of three essential oil
extracts (lavender, thyme and eucalyptus)
against T. urticae Koch affirmed that
lavender was the most efficient (Sertkaya et
al. 2010). Essential oils of lavender and
thyme showed a relationship between
essential oil content and activity of the
enzyme glutathione-S-transferase, non-
specific esterase and alkaline phosphatase as
well as inhibition of protease enzyme in the
two-spotted spider mite. The major essential
oil contents of lavender are α-bisabolol
oxide A (35.251%), and trans-α-farersene
(7.758%), while the main components of
thyme are terpinen-4-ol (23.860%), p-
cymene (23.404%) and sabinene (10.904%).
The major components of both plant extracts
may be responsible for changes in enzyme
activities of T. urticae. The present results
are in agreement with data cited by Kawka
(2004), who studied the effect of lavender
extracts from fresh and dry flowers on T.
urticae. Application of extracts on leaves
demonstrated greater mortality. It has been
claimed that increased activities of
detoxifying and antioxidant enzyme systems
in acaricides were responsible for this
resistance (Afify et al. 2011).
A decrease in proteinase enzyme, which is
involved in the biological system of defense
proves the presence of proteinase inhibitor
in the extracts as cited by Born et al. (2009),
Kant et al. (2008) and Azzouz et al. (2005),
in reports that demonstrate that the extracts
can induce defense gene expression of
proteinase inhibitor activity. Proteinase
inhibitors are proteins that inhibit digestive
enzymes in the gut of arthropod herbivores,
which can reduce their growth and
reproduction. Glutathione-S-transferases are
major enzymes involved in metabolic
resistance to insecticides, as well as in the
detoxification mechanisms of many
molecules and probably in the transport of
physiologically important lipophilic
compounds. Glutathione-S-transferases play
an important role in protecting tissue from
oxidative damage and stress (Gui et al.
2009, Ugurlu et al. 2007).
The changes in the activity of α, βesterase,
glutathione-S-transferase and alkaline
phosphatase and protease enzymes in target
site susceptibility are key biochemical
mechanisms of the development of active
233 International Journal of AgriScience Vol. 2(3): 228-236, March 2012
components of essential oils, which show
more potency against Tetranychidae. These
studies have laid a solid foundation for
further studies on biochemical mechanisms
of resistance in Tetranychus cinnabarinus
and other spider mites. However these
points need further investigation in the
future to prove these suggestions by using
individual components and to evaluate their
effects on enzyme activities of the two-
spotted spider mite. Even this suggestion
was approved by Ma et al. (2008), who
recognized bioactivity and the effect of
terpinen-4-ol on activities of some enzymes
in adult housefly (Musca domestica). The
results showed that the LD50 of terpinen-4-
ol was 23.91μg/insect. The poisoning
symptom of terpinen-4-ol could be divided
into the following four stages; excitation,
convulsion, paralysis and recovery stages.
The highest effect of terpinen-4-ol on
esterase activity was measured during the
recovery stage (8±0.009 μmol/20min).
Glutathione-S-transferase, monooxygenase
(P450) and esterases activity were detected
in resistance in the two-spotted spider mite
(Puinean et al. 2010). In contrast, no
sesquiterpenes were detected in the fresh
resin oil and it was constituted basically by
monoterpenes hydrocarbons (42.4%) and
oxygen-containing monoterpenes (27.7%),
of which α-phellandrene (13.9%) and
terpinen-4-ol (7.4%) were the major
components, respectively (Wendel et al.
2007). Conceivably, such a challenge has
forced the development of mechanisms for
survival and adaptation throughout evolution
and insecticide activity of these essential oils
against Anopheles stephensi (Prajapati et al.
2005). Furthermore, and in the above
context, induction of detoxifying enzymes
by a large number of toxicants has been
observed in arthropods (Cao et al. 2008).
The present results are in agreement with the
research of Wendel et al. (2007), in a study
that evaluated the acaricidal activity of some
essential oils against TSSM, such as fresh
and aged resin (Protium bahianum) results
showed higher oil yield 4.6% and 3.2%,
respectively. About 22 and 13 components
were identified in the oils from fresh and
aged resins, comprising 95.8% and 98.6%,
respectively. In the fresh resin oil,
monoterpenes (70%) were the major group
of constituents, mainly p-cymene (18.3%)
followed by hydrocarbons, such as α-
phellandrene (14.0%), tricyclene (11.4%)
and β-phellandrene (9.1%), while the aged
resin oil contained sesquiterpenes as the
major group with santalol acetate (83.1%) as
the principal component.
Treatment with chloroform extract from
Kochia scoparia enhanced SOD, POD and
CAT activities during the 24 hour period
following treatment (Wang et al. 2010, Cao
et al. 2007) and traditional Chinese plants
demonstrated toxicity to Tetranychus
cinnabarinus (Ren et al. 2009, Xiao et al.
2008) and even glucoside had an acaricial
effect (Ren et al. 2007). Acaricidal activities
of Wikstroemia chamaedaphne extracts
against Tetranychus were also reported.
Twenty-nine compounds were identified as
potential acaricidal agents against
Tetranychus cinnabarinus (Wang et al.
2010) and had an effect on the activity of
Tetranychus enzymes (Wang et al. 2009).
The essential oils from accessions of Lippia
sidoides Cham. (Verbenaceae) were
characterized by GC and GC/MS and
investigated for their acaricidal activity
against the two-spotted spider mite T.
urticae Koch (Cavalcanti et al. 2010).
In conclusion, three essential oils of
lavender, thyme and eucalyptus all
possessed the property of acaricidal activity
against T. urticae. However, further
investigation is needed to study the
components of these plants, which are
responsible for inhibiting the activities of
enzymes in the two-spotted spider mite.
International Journal of AgriScience Vol. 2(3): 228-236, March 2012 234
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