Industrial Crops and Products 32 (2010) 405410

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Industrial Crops and Products

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Repellency and toxicity of essential oils from the leaves and bark of Laurelia
sempervirens and Drimys winteri against Tribolium castaneum
Nelson Zapata a,b , Guy Smagghe a,
a
b

Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
Departamento de Produccin Vegetal, Facultad de Agronoma, Universidad de Concepcin, Avenida Vicente Mndez 595, Chilln, Chile

a r t i c l e

i n f o

Article history:
Received 17 December 2009
Received in revised form 1 June 2010
Accepted 4 June 2010

Keywords:
Tribolium castaneum
Essential oils
Safrole
Limonene
Methyl eugenol
Curcumene
-Pinene

a b s t r a c t
We report here on the repellent activity, as well as contact and fumigant toxicity, of four essential oils
extracted from the leaves and bark of Laurelia sempervirens and Drimys winteri against an important
stored-product insect pest: the red our beetle, Tribolium castaneum. The four oils tested had a very
strong repellent activity towards T. castaneum when tested in a lter paper arena test. After 4 h exposure
>90% repellency was achieved with L. sempervirens oils at low concentrations of 0.032 l/cm2 , while for
D. winteri oils concentrations of 310 times higher were needed to achieve this activity. Oils of both, L.
sempervirens and D. winteri, were found to be toxic towards T. castaneum when applied topically or by
fumigation. LD50 values by topical application of L. sempervirens oils were from 39 to 44 g/mg insect; for
D. winteri oils these were from 75 to 85 g/mg insect. By fumigation, LC50 values for L. sempervirens oils
were 1.61.7 l/l air, while these were 9.010.5 l/l air for D. winteri oils. In addition, with L. sempervirens
oils 50% of the tested beetles were killed at 100 <l/l air within 3.04.4 h, while with D. winteri oils the
LT50 values were 6.17.4 h. The results obtained are discussed in relation to the main constituents of the
oil extracts. In conclusion, the essential oils from the leaves and bark of L. sempervirens may be explored
as a potential natural insecticide for stored-product insect pests because of their high repellency and
insecticidal activities.
2010 Elsevier B.V. All rights reserved.

1. Introduction
The red our beetle, Tribolium castaneum Herbst (Coleoptera:
Tenebrionidae), is one of the most widespread and destructive
stored-product pest throughout the world. Beetles and larvae
feed on a very wide variety of dry vegetable substances, such
as milled cereal products (Rees, 2004). The application of various synthetic insecticides and fumigants to grain storage over the
years has led to a number of problems, including the development
of insecticide resistance in stored grain insect pests (Lorini and
Galley, 1999; Sousa et al., 2009). In addition, the increasing public
concern over pesticide safety and possible damage to the environment has resulted in increasing attention being given to natural
products for the control stored pests (Rajendran and Sriranjini,
2008).
In this context, many plant products have been evaluated for
their toxic properties against different stored grain pests, especially in the form of essential oils (Regnault-Roger, 1997; Rajendran
and Sriranjini, 2008). Essential oils are composed of complex

Corresponding author. Tel.: +32 9 2646150; fax: +32 9 2646239.

E-mail address: guy.smagghe@ugent.be (G. Smagghe).
0926-6690/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2010.06.005

mixtures of monoterpenes, biogenetically related phenols, and

sesquiterpenes, and are obtained through steam distillation of vegetative structures of many plants (Isman et al., 2007). Essential
oils are particularly abundant in some families of plant: Conifers,
Rutaceae, Umbelliferae, Myrtaceae and Labiatae, and are often
localized in specialized histological structures (Regnault-Roger,
1997).
Bioactivity of essential oils is directly related to its chemical
composition, which can vary dramatically, even within the same
species. Sources of compositional variability can include the plant
part extracted, phenological state of the plant, and time of year, as
well growth environment conditions (Angioni et al., 2006; Isman
et al., 2007). Bioactivity of essential oils is also affected by interactions among their structural components. Even minor compounds
can have a critical function due to additive action between chemical
classes and synergism or antagonism (Hummelbrunner and Isman,
2001; Sampson et al., 2005; Angioni et al., 2006; Bakkali et al.,
2008).
Essential oils possess acute contact and fumigant toxicity to
insects (Liu and Ho, 1999; Sahaf et al., 2008; Abdelgaleil et al.,
2009), repellent activity (Wang et al., 2006; Nerio et al., 2009),
antifeedant activity (Huang et al., 1997; Huang et al., 1999), as
well development and growth inhibitory activity (Tomova et al.,
2005; Waliwitiya et al., 2008). Since toxic mechanisms depend

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N. Zapata, G. Smagghe / Industrial Crops and Products 32 (2010) 405410

on phytochemical patterns, as yet as, not fully understood, but

the rapid action of essential oils or their constituents in some
pests is indicative of a neurotoxic mode of action (Isman et al.,
2007).
Drimys winteri J.R. Forster and G. Forster (Winteraceae) and
Laurelia sempervirens (Ruiz and Pavn) Tul. (Monimiaceae) are
native trees, widespread in the forest area of south and central
regions of Chile (Rodrguez et al., 1983). Different vegetative parts
of these trees have been used for medicinal purposes since ancient
et al., 2001). Their chemical
times by the Mapuche people (Munoz
characterization has focused on compounds for medicinal applications. As a consequence, very few studies have been done on
their interactions with herbivores or on their potential use as
biopesticides.
Phytochemical studies of wood and bark of L. sempervirens have
revealed a high content of alkaloids of the poryphynoid and bisben et al., 2001).
zylsoquinoline type (Cassels and Urza, 1985; Munoz
In addition, considerable amounts of essential oil in both leaf and

bark have been also reported (Munoz

et al., 2001; Bittner et al.,
2009). The L. sempervirens essential oil is mainly composed of safrole, a methylene dioxy that is also present in high quantities in
sassafras oil (FAO, 1995; Zapata et al., 2010). Essential oils from the
leaves of L. sempervirens have shown insecticidal, fungicidal and
acaricidal activity as well as having plant growth regulator activity
(Neira et al., 2004; Cspedes et al., 2006; Bittner et al., 2008, 2009).
D. winteri seems to be a well-defended plant since it contains a substantial amount of essential oil as well as drimane-type
sesquiterpenes, drimendiol, isotadeonal, isodrimeninol and polygodial which possess antibacterial, antifungal, antifeedant and
plant-growth regulatory properties (Brown, 1994; Barrero et al.,
2000; Jansen and Groot, 2004; Rodrguez et al., 2005; Zapata et al.,
2009, 2010).
As part of future strategies for stored-product insect control,
essential oils with repellent and/or insecticidal properties should
be studied. Therefore, the aim of the present work was to study
the repellent activity as well as contact and fumigant toxicity of
essential oils extracted from the leaves and bark of L. sempervirens
and D. winteri against the red our beetle, T. castaneum.

sidered dead when no leg or antennal movements were observed

when gently prodded with a brush.
2.3. Repellent activity
A choice bioassay system was used to evaluate repellency of
the four oils. Half lter paper disks of 8 cm diameter were treated
with 0.5 ml of acetonic solutions of the oils from the leaves or bark
of L. sempervirens (0.0010.032 l/cm2 ) or with the oils of D. winteri (0.020.20 l/cm2 ) and dried for 3 min under a fume extractor.
Half of the bottom of a Petri dish was covered with the treated lter paper, while the other half was covered with a lter paper disk
impregnated with acetone. Ten unsexed adults were put into each
Petri dish and the lid was sealed with in place with paralm. Six
replicates were run for each tested concentration, so that 60 adults
were assayed per concentration. The test was carried out under the
same environmental conditions described for the rearing. The numbers of insects on the two half paper disks were recorded after 2 and
4 h from the beginning of the test. Percentage of repellency (PR) was
calculated as follows: PR = (C T)/(C + T) 100, where C = numbers
of insects on the untreated area, and T = numbers of insects on the
treated area (Nerio et al., 2009).
2.4. Contact activity
A series of dilutions of essential oils extracted from the leaves
and bark of L. sempervirens (540%) or D. winteri (1580%) was prepared using acetone as a solvent. Aliquots of 0.5 ml of the dilutions
were topically applied onto the thorax of the insects with a Hamilton microapplicator (Bonaduz, Switzerland). Controls were treated
with acetone. Ten unsexed adults of T. castaneum were used for
each concentration and control, and the experiment was replicated
six times. Both treated and control insects were then transferred
to glass Petri dishes with culture media and kept under the same
environmental conditions described for the rearing. Mortality percentages were recorded after 24 h of treatment and LD50 and LD90
values were calculated according to Finney (1971).
2.5. Fumigant activity

2. Materials and methods

2.1. Plant material and essential oil extraction
The leaves and stem bark of L. sempervirens and D. winteri were
collected in December of 2008 in the South-Central Region of Chile
(36 51 S, 71 57 W). Fresh plant material was washed with distilled
water to remove any debris and then was crushed. Essential oil was
extracted from the plant samples using a Clevenger-type apparatus, where the plant material is subjected to hydrodistillation for
4 h. Anhydrous sodium sulphate was used to remove water after
extraction. Oil yield was calculated according to the dry weight of
the plant material used in the extractions. The extracted oils were
stored in a refrigerator at 4 C until use for the insect tests. The
chemical composition (qualitative and quantitative analysis) of the
four essential oils tested in this study is presented in Zapata et al.
(2010).
2.2. Insects
T. castaneum was obtained from a stock colony maintained in the
Laboratory of Agrozoology at Ghent University, Belgium. This insect
was reared on wheat our mixed with brewers yeast (10/1, w/w) in
a growth incubator at 2325 C and 6570% relative humidity (r.h.).
In the present study, adults of 1-week old were used with a mean
fresh weight of 1.86 0.09 mg. In all bioassays, insects were con-

To determine the fumigant toxicity of the oils and the exposure time required to kill 50 and 90% of the insects (LT50 and
LT90 values), lter papers (Whatman #1, cut into 2 cm diameter
pieces) were impregnated with an appropriate concentration of
essential oils extracted from the leaves and bark of L. sempervirens
(10100 l/l air) or D. winteri (25600 l/l air) without using
any solvent. The impregnated lter paper was then attached to
the undersurface of the screw cap of a 30 ml glass vial. The lids
were screwed tightly onto the vials, each of which contained ten
unsexed adults of T. castaneum. Six replicates were prepared for
each treatment including a control without any essential oil, and
were kept under the same environmental conditions described
for the rearing. Mortality was recorded after 2, 4, 6, 10 and 24 h
of exposure to the essential oils. Time-mortality data for each
experiment were analyzed via the method developed by Finney
(1971) with time as the explanatory variable.
Another experiment was designed in order to determine the 50
and 90% lethal concentration. Filter paper disks (Whatman #1 of
8 cm diameter) were impregnated with essential oils of L. sempervirens (0.58.0 l/l air) or D. winteri (4.032.0 l/l air). The treated
paper disk was then attached to the undersurface of the screw lids
of a 1500 ml glass ask. The lids were screwed onto the asks, each
of which contained ten unsexed adults. Replicates and incubation
conditions were the same described for the previous experiment.
Mortality was recorded after 24 h of exposure, LC50 and LC90 values
were calculated.

N. Zapata, G. Smagghe / Industrial Crops and Products 32 (2010) 405410

2.6. Data analysis

One-way analysis of variance (ANOVA) and the least signicant
difference (LSD) multiple range test were performed on the data
to determine signicant (P < 0.05) differences among concentrations (STSC Inc., Rockville, M.D). Probit analysis (Finney, 1971) was
conducted to estimate lethal doses (LD50 and LD90 ), lethal concentrations (LC50 and LC90 ) and lethal times (LT50 and LT90 ) with their
95% ducial limits by POLO Plus V 2.0 (LeOra Software Inc., Berkeley, CA); LD, LC and LT values were considered signicantly different
when their respective 95% ducial limits did not overlap.
3. Results
3.1. Essential oils
The yield of the essential oils extracted from the leaves and
bark of L. sempervirens were 2.79 and 6.26%, respectively, while
the yields of oil for D. winteri leaves and bark were 1.37 and 4.18%,
respectively. The density of essential oils from the leaves and bark of
L. sempervirens were 1.05 0.01 and 1.01 0.02 g/ml, respectively;
that of oils from the leaves and bark of D. winteri were 0.96 0.01
and 0.90 0.01 g/ml, respectively.
3.2. Repellent activity
The four essential oils tested were strongly repellent to T. castaneum and the concentrationresponse analyses were signicant
(Table 1). The repellent activity of the essential oils extracted from
L. sempervirens and D. winteri were signicantly inuenced by
the concentration applied and, interestingly, the activity was also
increased when insects were exposed for a longer time. Essential
oils extracted from the leaves or bark of L. sempervirens showed the
same repellent activity to T. castaneum. When applied at concentrations ranging from 0.001 to 0.032 l/cm2 during 4 h of exposure
the PR values for these two oils ranged from 43 to 93% and from
47 to 97%, respectively. The same tendency was observed with oils
from the leaves and bark of D. winteri: the PR values with these oils
at concentrations of 0.020.20 l/cm2 ranged from 50 to 97% and
from 53 to 100%, respectively. T. castaneum was particularly sensitive to the essential oils of L. sempervirens since >90% repellency was
obtained with low concentrations of 0.032 l/cm2 , while with the
D. winteri oil concentrations of 310 times this value were needed
to achieve similar PR values.
3.3. Contact
The essential oils extracted from the leaves and bark of L. sempervirens and D. winteri were found to be toxic to T. castaneum adults
when applied topically to the insects (Table 2). On the basis of LD50
values, T. castaneum was signicantly more susceptible (no overlap in 95% ducial limits) towards the essential oils extracted from
L. sempervirens (ducial limits 34.2949.05 g/mg insect) than the

407

Table 1
Repellent activity of essential oils from leaf and bark of Laurelia sempervirens and
Drimys winteri against Tribolium castaneum at different exposure time.
Dose (l/cm2 )

Repellency (%)
2h

4h

L. sempervirens leaf oil

0.001
36.67 3.33 a
0.002
43.34 6.15 ab
0.004
56.67 6.15 bc
0.008
63.34 8.03 cd
0.016
70.00 4.47 cd
0.032
76.67 8.02 d
(F = 6.08; df = 5,30; P < 0.001)

43.34 6.67 a
56.67 4.17 a
63.34 6.15 b
70.00 4.47 b
76.67 3.33 b
93.34 4.22 c
(F = 27.98; df = 5,30; P < 0.001)

L. sempervirens bark oil

0.001
40.00 5.16 a
0.002
50.00 4.47 a
0.004
53.34 6.67 a
0.008
70.00 6.83 b
0.016
76.67 3.33 b
0.032
83.34 6.15 b
(F = 9.24; df = 5,30; P < 0.001)

46.67 11.16 a
60.00 8.03 ab
66.67 8.43 abc
76.67 6.15 bcd
83.34 3.33 cd
96.67 3.33 d
(F = 6.22; df = 5,30; P = 0.001)

D. winteri leaf oil

0.02
0.04
0.08
0.12
0.16
0.20

36.67 3.33 a
43.34 6.15 ab
56.67 3.33 bc
70.00 4.47 cd
76.67 3.33 d
83.34 6.15 d
(F = 16.28; df = 5,30; P < 0.001)

50.00 4.47 a
63.34 6.15 ab
66.67 4.22 b
83.34 6.15 c
86.67 6.67 c
96.67 3.33 c
(F = 10.68; df = 5,30; P < 0.001)

D. winteri bark oil

0.02
43.34 6.15 a
0.04
56.67 3.33 ab
0.08
66.67 4.22 bc
0.12
73.34 6.67 cd
0.16
76.67 3.33 cd
0.20
86.67 4.22 d
(F = 10.17; df = 5,30; P < 0.001)

53.34 4.22 a
66.67 8.43 b
73.34 4.22 b
90.00 4.47 c
100.00 0.0 c
100.00 0.0 c
(F = 17.35; df = 5,30; P < 0.001)

Means within the same column followed by the same letter are not signicantly
different. ANOVA, LSD (P > 0.05).

oils extracted from D. winteri (ducial limits 69.3590.75 g/mg

insect). However, there was no signicant difference in contact toxicity between oil extracted from the leaves or bark of L. sempervirens
(overlap in 95% ducial limits). On the basis of LD90 values, only the
oil extracted from the bark of L. sempervirens was signicantly more
toxic to T. castaneum than the oils of D. winteri.
3.4. Fumigant activity
The four essential oils tested showed a strong fumigant activity against T. castaneum, and the time needed to cause 50% (LT50 )
and 90% (LT90 ) mortality decreased with increasing concentrations
of the oils (Tables 3 and 4). When insects were fumigated with
oils from the leaves or bark of L. sempervirens at a concentration of
10 l/l air, the lower and upper 95% ducial limits of the LT50 and
LT90 values were 8.14 and 10.00 h, and 14.76 and 22.95 h, respectively (Table 3). The lethal time values decreased signicantly when

Table 2
LD50 and LD90 values of essential oils from leaves and bark of Laurelia sempervirens and Drimys winteri applied by contact against Tribolium castaneum.
Essential oil

LD50 a (95% FL)

LD90 a (95% FL)

Slope SE

Chi square (2 )

df

L. sempervirens
Leaf
Bark

44.05 (39.0049.05)
38.94 (34.2943.11)

102.49 (87.62127.98)
78.7 (68.9595.53)

3.49 0.38
4.19 0.52

10.37
13.95

40
40

D. winteri
Leaf
Bark

84.05 (77.4190.75)
75.14 (69.3580.82)

162.47 (145.67187.40)
132.29 (120.45149.23)

4.48 0.39
5.21 0.46

18.82
14.71

40
40

LD: lethal dose; FL: ducial limits.

a
Concentration: g oil/mg insect.

408

N. Zapata, G. Smagghe / Industrial Crops and Products 32 (2010) 405410

Table 3
LT50 and LT90 values of essential oils from leaves and bark of Laurelia sempervirens applied as a fumigant against Tribolium castaneum.
Oil concentration (l/l air)

LT50 (h) (95% FL)

LT90 (h) (95% FL)

Slope SE

Chi square (2 )

df

Leaf oil
10
20
40
60
80
100

9.14 (8.4110.00)
7.99 (7.338.71)
6.48 (5.967.00)
5.82 (5.316.32)
5.07 (4.575.56)
3.96 (3.494.41)

18.7 (16.1522.95)
16.67 (14.4920.21)
12.27 (10.9814.23)
11.43 (10.2213.24)
10.93 (9.6712.80)
9.17 (8.0610.83)

4.13
4.01
4.62
4.37
3.84
3.51

0.40
0.38
0.43
0.39
0.34
0.32

22.31
16.27
10.51
16.20
15.25
26.54

40
40
40
40
40
40

Bark oil
10
20
40
60
80
100

8.8 (8.149.55)
7.47 (6.928.04)
6.62 (6.127.12)
5.6 (5.116.09)
4.06 (3.604.50)
3.42 (3.043.79)

16.84 (14.7620.21)
13.68 (12.2115.98)
11.9 (10.7213.71)
11.01 (9.8412.74)
8.91 (7.8910.41)
6.81 (6.097.85)

4.55
4.87
5.03
4.36
3.76
4.29

0.43
0.46
0.47
0.38
0.34
0.40

23.33
14.31
25.04
27.58
12.03
11.28

40
40
40
40
40
40

Chi square (2 )

df

LT: lethal time; FL: ducial limits.

Table 4
LT50 and LT90 values of essential oils from leaves and bark of Drimys winteri applied as fumigant against Tribolium castaneum.
Oil concentration (l/l air)

LT50 (h) (95% FL)

LT90 (h) (95% FL)

Slope SE

Leaf oil
25
50
100
200
400
600

11.18 (10.2412.34)
8.77 (8.139.50)
6.66 (6.057.32)
4.87 (4.325.42)
4.49 (3.974.99)
3.89 (3.434.33)

23.46 (19.9729.40)
16.42 (14.4619.60)
15.29 (13.2318.57)
12.2 (10.5514.77)
10.98 (9.5614.13)
8.91 (7.8411.19)

3.98
4.71
3.55
3.21
3.30
3.56

0.38
0.45
0.32
0.29
0.30
0.33

16.94
12.70
23.26
22.84
22.10
23.58

40
40
40
40
40
40

Bark oil
25
50
100
200
400
600

13.02 (11.9714.37)
9.25 (8.689.90)
6.84 (6.277.44)
4.59 (4.125.04)
3.86 (3.464.24)
2.78 (2.443.10)

24.25 (20.8630.00)
15.04 (13.5217.48)
13.97 (12.3116.57)
9.51 (8.4811.00)
7.53 (6.758.63)
5.32 (4.736.18)

4.74
6.08
4.13
4.05
4.41
4.56

0.46
0.62
0.38
3.56
0.40
0.47

12.49
20.29
18.44
15.46
18.70
9.16

40
40
40
40
40
40

LT: lethal time; FL: ducial limits.

Table 5
LC50 and LC90 values of essential oils from leaves and bark of Laurelia sempervirens and Drimys winteri applied as fumigant for 24 h against Tribolium castaneum.
Essential oil
L. sempervirens
Leaf
Bark
D. winteri
Leaf
Bark

LC50 a (95% FL)

LC90 a (95% FL)

Slope SE

Chi square (2 )

df

1.66 (1.351.99)
1.63 (1.322.00)

8.44 (6.3512.56)
8.61 (6.4312.98)

1.81 0.18
1.77 0.18

16.34
19.86

34
34

8.96 (7.5410.36)
10.45 (9.1611.71)

31.54 (25.3543.32)
24.86 (21.4130.43)

2.35 0.26
3.40 0.34

10.54
15.25

34
34

LC: lethal concentration; FL: ducial limits.

a
Concentration: l/l air.

insects were fumigated with the same oils at a concentration of

100 l/l air. At this concentration, the 95% ducial limits of the
LT50 and LT90 values were 3.04 and 4.41 h, and 8.06 and 10.83 h,
respectively.
The LT50 and LT90 values when T. castaneum were fumigated
with oils from the leaves and bark of D. winteri, are presented in
Table 4. The 95% ducial limits of the LT50 and LT90 values for the
lowest concentration tested (25 l/l air) were 10.24 and 14.37 h,
and 19.97 and 30.00 h, respectively. The 95% ducial limits of the
LT50 and LT90 values for the high concentration (600 l/l air) were
2.44 and 4.33 h, and 4.73 and 11.19 h, respectively.
The LC50 and LC90 values of the four essential oils tested are
shown in Table 5. T. castaneum was signicantly more sensitive to
the oils of L. sempervirens than to the oils of D. winteri. Oils extracted
from both leaf and bark showed the same insecticidal activity. The
95% ducial limits of the LC50 and LC90 values for the L. sempervirens
leaf and bark oils were 1.32 and 2.00 l/l air, and 6.35 and 12.98 l/l

air, respectively. While the 95% ducial limits for the same lethal
concentrations for the D. winteri leaves and bark oils were 7.54 and
11.71 l/l air, and 21.41 and 43.32 l/l air.
4. Discussion
A total of 16 and 20 compounds have been identied from the oil
of the leaves and bark of L. sempervirens, respectively (Zapata et al.,
2010). The main constituents in the leaves oil were safrole (82.41%)
and limonene (7.76%). Safrole was also the main constituent in oil
from bark (49.71%), and methyl eugenol (18.04%) was the second
main constituent, followed by limonene (13.49%). In the essential
oils extracted from the leaves and bark of D. winteri 32 and 16 compounds were identied, respectively. The main constituents in the
D. winteri leaves oil were more diverse with -curcumene (11.12%)
as a major compound and then a group of 5 compounds consisting
of 69%, i.e. limonene + myrcene, limonene, trans-caryophyllene, -

N. Zapata, G. Smagghe / Industrial Crops and Products 32 (2010) 405410

pinene, sabinene and 4-terpineol. The main constituents in the oil

from bark of D. winteri were -pinene (57.82%) and -curcumene
(11.22%) and -pinene + myrcene (7.37%).
The composition of the essential oils extracted from L. sempervirens showed a substantial similarity to other plants species of
this type as reported in literature with safrole being the main constituent (Bittner et al., 2008, 2009). In contrast, -pinene was the
main constituent of essential oil extracted from bark of D. winteri,
which concurs with that previously reported for oil from this plant
(Barrero et al., 2000). Essential oil extracted from the leaves of D.
winteri seems to be more complex than the other oils analyzed as
there is no clear predominance of a single compound. Terpineol
has been reported as the most abundant compound in essential
oils extracted from the leaves of D. winteri (Cspedes et al., 2006);
Terpineol was also present in signicant amount in the oil extracted
for this study.
The essential oils extracted from the leaves and bark of L. sempervirens and D. winteri were shown here to possess contact and
fumigant toxicity as well as repellent activity towards T. castaneum.
Many plant products, such as essential oils, have been screened for
their repellent activity against stored grain pests (Regnault-Roger,
1997; Cosimi et al., 2009; Nerio et al., 2009). Other studies have
shown that T. castaneum can also be repelled by essential oils from
Evodia rutaecarpa Hook F and Thomas (Liu and Ho, 1999), Ocimum
gratissimum L. (Ogendo et al., 2008) and Artemisia vulgaris L. (Wang
et al., 2006).
Based on our results, adults of T. castaneum were found to be
repelled by essential oils tested even at very low concentration. L.
sempervirens oils show a similar repellent activity compared with
oils from D. winteri, but at concentration 310 times lower. We
believe the strong repellent activity of L. sempervirens oils that is
presented here could be due to safrole, the main constituent of
these oils (>50%). In previous research, this compound has shown
to be a powerful repellent towards cockroaches of Periplaneta americana L. (Ngoh et al., 1998) and also a feeding deterrent for adults of
the weevil Sitophilus zeamais Motsch (Huang et al., 1999). In addition, safrole is the most effective insect repellent among several
essential oil compounds tested, including -pinene (Ngoh et al.,
1998), that is one of the main constituents of the D. winteri oils
here tested. However, to clarify repellent activity of these pure
constituents against T. castaneum future studies should be done.
The essential oils assayed were not only active repellents
against T. castaneum, but they were also effective insecticides. Oils
extracted from both L. sempervirens and D. winteri when topically
applied on T. castaneum adults have shown medium lethal concentrations comparable to those reported previously for the Elletaria
cardamomum L. oils when applied by contact against S. zeamais and
T. castaneum (Huang et al., 2000). In this study, essential oils from L.
sempervirens were signicantly more active than oils from D. winteri
when applied topically against T. castaneum, however, the lethality
of the oils extracted from the same plant are equal, regardless of
whether they are derived from the leaves or bark.
The toxic effect of essential oils, apart from the variability of
phytochemical patterns, involves several other factors. The point
of entry of the toxin is one of them. Commonly, essential oils can
be inhaled, ingested or skin absorbed by insects (Regnault-Roger,
1997). Insect have been shown to be very sensitive for topical or
contact application of essential oils and their constituents; here
T. castaneum was not an exception (Huang et al., 1997, 2002;
Abdelgaleil et al., 2009). It is not surprising that strong activity was
observed in the oils extracted from the leaves and bark of L. sempervirens, when applied topically to T. castaneum, if we assume safrole
is the major compound involved. Safrole is also the main compound (>82%) present in the essential oil of Piper hispidinervum C.
DC., which showed high insecticidal activity when applied topically
on Spodoptera frugiperda Smith larvae (Lima et al., 2009), Tenebrio

409

molitor L. adults (Fazolin et al., 2007) and S. zeamais adults (Estrela

et al., 2006).
From the standpoint of pest control, one of the most valued
properties of essential oils is their fumigant activity against insects,
since it may involve their successful use to control pests in storage without having to apply the compound directly to the insects.
Based on the results from fumigant bioassays the four essential
oils tested showed high toxicity when they were applied against
T. castaneum with insecticidal activity dependent on oil concentration and exposure time. When insects were fumigated for 24 h with
essential oils from D. winteri, a concentration near to 10 l/l air oil
was necessary to cause 50% mortality (LC50 ), while a concentration
of 1.32 l/l air was enough to cause equal mortality when essential oils from L. sempervirens were used. Fast insecticidal activity by
the vapors of oils from L. sempervirens also became evident when
determining the medium lethal time values (LT50 ). This was conspicuous when insects were exposed at a concentration of 100 l/l
air of oil from L. sempervirens, where 50% of the population died
within approximately 4 h. However, when insects were exposed to
the same concentration of D. winteri oils, an exposure of 7 h was
necessary to kill the same proportion of the insect population.
Previously, the fumigant activity of essential oils from L. sempervirens has also been reported for the bean weevil, Acanthoscelides
obtectus Say. When it was applied at 8 l/l air no insects survived
after 96 h of exposure (Bittner et al., 2008). Similarly, safrole that
is the main compound of L. sempervirens oil, also showed high
insecticidal activity when applied by fumigation against S. zeamais and T. castaneum (Huang et al., 1999) and Sitophilus oryzae
L. adults (Kim and Park, 2008). Safrole has also shown strong fumigant toxicity against the cockroach P. americana (Ngoh et al., 1998),
Bemisia tabaci Gennadius and Frankliniella occidentalis Pergande
(Kostyukovsky et al., 2002).
This study demonstrated repellent and contact bioactivity as
well as an outstanding fumigant activity of four essential oils
extracted from two plants belonging to the Chilean native ora.
Among the oils assayed against T. castaneum, those which came
from L. sempervirens are the most promising, and could be considered for practical applications for stored-food pest control. But
before use, it is necessary to study fumigant effectiveness under
outdoor conditions, considering variables such as temperature and
relative humidity as well as their toxicity to humans and their persistence in the environment.
Acknowledgments
Authors thank G. Belmar for assistance in the oil extraction. N.
Zapata is grateful to Proyecto Fondecyt no. 11070060, and to EMCW
of the European Community Mobility Program for a postdoctoral
research scholarship in Ghent University.
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