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Insecticidal and repellent activities of Citrus reticulata, Citrus limon and Citrus aurantium essential oils on Callosobruchus maculatus

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In this research, fumigant toxicity of Citrus reticulata Blanco (Rutaceae), Citrus limon L. (Rutaceae) and Citrus aurantium L. (Rutaceae) peel essential oils was examined on adults of Callosobruchus maculatus (F.) at 27±1°C and 65±5% RH in darkness. The oils were extracted from the fruit peels using water steam distillation. A Y-tube olfactometer was used to examine the repellency of the essential oil. Adults (1-7 days old) of cowpea beetles were introduced individually into the introduction chambers. The LC 50 values (%95 fiducial limits) for C. reticulata, C. limon and C. aurantium were estimated to be 8.70 (8.30-9.15), 7.21 (6.79-7.71) and 6.33 (5.88-6.88) µl/l air, respectively. LC 50 values indicated that C. maculatus was significantly less sensitive to C. reticulata. Also C. reticulata was significantly more repellent to C. maculatus. The findings showed the strong insecticidal and repellent activity of Citrus peel essential oils and their potential role as fumigants for stored product insects.
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Integrated Protection of Stored Products
IOBC/wprs Bulletin Vol. 69, 2011
pp. 289-293
289
Insecticidal and repellent activities of Citrus reticulata, Citrus limon
and Citrus aurantium essential oils on Callosobruchus maculatus
Mahdieh Saeidi
1
, Saeid Moharramipour
1
*, Fatemeh Sefidkon
2
, Sirous Aghajanzadeh
3
1
Department of Entomology, Faculty of Agriculture, Tarbiat Modares University, P.O.Box
14115-336, Tehran, Iran;
2
Research Institute of Forests and Rangelands, P. O. Box 13185-
116, Tehran, Iran;
3
Citrus Institute, Ramsar, Iran
* Corresponding author e-mail: moharami@modares.ac.ir
Abstract: In this research, fumigant toxicity of Citrus reticulata Blanco (Rutaceae), Citrus limon L.
(Rutaceae) and Citrus aurantium L. (Rutaceae) peel essential oils was examined on adults of
Callosobruchus maculatus (F.) at 27±1°C and 65±5% RH in darkness. The oils were extracted from
the fruit peels using water steam distillation. A Y-tube olfactometer was used to examine the
repellency of the essential oil. Adults (1-7 days old) of cowpea beetles were introduced individually
into the introduction chambers. The LC
50
values (%95 fiducial limits) for C. reticulata, C. limon and
C. aurantium were estimated to be 8.70 (8.30-9.15), 7.21 (6.79-7.71) and 6.33 (5.88-6.88) µl/l air,
respectively. LC
50
values indicated that C. maculatus was significantly less sensitive to C. reticulata.
Also C. reticulata was significantly more repellent to C. maculatus. The findings showed the strong
insecticidal and repellent activity of Citrus peel essential oils and their potential role as fumigants for
stored product insects.
Key words: Citrus reticulata, Citrus limon, Citrus aurantium essential oil, Callosobruchus maculatus
Introduction
Infestation of grain by various storage-product pests may occur at various stages from time of
harvest to consumption by consumers. Synthetic organic chemicals have been used as
effective means of stored-product pest control for many years. Many compounds have been
and will be phased out because of their toxicity to humans, resistance problems in insects and
environmental concerns. Fumigation has played a significant role in controlling stored-
product insect pests. In many storage systems, methyl bromide and phosphine are widely used
as fumigants. EPA (2001) has proposed elimination of the production of methyl bromide,
because of its ozone depletion potential. Additionally, some stored-product insects are found
to have developed resistance to methyl bromide and phosphine (Champ and Dyte, 1977;
Subramanyam and Hagstrum, 1995). Because of the drawbacks in continued use of today’s
conventional fumigants effort is needed for development of new compounds to replace those
currently used. Essential oils are potential source of alternative compounds to currently used
fumigants. Essential oils have low toxicity to warm blooded animals, high volatility, and
toxicity to stored-grain insect pests (Shaaya et al., 1991; Regnault-Roger et al., 1995). These
problems have highlighted the need for the development of alternative products to
conventional insecticides such as natural extracts derived from plants. Many types of spices
and herbs are known to possess insecticidal activities (Tripathi et al., 1999) especially in the
form of essential oils (Shaaya et al., 1991). They do not leave residues toxic to the
environment and have medicinal properties for humans with lower toxicity to mammals
(Duke, 1985). Essential oils of citrus peels proved to reduce oviposition or larval emergence
through parental adult mortality (Don-Pedro, 1996; Elhag, 2000). More recently fumigant
290
activity of three citrus species including Citrus sinensis Osbeck and Citrus medica L. on
callosobruchus maculatus (F.) has been demonstrated by Moravej et al. (2008).The present
study was undertaken to investigate the potential effects of essential oils extracted from the
C. reticulata, C. limon and C. aurantium on C. maculatus. However, this is the first study
demonstrating that three essential oils have both insecticidal and repellency activities against
C. maculatus.
Material and methods
Insect rearing
Callosobruchus maculatus, was reared on mungbean pulses. The cultures were maintained in
the dark in a growth chamber set at 27±1°C and 65±5% RH. Adult insects, 1-7 days old were
used for fumigation toxicity tests. All experimental procedures were carried out as the same
environmental condition as the culture.
Plant materials
Fruits of C. reticulata (Klemantin cultivar), C. limon (Lisbone cultivar) and C. aurantifolia
were collected at the ripening stage from Ramsar, in December 2008. All the trees were
cultivated in the experimental fields of the Citrus Experimental Institute of Iran.
Extraction of essential oil
The essential oils were extracted from Fresh rind tissue (albedo and flavedo) of fruits by
water steam distillation using a Cleavenger-type apparatus, until there was no significant
increase in the volume of the oil collected. The essential oils were dried over anhydrous
sodium sulfate and were stored in glass tubes at 4°C in refrigerator, until they were used. The
oil of C. reticulata, C. limon and C. aurantium were yielded 1.71, 2.38 and 1.46% w/w on a
wet weight basis.
Bioassays with adults of C. maculatus
To assess 50 and 95% lethal concentration of adult insects, different dilutions were prepared
to evaluate mortality of insects after a preliminary dose setting experiment. Concentrations of
the C. reticulata, C. limon and C. aurantifolia oils were 6.45, 7.25, 8.06, 8.87, 9.67, 10.48µl/l
air, 4.43, 5.24, 6.04, 6.85, 7.66, 8.46, 9.27µl/l air and 7.14, 8.92, 10.71, 12.5, 14.28, 16.07,
17.85µl/l air respevtively. Control insects were kept under the same condition without any
essential oil. Each dose was replicated five times. The number of dead and live insects in each
bottle was counted 24h after initial exposure to the essential oil. The dead insects were
monitored for at least 48h after recording the data and no affected insects recoverd. Probit
analysis (Finney, 1971) was used to estimate LC
50
and LC
95
values with their fiducial limits
by SAS 6.12 (SAS institute, 1997). Calculated LC
50
values were considered significantly
different when their 95% fiducial limits did not overlap.
Repellency bioassay
Responses of T. confusum to the citrus essential oils were tested using a glass Y-tube
olfactometer. A Y-tube olfactometer that provide insects with one of two choices are
commonly used to measure unit olfactory responses. An introduction chamber with 5.5cm
length and 2cm diameter is located at the base of the Y-tube. Each arm of the Y-tube contains
a glass vial with 26.5cm length and an opening of 2cm in diameter which traps insects
moving into each chamber. Essential oil-treated food is placed in one chamber of the
291
olfactometer and the other (control) chamber contained a solvent-treated food (only acetone).
The end of glass vials were connected to two other glass vials, each containing charcoal
pellets. An air flow was first purified by passage through glass vials filled with charcoal
pellets and was then led into the vials containing essential oil-treated and acetone-treated as
control.
Test solutions were prepared by dissolving 1, 3, 5 and l of three essential oils in 50µ l
acetone. One microlitre of essential oil was applied on 2g of wheat, as uniformly as possible.
In the control, the food was treated with acetone only. Thirty unsexed adults (1-7 days old) of
flour beetles were introduced into the release base chamber individually. After 20 min, the
number of beetles in each of treated and control chamber was counted and the percentage
repellency (PR) values were computed using the formula of Liu et al. (2005):
100×
=T
EC
PR%
where C and E is the number of insects in control and treatment respectively; T is the total
number of responded insects.
Results and discussion
The results of probit analysis showed that
C. maculatus
adults were comparatively less
susceptible to
C. reticulata
(LC
50
=8.70µl/l air) oil than
C
.
aurantium
(LC
50
= 6.33µl/l air)
and
C
.
limon
(LC
50
= 7.21µl/l air) oils (Table 1). In the present study, the repellency of
C. reticulata
,
C. limon
and
C. aurantium
were evaluated against
C. maculatus
adults
.
The
essential oil of
C. reticulata
strongly repelled the
C. maculatus
in all concentrations.
Therefore, repellency of
C. reticulata
was significantly higher than
C. limon
and
C. aurantium
. The adult insects were exposed to the concentration of 1, 3, 5 and 7
µ
l/l air to
estimate repellent activities. Repellent values for
C. reticulata
was estimated to be 26.66,
33.33, 33.66 and 40% respectively.
Citrus reticulata
essential oil was significantly more
repellent to
C. maculatus
at 7
µ
l/l air (Table 2). The findings showed the strong repellent role
of
Citrus reticulata
oil. In general, the repellency increased with increasing concentration of
essential oils in all cases (Table 2).
In another study, three essential oils were tested for their fumigant toxicity and
repellency against adults of
C. maculatus
. The insecticidal activity varied with plant-derived
material and concentration. The chemical constituents of many plant essential oils are mainly
composed of monoterpenoids (Coats et al., 1991; Regnault-Roger and Humraoui, 1995; Ahn
et al., 1998). Monoterpenoid compounds have been considered as potential pest control agents
because they are acutely toxic to insects and possess repellent (Watanabe et al., 1993) and
antifeedant properties (Hough-Goldstein, 1990).
The LC
50
values of
C. reticulate
,
C. limon
and
C. aurantium
oils estimated in this study,
it could be concluded that these essential oils to be less toxic than
Artemisia sieberi
Besser oil
(Negahban et al., 2007). Our results did not show that fumigant toxicity is necessarily
correlated with the high repellency. As similar to our results in this study, Talukder and
Howse (1993) reported that in spite of high toxicity of pithraj (
Aphanamixis polystachya
Wall
and Parker) seeds against
Callosobruchus chinensis
(L.), it exhibited a weak repellency. Our
findings suggest that there may be different modes of action of the oil on insecticidal activity
and repellency.
Increased public concern over the residual toxicity of insecticides applied to stored grain,
the occurrence of resistant insect strains and the necessary precautions to work with
traditional insecticides calls for new approaches to control stored-product insect pests
292
(Yildirim et al., 2001). Our observations showed that fumigant activity of
C. reticulata
,
C. limon
and
C. aurantium
oils were characterized by hyperactivity, convulsion, paralysis and
fast knock down followed by death. Needless to say, that there is an urgent need for
environmentally safe alternatives to conventional fumigants, for the control of stored product
insects, namely phosphine and methyl bromide.
Table 1. Fumigant toxicity of
Citrus reticulata, Citrus limon and Citrus aurantium on
Callosobruchus maculatus
χ
2
df Slope ± SE LC
95
l/l air)
LC
50
l/l air)
Essential oil source
0.772 3 1.23 ±8.06 17.2-12.34 (13.92) 9.15-8.30 (8.70) C. reticulata
5.043 5 0.82±5.81 18.17-11.81 (13.84) 7.71-6.79 (7.21) C. limon
0.610 3 0.85 ±5.02 20.17-10.92 (13.45) 6.88-5.88 (6.33) C. aurantium
Table 2. Repellent effects of the essential oil from
Citrus reticulata, Citrus limon
and
Citrus
aurantium
against
Callosobruchus maculatus
P-value %
Repellency
χ
2
Number of insects Concentration
l)
Essential oil
C
1
T
1
NR
1
0.046 26.66 4.0
12 14 4 1 C. reticulata
0.025 33.33 5.0
15 10 5 3
0.028 33.66 4.841 18 5 7 5
0.011 40 6.54 17 8 5 7
0.491 10 0.47
11 11 8 1 C. limon
0.157 20 2.00
12 12 6 3
0.090 23.33 2.88
12 13 5 5
0.012 36.66 6.25
20 1 9 7
0.491 10 0.47
11 11 8 1 C. aurantium
0.275 16.66 1.19
13 9 8 3
0.020 30 5.40
12 15 3 5
0.012 36.66 6.25
20 1 9 7
1
NR: No response, T: Treatment, C: Control
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