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Research Article
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Fumigant Toxicity of Citrus Oils Against Cowpea Seed Beetle Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) |
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G. Moravvej
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S. Abbar
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ABSTRACT
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In the present study, the effects of volatile components of Citrus paradisi, C. aurantium, C. limonium and C. sinensis peel essential oils were investigated on the cowpea adult bruchid, Callosobruchus maculatus (F.). The oils were extracted from the fruit peels using hydrodistillation. The results indicated that the citrus oils had high fumigant activity against adult beetles. The mortality of 1-2 day-old adults increased with concentration and exposure time from 3 to 24 h. The oil of C. paradisi was more effective than those of C. aurantium and C. limonium (The LC50 values were 125, 145 and 235 &Amp;plusmn;1 L-1 at 24 h exposure, respectively). The oil of C. sinensis proved to be least toxic (LC50 = 269 &Amp;plusmn;1 L-1). The results suggested that citrus peel oils can be used as potential control measure against cowpea beetles.
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INTRODUCTION
The cowpea seed beetle, Callosobruchus maculatus (F.)
is a field-to-store pest of pulses and the level of field infestation
is a major factor that influences the bionomics of this bruchid in storage
(Olubayo and Port, 1997; Nahdy et al., 1998). According to Rees
(2004), loss of seed material is considerable-each adult Callosobruchus
emerging from a cowpea (Vigna unguiculata) would have consumed
about 25% of the seed from which it emerged. Damaged seed often does not
germinate or germinate well. Heavy infestations of bruchids can cause
heating of commodity which results in quality loss and mould growth. Currently,
only two fumigants, methyl bromide and phosphine, are widely used against
stored product insect pests (Perry et al., 1998). The use of methyl
bromide is being restricted because of its potential to damage the ozone
layer (Butler and Rodriguez, 1996; MBTOC, 1998). The future use of phosphine
could be threatened by the further development of resistant strains of
pests (Bell and Wilson, 1995; Daglish and Collins, 1999). Therefore, there
is an urgent need to develop safe alternatives to conventional insecticides
and fumigants to protect stored-products from insect infestations.
In the search for alternatives to conventional fumigants,
essential oils extracted from aromatic plants have been widely investigated
and their toxicity to stored-product insects has been of special interest
during the last decade (Shaaya et al., 1993; Ho et al.,
1997; Obeng-Ofori and Reichmuth, 1997; Obeng-Ofori et al., 1997;
Huang et al., 2002). The fruit peels of some Citrus species
have been reported to have insecticidal properties against insect pests
(Don-Pedro, 1985; Onu and Sulyman, 1997; Elhag, 2000). The major active
component of citrus oils is limonene. Insecticidal activity of limonene
has been successfully applied for the control of insect parasitoids of
pet animals. (+)-limonene was toxic to malation-resistant fleas (Collart
and Hink, 1986) and to all life stages of the cat flea, Ctenocephalides
felis (Hink and Fee, 1986). Weekly application of (+)-limonene reduced
flea and tick infestation by 80% in dog and cat, with no adverse effects
on blood composition or liver or kidney function (Tonelli, 1987). Limonene-treated
water had adverse effect on oviposition of female mosquitoes (Kassir et
al., 1989).
The toxicity of powdered sun-dried orange and grapefruit
peels to C. maculatus has been demonstrated (Don-Pedro, 1985).
In another studies, the essential oils of citrus peels proved to reduce
oviposition or larval emergence through parental adult mortality (Don-Pedro,
1996; Elhag, 2000). Studying on the toxicity of essential oils from various
plant species, Papachristos and Stamopoulos (2002) demonstrated the fumigant
activity of Citrus sinensis for males and females of the bruchid,
Acanthoscelides obtectus (Say).
The above studies were limited only to one or two Citrus
species and conducted mainly using powdered fruit peels. In the present
study, the activities of volatile fractions of essential oils extracted
from the fruit peels of four different Citrus species on the cowpea
adult bruchid, C. maculatus were investigated.
MATERIALS AND METHODS
Rearing of test insects: The study was undertaken in Entomology
laboratory of Dept. of Plant Protection, Ferdowsi University of Mashad,
Iran. A culture of non-flight form of the cowpea weevil, C. maculatus
was established on the seeds of unshelled brown cowpea, V. unguiculata
in wide-mouthed plastic jars under laboratory conditions (32±6°C,
45±5% RH and 12-12 h light-dark cycle). Adding new seeds and removing
very old ones to and from the main culture monthly prevented crowding
of the bruchids and so no flight form was developed. One to two days old
adults were used for all bioassays.
Extraction of essential oil: The citrus fruits were collected
from the central local market of Mashad, Iran during November and December
2006. The essential oils were extracted from fresh rind tissue (albedo
and flavedo) of fruits by water steam distillation using a Clevenger apparatus.
The Citrus species were C. sinensis Osbeck., C. aurantium
Risso., C. limonium Risso. and C. paradisi Macf. (Rutaceae).
About 1.5 mL oil was extracted per 100 g fresh peel. Extracted oils were
stored at 5°C until the onset of bioassays.
Bioassays: The bioassays were conducted following the preparation
of essential oils as described by Rahman and Schmidt (1999) and Keita
et al. (2001) with slight modifications. The 2 cm diameter pieces
of filter paper (Whatman No. 1) were impregnated with six different oil
concentrations (including 0 μl L-1 as control) to give
the equivalent concentrations in air of 148-555 μl L-1
for C. limonium and C. sinensis, 74-370 μl L-1
for C. aurantium and 74-444 μl L-1 for C. paradisi.
The range of concentrations had been chosen on the basis of a number of
preliminary trials. The filter paper was attached to the undersurface
of the screw cap of a glass vial (volume 27 mL). The cap was screwed tightly
onto the vial containing 5 pairs of one-to 2 day-old C. maculatus
adults. Six replicates of each control and treatment were set up. Mortality
was recorded after 3, 6, 9, 12 and 24 h from the commencement of exposure.
When no leg or antennal movements was observed, insects were considered
dead.
Data analysis: Mortality data of adults at 24 h exposure time
for each plant species were analysed with the probit model (Finney, 1971)
using a Maximum Likelihood Program (POLO-PC, LeOra Software, Berkeley,
California). The program POLO-PC calculates a theoretical natural response
for each experiment, based on the pattern of mortality at all concentration
levels, including controls. The results include estimate of the LC50
(and other LCs if required) and the 95% confidence limits, slope and intercept
of probit mortality regression and the relevant statistical tests (such
as t-ratio, g factor and heterogeneity). For comparison of the probit
mortality lines of treatments, the program also provides the likelihood
ratio tests of equality and parallelism. The data obtained from repeated
experiments were pooled for probit analysis, after acceptance of the hypothesis
of equality following likelihood ratio tests (Russel et al., 1977).
Estimated median lethal concentration to kill 50% of aphids was expressed
as LC50 (μL oil per unit air liter). The resistance ratio
and 95% confidence limits of this ratio were determined between adults
from different treatments and comparisons were made based on the procedure
described by Robertson and Preisler (1992). The estimates of parameters
needed for computing confidence limits of the resistance ratio were provided
by individual probit analysis in the POLO-PC output.
RESULTS
The essential oil vapors of the peel of four Citrus species showed
variable toxicity to adults of C. maculatus, depending on concentration
and exposure period (Table 1). There was no mortality
in the presence of 148 μl L-1 of C. sinensis oil
at 3 and 6 h exposure. At 3 h exposure, the maximum rates of mortality,
24, 29 and 40%, were achieved in the presence of 400, 370 and 444 μl
L-1 of C. limonium, C. aurantium and C. paradisi
oils, respectively. At 12 h exposure, the minimum rates of mortality of
5 and 25% in adults were attained by the presence of 148 μl L-1
of C. sinensis and C. limonium oils, respectively. The essential
oils of C. paradisi and C. aurantium achieved the mortalities
lower than 16% in the presence of 74 μl L-1 for 12 h exposure.
The essential oils of C. sinensis and C. limonium achieved
respectively the mortalities of 96 and 91% against the adults, at the
highest concentration and exposure period investigated (556 μl L-1
air, 24 h). The essential oils of C. paradisi and C. aurantium
were more active and achieved, respectively 96 and 84% mortalities after
24 h exposure at much lower concentrations (444 and 370 μl L-1
air, respectively). In all bioassays, the control mortality did not exceed
1.7% at 3 h and 3.3% at 24 h exposure. These control mortalities were
quite below the recommendation (30%) made by the FAO (Anonymous, 1970).
Mortality data were adjusted for natural mortality in
controls according to Abbott (1925) formula and within each oil concentration,
were plotted against exposure periods. The results showed that there were
positive and linear significant relationships between percent mortality
of adults and duration of exposure to the
Table 1: |
Mean percent (±SE)
mortality in the adults of C. maculatus exposed for various
periods to four citrus essential oils at different concentrations
(n = 6)A |
 |
A:
Oils were applied to 2 cm filter papers held in 27 mL vials |
Table 2: |
Analysis results
of linear regressions of C. maculatus mortality data on
exposure periods A in various concentrations of four
citrus essential oilsB |
 |
A:
Mortality data at each exposure period was a mean of six replicates,
B: Oil applied to 2 cm filter papers held in 27 mL
vials. Exposure periods were 3, 6, 9, 12 and 24 h |
essential oil vapors within all concentration levels and plant species
(p<0.05), although not significantly within concentrations of 285 μl
L-1 of C. limonium (p = 0.054); 111 and 167 μL
L-1 of C. aurantium (p = 0.064 and 0.053, respectively)
and 444 μl L-1 of C. paradisi (p = 0.071). Coefficients
of determination (R2) indicated that between 62-95% of the
variation in the rates of adult mortality was explained by duration of
exposure to essential oils. Within each essential oil, the slopes of regressions
of mortality rates on exposure times were smaller in low concentrations
than those in high concentrations (Table 2).
Table 3: |
Fumigant toxicity of four citrus essential
oil vapors to C. maculatus adults A |
 |
A:
Oil applied to 2 cm filter papers held in 27 mL vials. Exposure
period was 24 h, B: n = Total number of one-to 2 day
old adult insects tested (including control), C: CL
= Confidence Limits |
However, as different concentrations were applied for the four citrus
oils, more appropriate comparisons could be obtained using probit analyses
of data. The dose-mortality responses of C. maculatus adults to
citrus essential oils were compared in terms of differences in slope and/or
intercept of probit regressions and LC50s and LC90s.
The slope values in fumigant toxicity were in the range of 2.88-5.61.
The heterogeneity factors less than 1.0 for C. sinensis and C.
paradisi oils indicated that the results were found to be within the
95% confidence limits, so no correction factor (g) was required. The heterogeneity
factors more than 1.0 for C. limonium (1.35) and C. aurantium
(2.88) required to use g factor for correcting of the respective LC50s
values. The regression test (t ratio) was greater than 1.96 for all essential
oils and the potency estimation test (g factor) was less than 0.5 at all
probability levels for all essential oils, except at 99% level for C.
limonium (0.65) and C. aurantium (1.08) (Table
3).
The slopes of the four probit morality regressions for the essential
oils were significantly different, as revealed by rejecting the likelihood
ratio test of parallelism (χ2 = 24.42, df = 3, p<0.001),
suggesting that at least the slope of one of the probit mortality regressions
would be significantly different from the others. Further likelihood ratio
tests between the paired combinations concerned revealed that the slope
of the probit mortality line for the oil of C. limonium was not
significantly different from those of C. aurantium (χ2
= 0.496, df = 1, p = 0.481) and C. paradisi (χ2
= 0.025, df = 1, p = 0.876). Similar result was obtained when comparing
the slopes of the probit mortality lines between the two latter oils (χ2
= 0.334, df = 1, p = 0.563). In contrast, the slope of the probit mortality
line for the oil of C. sinensis was significantly greater than
those of C. limonium (χ2 = 14.44, df = 1, p<
0.001), C. aurantium (χ2 = 22.30, df = 1, p<0.001)
and C. paradisi (χ2 = 16.62, df = 1, p<0.001)
(Table 3).
The intercepts of probit morality regressions for the
four essential oils were significantly different, as revealed by rejecting
the likelihood ratio test of equality (χ2 = 135.97, df
= 6, p<0.001), suggesting that at least the
Table 4: |
LC90s
ratios and their 95% confidence limitsA calculated
for comparing fumigant toxicity of citrus essential oils to the
adults of C. maculatusB |
 |
A:
The lower and upper 95% CL were calculated according to the procedure
described by Robertson and Preisler (1992), B: Italic,
bold and bracket-enclosed values show LC90 (μl
L-1 air), the ratio of LC90s concerned and
its 95% confidence limits, respectively, *: Significant difference
between crossed LC90s at 5%; ns: Non-significant |
Table 5: |
LC50s
ratios and their 95% confidence limitsA calculated
for comparing fumigant toxicity of citrus essential oils to the
adults of C. maculatusB |
 |
A:
Lower and upper 95% CL were calculated according to the procedure
described by Robertson and Preisler (1992), B: Italic,
bold and bracket-enclosed values show LC50 (μl
L-1 air), the ratio of LC50s concerned and
its 95% confidence limits, respectively, *: Significant difference
between crossed LC50s at 5%; ns: Non-significant |
intercept of one of the probit mortality regressions would be significantly
different from the others. Further likelihood ratio tests of equality
indicated that the intercepts between all possible paired combinations
significantly differed from each other (p<0.001), except those of C.
aurantium and C. paradisi (χ2 = 4.06, df =
2, p = 0.131) (Table 3).
The above differences in parameters of the probit mortality regressions
between experimental treatments were reflected in the LC90
or LC50 estimates (Table 3-5).
Based on the LC90 values, the essential oil of C. limonium
showed the least and that of C. paradisi the highest fumigant toxicity
to the adults of C. maculatus. However, comparisons of LC90s
among the four essential oils using their ratios and 95% confidence limits
indicated that the toxicity of C. sinensis oil did not significantly
differ from the toxicity of C. aurantium oil. Also the toxicity
of the latter was not significantly different from that of C. paradisi
oil Comparison between all other paired oils showed significant differences
in their toxicity to C. maculatus adults (Table 4).
In contrast, based on the LC50 values, the essential oil of
C. sinensis showed the least fumigant toxicity to the adults of
C. maculatus, although not significantly different from the oil
of C. limonium. Moreover, the fumigant toxicity of C. sinensis
oil was significantly lower than that of C. aurantium oil. The
results of comparison between the other paired combinations of essential
oils were similar to those obtained by LC90 values (Table
5).
DISCUSSION
Over 120 plants and plant products have been shown to
have insecticidal or deterrent activity against stored product pests (Dale,
1996). Currently many farmers in parts of Africa and Asia use some of
these botanicals to protect their legumes from attack by bruchids, with
varying degrees of success (Don-Pedro, 1990; Singh, 1990; Dharmasena,
1995; Dharmasena et al., 1998). However, the number and quality
of plants used by farmers is often limited by their availability (Dharmasena,
1995).
Rutaceae is a large family containing 130 genera in seven
subfamilies, with many important fruits and essential oil products. Lemon
essential oil has the highest value of all essential oils imported to
the USA and is widely used as flavoring agent in bakery, as fragrance
in perfumery and also for pharmaceutical applications (Weiss, 1997). The
essential oils extracted from Citrus genus contain a high percentage
of monoterpenes. Its major component is limonene. Analysis of the toxicity
data in the present study showed that the essential oil vapors from citrus
peels exhibited a variable toxic action against the adult stage of C.
maculatus, depending on plant species, concentration and exposure
period. Studying on thirteen essential oils related to seven plant families,
Papachristos and Stamopoulos (2002) observed varying toxicities among
different plant species to the bruchid, A. obtectus. A similar
positive relationship between the rate of mortality and exposure time
obtained in our study has also been demonstrated in bioassays of many
insects with various toxicants (Robertson and Preisler, 1992; Perry
et al., 1998; Tunc et al., 2000; Sanon et al., 2002).
The essential oil vapor of C. paradisi exhibited the most toxicity
to the cowpea adult beetles as demonstrated by the lowest of both LC50
and LC90 estimates at 24 h exposure. Based on the LC50
values, the fumigant toxicity of the essential oils in descending order
was C. paradisi, C. aurantium, C. limonium and C.
sinensis. Similar results were obtained based on the LC90
values, except for the last two oils in which C. sinensis oil was
significantly more toxic than C. limonium oil.
Studying on the effects of essential oils from various
plant species on the bruchid, A. obtectus, Papachristos and Stamopoulos
(2002) reported that fumigant LC50s values of C. sinensis
at 24 h exposure for males and females were 11.4 and 19.5 μl L-1
air, respectively. These values were much lower than the LC50
value 269 μl L-1 of the same plant source obtained in
the present study for C. maculatus adults. The observed difference
between our results and those of Papachristos and Stamopoulos (2002) seems
to be reasonable because of different species and size of insects and/or
methodology of oil extraction concerned, as shown in similar experiments
with various stored product pests and essential oil vapors (Huang et
al., 2000; Sanon et al., 2002). Don-Pedro (1996) attributed
the mortality of C. maculatus adults on citrus peel-treated grains
to the fumigant activity of the vapor released by peels. He indicated
that grains treated by 7 mL oil kg-1 seed caused 100% mortality
1 h after application. LD50 values of orange and grapefruit
peels admixed with cowpea grains on adult C. maculatus were 4 and
5.62 g peel per 100 g cowpea, respectively. The LC50 values
of C. maculatus adults for citrus essential oil vapors (125-269
μl L-1) in the present study were much lower than the
value reported by Sanon et al. (2002) for the oil
of Bosica senegalensis Lam. (Capparaceae) (480 μg L-1).
The slope value of probit mortality regression in descending
order was C. sinensis, C. limonium, C. paradisi
and C. aurantium (Table 3). However, the likelihood ratio tests
showed that only was the slope for the essential oil of C. sinensis
significantly greater than others and that the slopes for the last three
essential oils were similar. A steep slope value indicates that there
is a large increase in the mortality of insects with relatively small
increase in the concentration of toxicant (Robertson and Preisler, 1992;
Perry et al., 1998; Tiwari and Singh, 2004) that was true for the
fumigant toxicity of C. sinensis essential oil within the experimental
conditions of present study. The lower toxicities (the higher LC50s)
of C. sinensis and C. limonium compared to two other species
were due to their range of higher concentrations needed for calculations
of probit regressions. However, the steep slope of probit mortality regression
of C. sinensis was reflected in its higher toxicity to adults when
comparison was made in terms of LC90 values, being significantly
lower in C. sinensis (454 μl L-1 air) than in C.
limonium (589 μl L-1 air) (Table 4). When the value
of t-ratio is greater than 1.96, the regression is significant.
Values of heterogeneity factor less than 1.0 denote that
in the replicate tests of random samples, the concentration response line
would fall within 95% confidence limits and thus the model fits the data
adequately, the case that was not true for C. limonium and C.
aurantium oils (Table 3). The index of significance of potency estimation
g indicates that the value of the LC50 or LC90 was
within the limits at 95% probability level (Table 3) as it is less than
0.5 (Robertson and Preisler, 1992; Tiwari and Singh, 2004).
According to the report of Regnault-Roger (1997), the
essential oils represent a market estimated at $700 million and a total
world production of 45000 t. Nearly 90% of this production is focused
on 15 products, particularly mints (Mentha piperita, M. arvensis
and M. spicata) and citrus (orange, lemon and lime). The advantages
of using citrus peel oil as a grain protectant are: It can be easily extracted
from peels by water steam distillation, it may have very low toxicity
to mammals since citrus oil is one of the popular flavorings or fragrances
(as food ingredients) and consumed by people in various parts of the world,
as the essential oil is volatile, it can potentially be used as a fumigant
and it is cost-effective and its application is easy. However, as the
essential oils are intended to be used like fumigants to disinfest commodities,
they should have the ability to kill all stages of insects. Moreover,
aromatic plants contain, in general, essential oils at concentrations
of 1-3% (Cakir, 1992) so large quantities of plant material have to be
processed in order to obtain essential oils in quantities sufficient for
commercial scale tests. The observed fumigant activity in the present
study shows that essential oils are sources of biologically active vapor
that are potentially efficient insecticides. Consequently, the possibility
of employing these natural fumigants to control insects in stored products
can be worthy of further investigations.
ACKNOWLEDGMENTS
Thanks are due to the Faculty of Agriculture for assistance
to conduct these experiments as part of M.Sc. studies of S. Abbar. We
thank M. Azizi-Arani and S. Hatefi for their technical support and the
anonymous reviewers for their constructive comments on the manuscript.
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