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Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2899
Bio-insecticidal effects of plant extracts and oil emulsions of
Ricinus communis L. (Malpighiales: Euphorbiaceae) on the
diamondback, Plutella xylostella L. (Lepidoptera: Plutellidae)
under laboratory and semi-field conditions
Tounou Agbéko Kodjo*, Mawussi Gbénonchi, Amadou Sadate, Agboka Komi, Gumedzoe Yaovi
Mawuena Dieudonné and Sanda Komla
Ecole Supérieure d’Agronomie (ESA), Université de Lomé (UL), BP 1515 Lomé-Togo;
*Author for correspondence, Email: ktounou@gmail.com
Original Submitted In 30
th
May 2011. Published online at www.biosciences.elewa.org on July 11, 2011.
ABSTRACT
Objective: The objective of this study is to evaluate the potential of using Ricinus communis leaf, root, seed
kernel crude extracts and oil emulsion to control the diamondback moth, Plutella xylostella (L.)
(Lepidoptera: Plutellidae).
Methodology and results: The effect of different treatments of R. Communis plant extract (20%) and oil
emulsion (5% and 10%) and their persistence (0, 3 and 5 days after application; DAA) on mortality and
oviposition behaviour of P. xylostella were tested in laboratory and field cage experiments. In general, R.
communis products have strong larvicidal effect on P. xylostella, with 100% mortality recorded on 3
rd
instar
larvae treated with 10% oil emulsion in both ingestion and contact toxicity tests. Aqueous extracts were
significantly less toxic with the highest mortality rates (67.49 ± 1.98% and 70.86 ± 0.85%) recorded with
seed kernel extract and the lowest with the root extract (53.98 ± 1.21% and 54.87 ± 1.88%), in topical
toxicity and ingestion toxicity experiments, respectively. The adult emergence was significantly affected
with the lowest emergence rate recorded in 5% oil emulsion, 57.72 ± 72% and 49.98 ± 0.98% in topical
toxicity and ingestion toxicity tests, respectively. No significant different was noted between Dursban and
aqueous extract treatments. Among emerged adults from larvae treated with oil and aqueous extracts, a
44–79% abnormal development as wings and legs deformation were observed. The sex ratio was skewed
in favour of males among F1 progeny from Durban and R. communis treated insects and in favour of
females in controls. In field-cage experiments, treated plants had strong larvicidal and oviposition deterrent
index on P. xylostella. The oviposition deterrence index was highest with castor bean oil at a concentration
of 10%. Diamondback females clearly discriminated between plants sprayed with R. communis products
and those with water. Treating diamondback-infested cabbage plants with plant extracts and oil emulsions
resulted in more than 59% mortality 7 DAA. Experiments on the residual effects revealed a significant
decrease in larval mortality with time between the botanical application and insect release.
Conclusion and application of findings: In view of the low oviposition rates, oviposition deterrent, immature
mortality, and the relatively low persistence of the toxic ricin oil, it can be expected that the use of R.
communis product be suitable for P. xylostella population density reduction in the field.
Key words: botanicals, Ricinus communis, integrated pest management, Oviposition, Plutella xylostella.
Journal of Applied Biosciences 43: 2899 – 2914
ISSN 1997–5902
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2900
INTRODUCTION
The diamondback moth, Plutella xylostella (L.)
(Lepidoptera: Plutellidae), is regarded as the most
destructive insect pest of Brassicaceae crops that
requires US$1.0 billion globally in estimated
annual management costs (Talekar and Shelton
1993) in addition to the crop losses. The larvae are
voracious defoliators with a potential to destroy the
entire crop if left uncontrolled (Verkerk & Wright,
1996). Currently, available control option against
P. xylostella relies on the application of chemical
insecticides (pyrethrenoids and
organophosphates) on the basis of spray schedule
(Castelo Branco & Gatehouse, 2001). However,
synthetic pesticides, while valued for effectiveness
and convenience control of P. xylostella, pose
certain problems, including phytotoxicity and
toxicity to non-target organisms, environmental
degradation and health hazards to farmers and
insect resistance. Plutella xylostella is known to
develop multiple- and cross-resistance to nearly all
groups of pesticides applied in the field including
new chemistries such as spinosyns, avermectins,
neonicotinoids, pyrazoles and oxadiazines (Sarfraz
& Keddie 2005; Sarfraz et al. 2005).
So far attempts for biological control of P.
xylostella as alternative to chemicals have mainly
concentrated on the use of parasitoids, predators
and pathogens. Parasitoids are the key group of
natural enemies of DBM and over 130 species
including hymenopteran, have been widely
reported to attack various life stages of DBM (for
more detail see Sarfraz et al., 2005). In periurban
areas of Benin, Anomma nigricans (Illiger)
(Hymenoptera: Formicidae) have been reported as
an important regulator of P. xylostella (Goudegnon
et al. 2004). Moreover, several pathogens
including fungi (Furlong, 2004), bacteria (Braun et
al., 2004) and viruses (Cherry et al., 2004).
Although increased efforts are made worldwide to
develop integrated pest management programs,
principally based on manipulation of natural
enemies to control agricultural pests, these are
often not affordable to African peasant farmers.
Therefore, the development of alternative control
methods for P. xylostella based on botanical
pesticides, which are locally available, is of
paramount importance to sustain the successful
use of biological control against DBM in Africa.
This group of pesticides is easily biodegradable
and their use in crop protection is a sustainable
alternative to synthetics (Immaraju, 1998; Juan &
Sans, 2000; Carpinella et al., 2002; Roy et al.,
2005; Isman, 2006; Asogwa et al., 2010).
Botanicals have been successful against a number
of agricultural pests in Africa (e.g., Barbouche et
al., 2001; Kétho et al., 2002; Lee et al., 2002;
Bruce et al., 2004; Sanda et al., 2006; Ogendo et
al., 2008, Agboka et al., 2009). In Togo, Dreyer
(1987) demonstrated good efficacy of crude extract
of neem seed against the larva of P. xylostella.
However, the difficulty in collecting neem seed and
the unavailability of locally made and ready-to-use
botanical based pesticides did not allow the
practical application of these alternatives. Apart
from neem products, other extracts derived from
indigenous plants including Cymbopogon
schoenanthus L., Ocimum basilicum Basil
(Labiae), Hyptis suaveolens L. (Lamiaceae) and
the fish bean Tephrosia vogelii Hook F.
(Leguminosae) recently gained attention with
regard to their insect pest control potential (Kétho
et al., 2002; Sanda et al., 2006; Agboka et al.,
2009). In Togo only few studies have been
conducted on insecticidal activities of plant extracts
although many plants are known to possess
potential for insect pest management. Kétho et al.
(2002) have demonstrated the insecticidal activity
of the C. schoenanthus against Callosobruchus
maculatus L. (Coleoptera: Pteromalidae). Sanda et
al. (2006), reported high toxicity of Cymbopogon
schoenanthus L. in controlling P. xylostella under
field conditions. Other botanical insecticide include
Ocimum basilicum Basil (Labiae) and the physic
nut Jatropha curcas L. (Euphorbiaceae). In order
to offer more choice in pesticide plants to the user,
there is need to assess other indigenous botanical
pesticide, especially those with a presumably
better potential of extension. Such plants, in
addition to their inherent pesticidal effectiveness
must be rustic, perennial and easily cultivated. One
of these candidates may be the castor bean plant,
Ricinus communis L. (Malpighiales:
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2901
Euphorbiaceae), a wild growing plant in all
ecological areas in Togo and other parts of the
world (Weiss, 2000). Castor bean plant contains
ricin toxin, one of the most toxic and easily
produced plant toxins worldwide (Thomas et al.,
1980; Bojean, 1991; Ogunniyi, 2006). However, to
the best of our knowledge, very few studies have
been conducted to investigate the pesticidal
activity of R. communis. The very limited data on
toxicity in target insects comprise mainly
information on aqueous extract of castor bean
products rather than on its oil. Aunty et al. (2006)
demonstrated high larvicidal activity of aqueous
extracts from leaves of R. communis against four
mosquito species, Culex pipiens (L.), Aedes
caspius (Pallas), Culiseta longiareolata (Aitken)
and Anopheles maculipennis (Meigen). No data on
the effects of R. communis on agricultural pests is
available. Therefore, the present work aimed at
assessing the efficacy of aqueous extracts and oil
emulsion of castor bean plant in controlling P.
xylostella.
MATERIALS AND METHODS
Plant materials: The cabbage variety KK CROSS, of
Japanese origin, widespread in Togo, was purchased
from the local suppliers and used in all experiments.
Different parts of castor bean plant (leaf, root and seed
kernel) used were harvested from plants growing on the
area of the Université de Lomé campus.
Insect culture : A laboratory culture of P. xylostella
originating from specimens collected on field-grown
cabbage plants at the market gardening site near the
“Port Autonome de Lomé”, was established at the
“Station d’Expérimentations Agronomiques de l’Ecole
Supérieure d’Agronomie de l’Université de Lomé”
(06°17’N, 001°21’E) following the protocol developed
by Ibrahimi et al. (2009). The insects were continuously
reared in cages for three generations on cabbage
variety KK CROSS plant. Cabbage plants at eight to
ten-leaf stage were placed inside oviposition cages
containing 8-10 pairs of DBM. The oviposition cage
consisted of transparent cubic Plexiglas's container (30
× 30 × 30 cm), with a fine nylon mesh installed on the
top-side. A small cotton-wool wick soaked in 10%
honey solution was placed in each oviposition cage as
a source of carbohydrate for adults. In other to
synchronize insect rearing, adult DBM were kipped on
the plants for 12 hr. Early 3
rd
instar larvae were used in
all experiments; early 2
nd
instars larvae were found to
be too fragile mostly dying during handling.
Preparation of aqueous extracts of castor bean
leafs, root and seed kernel: Extraction of castor bean
leaf, root and seed kernel was carried out as simple as
possible for easy adoption by the peasants growers.
Hence, crude aqueous extracts of each plant organ at
20% were prepared by soaking 200g pounded fresh-
plant material, respectively, in 1l of tape water, and left
for 12 hr and then filtered through muslin cloth. A
solution of emulsifier was added to each preparation
before both laboratory and field trials.
Extraction and formulation of castor bean seed
kernel oil: Ripe castor bean seed were sun-dried
before oven drying at 40°C for 72 hr. Grains obtained
after manual decortications, were ground using a pestle
and mortar. The toxic glucoprotein, ricin, is known to
remain in the seed cake and is not transferred to the oil
fraction when extracted by cold pressing of the seeds
(Cosmetic Ingredient Review Expert Panel, 2007).
Therefore, oil extraction was done using ethanol as
solvent. Castor bean seed powder obtained was
moistened and the paste is modelled in sticks of 150g
each, and oil extracted with 90% ethanol in a soxhlet
extractor. The excess solvent was removed under
reduced pressure; and the oil was placed in desiccators
to remove any remaining water and stored at 4 ± 2°C.
The amount of oil obtained from 150 g paste of castor
bean seed was 26.75 ml weighing 24.6 g. Castor bean
oil was thereafter formulated with a water and
emulsifier solution (soap without any detergent) to
prepare the different concentrations of oil emulsion for
both laboratory and field-cage trials.
Laboratory tests
Topical and ingestion toxicity of castor bean plant
extracts and oil emulsion to third instar Plutella
xylostella larvae: In topical toxicity experiment, 3
rd
instar larvae were topically treated with castor bean
leaf, root and seed kernel aqueous extracts at a
concentration of 20%, with oil emulsions at
concentrations of 5 and 10% or with the synthetic
pesticide Dursban (5% active ingredient, prepared by
diluting 5g of product in 10ml tape water), widely used
insecticide for diamondback control in the experimental
zone, by applying the solution on the anterior pronotum
of the larvae using a micro-litter syringe. Hundred and
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2902
five (105) 3
rd
instar P. xylostella larvae were inoculated
per treatment in group of 15 individuals. A further 105
3
rd
instar larvae were topically treated with emulsified
water, 1% ethanol and untreated check, referred to as
control 1, control 2 and control 3, respectively. Insects
were thereafter transferred to clean plastic Petri dishes
(150 x 20 mm
2
) containing untreated 9 cm diameter
cabbage leaf disc and incubated in seven groups of 15
for 7 days at 25 ± 1°C, 75 ± 5 % r.h. .
In ingestion toxicity test, discs (9 cm in diameter) cut
from cabbage leaves were immersed individually in the
different concentrations of oil or aqueous extracts for
one minute. The discs were left to dry at ambient
temperature for 5 min, and transferred to clean plastic
Petri dishes (150 x 20 mm
2
). Test insects were
inoculated in group of 15 individuals by feeding on
treated leaf disc for 24 h. The experiment set up is
similar as in topical toxicity test and larvae fed with
cabbage leaf discs treated with emulsified water, 1%
ethanol in water and untreated check served as
controls.
In both topical application and ingestion tests, untreated
leaf discs were used to feed the insects and changed
every 24 hours. Following this methodology, P.
xylostella larvae could be kept on fresh leaves
throughout the whole experimental period of seven
days. The experiment was repeated three times and
larval mortality was assessed at 24-h intervals. To
assure independence of values, each group of 15
insects per treatment was assessed only once, thus
group 1 was assessed at 24 h only, group 2 at 48 h and
so on to group 7 at 7 days. Insects that survived and
developed to pupae were followed until adult
emergence and percent emergence, moth deformities,
sex-ratio of adult moths and longevity were determined
for each treatment.
Field-cage experiments
Experiment 1: Direct treatment of diamondback
moth infested cabbage plant in cage experiments
The experiment was conducted to evaluate the efficacy
of castor bean plant extracts and oil in controlling P.
xylostella under semi-field conditions. Experiments
were carried out at the experimental station of the
“Station d’Expérimentations Agronomiques de l’Ecole
Supérieure d’Agronomie de l’Université de Lomé”
located in the southern guinea Savannah region of
Togo. The tests were carried out in the field in wooden
insect cages (50 x 50 x 90 cm) with four mesh sides
(the two lateral and the front and the top side), and the
remaining two sides made of wood. Five plastic pots
(11 x 7.5 x 8.5 cm), with one cabbage plant at eight-leaf
stage, were used per cage. Inside each cage, 60 to 90
P. xylostella larvae were released onto plants and the
cages were maintained at ambient conditions.
Treatments consisted of applying per plant 10 ml of
aqueous extract of R. communis root, leaf, and seed
kernel at a concentration of 20%, oil emulsion of R.
communis at concentrations of 5 and 10% and of the
synthetic pesticide Dursban, twenty-four hours after
releasing the insects. Plants were sprayed with a hand
sprayer covering both sides of the leaves. Emulsified
water, 1% ethanol in water and untreated check served
as control. Treatments were replicated three times and
mortality was scored daily for seven days. Insects that
died immediately after spraying were not included in the
analyses.
Experiment 2: Residual activity of the botanical
treatments
The residual activity of the R. Communis extracts and
oil on DMB larvae was evaluated over time. Healthy
cabbage plants in the field at eight-leaf stage were
sprayed with castor bean aqueous extracts, oil
emulsion, Dursban, at their respective concentrations
and control solutions. Treated plants, all sprayed at the
same time, were transferred to the insect cages
immediately (0 h) or three (72 h) and five days
thereafter. Inside each cage, 60 to 90 larvae were
released onto plants and held at ambient conditions.
Each treatment consisting of one cage with 5 cabbage
plants was replicated three times. The 3 levels of
control as mentioned above were considered and
insects were monitored daily for 7 days and mortality
was recorded.
Experiment 3: Effect of different treatments on the
oviposition behaviour of P. xylostella.
No-choice experiment: In no-choice experiment,
cabbage plants, produced singly in pots, were treated
at ten-leaf stage as described in the previous sections
with oil emulsion at 10%, aqueous extracts at 20% and
Dursban 5%. Once treated, plants were transferred
immediately to oviposition cages. Each treatment
consisted of three cages with one potted cabbage plant
each, and control 1, control 2 and control 3 as
described above were used for comparison.
Approximately 1 h after spraying the plants, one 1-day
old adult P. xylostella pair was released inside the cage
for oviposition.
Choice experiment: The choice experiment was
conducted to determine if females DBM could
successfully discriminate between plants treated with
R. communis products and untreated ones. In the no-
choice tests, the DBM did not discriminate between
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2903
untreated plants and plants treated with emulsifier
water and with 1% ethanol (for details see Results).
Hence females P. xylostella were given the choice
between cabbage plants treated with R. communis
aqueous extracts, oils emulsion or Dursban at the same
concentration as in no-choice experiment and untreated
control. Each combination (treated vs. untreated) was
repeated three times. To reduce bias between treated
and untreated plants inside each cage, the pots were
placed in the diagonally opposed angles of each cage.
Two newly emerged pairs of P. xylostella were released
in the centre of each cage and provided with a 10%
sucrose solution as food source.
In both no-choice and choice experiments, plants were
gently shaken four days after releasing the moths, to
remove surviving moths and eggs deposited on the
cabbage were tagged and counted under a binocular
microscope. The number of eggs laid per plant as well
as oviposition deterrence index (ODI) (choice
experiment) for the aqueous extracts, oils and Dursban
was calculated using the formula: [(C - T)/(C + T)] * 100
(Akhtar and Isman, 2003), with C being the number of
eggs laid on the untreated control plants and T the
number of eggs laid on the treated plants.
Statistical analysis: Median survival times (MST) were
calculated using SPSS (SPSS, 1999). The efficacy of
the different treatments was compared using the final
mortalities (i.e. final cumulative mortalities). Differences
in mortality rates, rate of pupation, adult emergence,
sex ratio, adult deformities and adult longevity, eggs
per plant and oviposition deterrent index were analyzed
by analysis of variance (ANOVA) and means were
separated using Student-Newman-Keuls test (PROC
MIXED procedure, SAS institute, 1997) and the
probability level was set at α = 0.05. Percentage data
and ratios were arcsine-transformed before analysis.
RESULTS
Laboratory tests
Topical and ingestion toxicity of castor bean plant extracts and oil emulsion to third instar larvae: In both
topical and ingestion tests, aqueous extracts and oil
emulsion of castor bean plant caused significantly
higher P. xylostella mortalities than the control (P <
0.0001, Tables 1 and 2). Oil emulsion was the most
active against P. xylostella larvae followed by the
Dursban, both being significantly more active compared
to the aqueous extracts. In both tests, no difference
was noted among the 3 levels of control. Dead larvae
were characterized by burned cuticle in R. communis
treatment particularly in oil emulsion treatments. Among
aqueous plant extracts, the seed kernel induced
significantly higher mortality when compared to the
roots and leave extracts (Tables 1 and 2). In oil
treatment, large number of larvae died shortly after
getting in contact with or ingesting the castor bean oil.
The median survival time (MST) of P. xylostella was the
lowest in oil emulsion treatments and the highest in the
3 controls (Tables 1 and 2).
Table 1: Mean Plutella xylostella 3
rd
instar mortality and Median Survival Time (MST) in topical application of
aqueous extracts and oil of castor bean, Ricinus communis.
Treatments Larval mortality (% ± SE)
a
MST(days ± SE)
a
Seed kernel extract (20%) 67.49 ± 1.98c 3.01 ± 0.09b
Roots extract (20%) 53.98 ± 2.41d 4.74 ± 0.57c
Leaf extract (20%) 58.98 ± 1.73d 3.86 ± 0.17b
Oil (5%) 82.19 ± 2.71b 1.05 ± 0.03a
Oil (10%) 100.00 ± 0.00a 1.02 ± 0.01a
Dursban (5%) 88.46 ± 2.97b 3.12 ± 0.77b
Control 1 (Emulsified water) 3.79 ± 1.07e >7
d
Control 2 (1% ethanol in water) 3.28 ± 0.85e >7
d
Control 3 (untreated check) 2.22 ± 0.95e >7
d
df 8,18
F 1280
P < 0.0001
a
Means in a column followed by the same are not significantly different (P < 0.05; Student-Newman-Keuls test).
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2904
Table 2: Mean mortality of 3
rd
instar larvae of Plutella xylostella and Median Survival Time (MST) in ingestion toxicity
tests of aqueous extracts and oil of castor bean.
Treatments Larval mortality (% ± SE)
a
MST(days ± SE)
a
Seed kernel extract (20%) 70.86 ± 0.85c 3.16 ± 1.03b
Root extract (20%) 49.89 ± 3.02d 4.12 ± 0.87b
Leaf extract (20%) 51.87 ± 0.88d 3.78 ± 0.79b
Oil (5%) 89.58 ± 1.90b 1.04 ± 0.04a
Oil (10%) 100.00 ± 0.00a 1.01 ± 0.01
c
a
Dursban (5%) 88.46 ± 2.97b 3.07 ± 0.82b
Control 1 (Emulsified water) 0.00 ± 0.00e >7
d
Control 2 (1% ethanol in water) 0.00 ± 0.00e >7
d
Control 3 (untreated check) 0.00 ± 0.00e >7
d
df 8,18
F 1975
P < 0.0001
a
Means in a column followed by the same letter are not significantly different (P < 0.05; Student-Newman-Keuls test).
Percent adult emergence, percentage of adults without
deformities as well as longevity of adult P. xylostella
were significantly affected by the previous treatments
on larvae (Tables 3 and 4). While all adult moths that
emerged in untreated, 1% ethanol and emulsified water
present normal morphological traits, significantly higher
proportion of adults emerging from R. communis
treatments presented abnormal development as wings
and legs deformation (Tables 3 and 4). In general, P.
xylostella appears to be more sensitive to castor bean
oil than to aqueous extracts. There was no difference
between females and male longevity (P > 0.05),
however when treated with castor bean products,
longevity of both males and females adult P. xylostella
was significantly reduced as compared to the controls
(Tables 3 and 4). The sex ratio of emerging moths was
male-biased for the R. communis products and
Dursban and female biased in untreated, 1% ethanol
and emulsified water controls (Tables 3 and 4), and did
not vary in ingestion test (Table 4). In topical application
test however the sex ratio was significant affected by
treatments (Table 3).
Field-cage experiments:
Experiment 1: Direct treatment of diamondback
moth infested cabbage plant in cage experiment:
The highest mortality of DBM was obtained in plants
treated with oil emulsion followed by Dursban (Table 5).
No significant differences in mortality were found
between the root and leaf extracts, both being
significantly less toxic compared to seed kernel extract
(Table 5). The majority of dead insects were recorded
during the first 4 days after the applications, i.e., 76.0 ±
1.2%, 61 ± 2.1%, 55 ± 1.5) and 53 ± 1.7) for Dursban,
seed kernel, leaf and root aqueous extracts,
respectively. The median survival time (MST) of P.
xylostella was significantly higher in the 3 control
treatments than in aqueous extract and Dursban
treatments. Lower MST values were registered in
Dursban treatment compared to aqueous extract
treatments (Table 5)
Experiment 2: Residual activity of the botanical
treatments
Except when P. xylostella larvae were released
immediately after spraying, no further mortality was
recorded in the 3 levels control treatments (i.e.,
untreated check, emulsifier solution and 1% ethanol in
water treatments) (Table 6). In all treatments, dead
insects were found in most cases attached to the leaf
surface. As in laboratory tests, insects that died on
plant treated with R. communis products showed
clearly burned cuticle. Significant effect of both
treatments and time from application to insect release
was observed (F = 1986; df = 8; P < 0.0001 and F =
4093; df = 2; P < 0.0001, respectively) with significant
interactions between the treatments and days after
application (F = 347.773; df = 16; P < 0.0001). P.
xylostella mortality was always significantly higher in R.
communis products treatments than in the control
treatments (P < 0.0001). For aqueous extract
treatments on 3- and 5-day-old spray residues, except
for seed kernel extract, similar mortality rates were
recorded, which were significantly lower than those on
fresh residues. Significant decrease in larval mortality
was noted over time for oil emulsion, seed kernel
extract and Dursban treatments (Table 6).
.
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2905
Table 5: Effect of different castor bean extracts and oil on DBM infesting cabbage plants in field cage experiments.
Treatments
b
Mortality (% ± SE)
a
MST
c
(days ± SE)
Seed kernel extract (20%) 67.15 ± 0.35c 2.61 ± 0.01c
Root extract (20%) 60.92 ± 3.41d 3.46 ± 0.08d
Leaf extract (20%) 58.76 ± 3.41d 3.50 ± 0.24d
Oil (5%) 97.92 ± 1.41a 1.02 ± 0.02b
Oil (10%) 100.00 ± 0.00a 0.73 ± 0.01a
Dursban (5%) 82.85 ± 3.41b 2.90 ± 0.08c
Control 1 (Emulsified water) 5.45 ± 0.37e >7
d
Control 2 (1% ethanol in water) 2.22 ± 0.17e >7
d
Control 3 (untreated check) 3.45 ± 0.37e >7
d
df 8,18
F 16,029
P 0.004
a
Seven days cumulative mortality;
b
means in the same columns followed by the same letter are not significantly
different (P < 0.05; Student-Newman-Keuls test);
c
median survival time;
d
MST exceeded the observational
period.
Table 6: Mortality (% ± SE) of 3rd instar of DMB on cabbage plants at different time intervals after treatment with
different castor bean extracts and oil in field-cage experiment.
Mortality (% ± SE)
a
Days after application
Treatments 0 3 5
Seed kernel extract (20%) 51.72 ± 3.20Ba 23.41 ± 1.72Cb 14.11 ± 1.47Bc
Root extract (20%) 48.44± 3.1Ba 19.94 ± 0.48Db 14.07 ± 0.78Bb
Leaf extract (20%) 42.44± 3.1Ba 17.94 ± 0.48Db 12.07 ± 0.78Bb
Oil (5%) 95.92 ± 1.31Aa 47.92 ± 2.22Bb 9.92 ± 2.17Cb
Oil (10%) 100.00 ± 0.00Aa 51.41 ± 1.72Bb 12.14 ± 1.47Bb
Dursban (5%) 75.21± 1.09Aa 65.56 ± 2.24Ab 29.15 ± 1.15Ac
Control 1 (Emulsified water) 6.87 ± 1.17Ca 0.00 ± 0.00Eb 0.00 ± 0.00Db
Control 2 (1% ethanol in water) 5.09 ± 0.92 Ca 0.00 ± 0.00Eb 0.00 ± 0.00Db
Control 3 (untreated check) 3.48 ± 1.04Ca 0.00 ± 0.00Eb 0.00 ± 0.00Db
df 8,18 8,18 8,18
F 1069 996.718 206.763
P < 0.0001 < 0.0001 < 0.0001
a
Means within columns followed by the same upper case and within rows followed by the same lower case letter are
not significantly different (P < 0.05; Student-Newman-Keuls test).
Experiment 3: Effect of different treatments on the
oviposition behaviour of P. xylostella.
No-choice experiment: Diamondback females laid
significantly less eggs on plants treated with castor
bean products, than on those in the 3 control
treatments (Figure 1), with the lowest number of eggs
recorded on plant sprayed with castor bean oil (i.e.,
14.75 ± 0.61) (df = 7, F = 855.739 and P <0.0001).
Similar number of eggs was recorded on plants treated
with Dursban (75.75 ± 7.86), root extract (76.63 ± 2.38)
and leaf extract (79.50 ± 0.50) a(Figure 1). No
significant differences were found between the control
treatments (Figure 1).
Choice experiment: Diamondback females clearly
discriminated between plants sprayed with R.
communis products and untreated ones (Figure 2).
Castor bean plant extracts and oil emulsion treatments
significantly reduced oviposition on the plants offered.
More eggs were laid by P. xylostella on plants treated
with water than those treated with R. communis
products and Dursban (df = 4, F = 109.371, P < 0.0001;
Figure 2).
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2906
The ODI was significantly higher in R. communis
treated plants compared to the control (Figure 2),
indicating complete oviposition deterrent effects by
castor bean plant oil and extracts at the tested
concentrations. The highest oviposition deterrent
effects of castor bean plant were noted with oil
emulsion followed by the seed kernel extract. No
difference was found between root, leave extracts and
Dursban treatments (Figure 2).
Con trol 1 (Em ulsifie d water)
Con trol 2 (1% eth anol in water)
Control 3 (u ntreated ch eck )
Dursban (5% )
Root extract (2 0%)
Leaf extract (20% )
Se ed kern el e xtra ct (20%)
Oil ( 10%)
Number of eggs per plant (mean ± SE)
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
T r e a tm e n t s
aa
a
bbb
c
d
Figure 1: Mean (± S.E.) number of eggs laid by Plutella xylostella females per plant in no-choice experiment in field
cage experiments. Different letters above bars indicate significant differences between means at P = 0.05 (Student–
Newman and Keuls test).
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2907
Treatments
oil (10%)
Seed kernel extract (20%)
leaf extract (20%)
Root extract (20%)
Dursban (5%)
Mean OID (%)
0
20
40
60
80
a
b
cc
c
Figure 2: Oviposition deterrence index (ODI) of different extracts and oil of castor bean.
Different letters above bars indicate significant differences between means at P = 0.05 (Student-Newman-Keuls test).
DISCUSSION
The results of the present study showed that castor
bean plant extracts and oil could be toxic to larvae of P.
xylostella through contact and ingestion. Plant based
pesticides have been found to exhibit larvicidal effects
(e.g., Kétho et al., 2002; Sanda et al., 2006; Ogendo et
al., 2008; Agboka et al., 2009). Under field conditions,
Sanda et al. (2006), find that Cymbopogon
schoenanthus L. could significantly reduced larval
population of P. xylostella. Torres et al. (2001) report
100% mortality against P. xylostella larvae with 10%
aqueous solution of the wood bark of A. pyrifolium in
Brazil. Using aqueous extracts from leave of R.
communis, Aouinty et al. (2006) have reported high
larvicidal activity against 2
nd
and 4
th
instar larvae of four
mosquito species, Culex pipiens (L.), Aedes caspius
(Pallas), Culiseta longiareolata (Aitken) and Anopheles
maculipennis (Meigen). The high mortality rate of P.
xylostella larvae in both laboratory and cage
experiments could be due to toxic effect of the plant.
Castor bean oil and pure compounds of R. communis
have been reported to exhibit high toxic effects in target
animals (Olsnes, 2004; Bigalke & Rummel, 2005;
Kumar et al., 2007; He et al., 2007). The toxicity of the
plant is ascribed to the presence of ricin, a water-
soluble glycoprotein concentrated in the seed
endosperm but present in lesser concentrations in other
parts of the plant and reputed to be one of the most
poisonous of the naturally occurring compounds (Darby
et al., 2001; Frederiksson et al., 2005; Kozlov et al.,
2006; El-Nikhely et al., 2007). This fact explained the
comparatively high effects recorded in oil and seed
kernel extract compared to the leave and root extracts.
According to Tokarnia et al., 2002, whilst the seeds are
the primary source of toxin, the rest of the plant may
also be considered to be slightly toxic. The mechanism
of toxicity has been elucidated in great detail. Following
uptake into cells by endocytosis, ricin causes acute cell
death by inactivation of ribosomal RNA, inhibiting
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2908
protein synthesis (e.g., Roberts & Smith, 2004;
Utskarpen et al., 2006; Parikh et al., 2008). Although
the high toxicity of ricin could explain the high larval
mortality recorded in this study, the burning effect of the
R. communis products could be an important factor in
the host death particularly in contact toxicity tests.
In the present work, a large number of adult died during
emergence, exhibiting abnormal development.
Morphological alterations are common in insects
treated with botanical based insecticides and have
been attributed to the reduction in the concentration of
ecdysone (a steroidal prohormone of the major insect
moulting hormone 20-hydroxyecdysone) (Schmutterer,
1990; Mordue-Luntz and Nisbet, 2000) or to its delayed
release into the circulatory fluid (Jacobson et al., 1983;
Marco et al., 1990).
The results from the cage study corroborated previous
laboratory data on the high toxicity of botanical oil
emulsion to P. xylostella larvae, thus confirming the
high potential of R. communis components for
controlling the diamondback. Our results showed that
direct treatment of diamondback-infested bean plants
can be very effective for control of P. xylostella. The
majority of diamondback larval mortality is probably a
result of direct contact of the applied toxin on the larva.
However, the significant decline in larval mortality in the
residual study indicated a strong decline in activity of
the toxin applied to leaf surfaces. Increasing time after
application resulted in significantly decreasing
diamondback larval mortalities, showing a probable
decrease in toxin activity under field conditions. Ricin is
known to quickly disappear when exposed to UV
radiation and high temperatures (Martinez-Herrera et
al., 2006). A number of factors including high
temperature for detoxifying castor seed meal (e.g.,
100°C for 30 min; 120°C for 25 min) have been
investigated, and have been reviewed by Anandan et
al. (2004). In addition to the toxic effect on larvae,
castor bean products acted as ovipositional deterrent
as it was shown in the ovipositional test. P. xylostella
adult tended not to oviposit on cabbage plants treated
with the oil and, to certain extend, on plant treated with
aqueous extracts, indicating a repellent effect.
Ovipositional deterrent effects of botanical pesticides
on many crop pests including Lepidopteran,
Homopteran and Dipteran species (Singh & Singh,
1998; Bruce et al., 2004; Showler et al., 2004;
Greenberg et al., 2005; Hossain & Poehling, 2006;
Seljåsen & Meadow, 2006; Adebowale and Adedire,
2006; Boateng & Kusi, 2008; Agboka et al., 2009) have
been reported. According to Udayagiri and Mason
(1995), chemical cues play a major role in host
selection. Similar results have been observed by Bruce
et al. (2004), who found that application of neem oil at
0.075 ml per maize plant leads to a reduction in the
number of eggs laid by Sesamia calamistis Hampson
(Lepidoptera: Noctuidae) and Eldana saccharina
Walker (Lepidoptera: Pyralidae) of 88 and 49%,
respectively, compared with the control. Oviposition
deterrence was also observed in Mussidia nigrivenella
(Lepidoptera: Pyralidae) by Agboka et al. (2009) who
report significantly higher oviposition deterrence index
with neem oil at 2.5 and 5%, Jatropha curcas at 5%
and Hyptis suaveolens at 20%. Lowery and Isman
(1993) suggest that this deterrence resulted from a
variety of compounds working in concert with another,
producing different behavioural responses, which vary
in magnitude between species.
In conclusion, the potential of R. communis in P.
xylostella control has good prospects. However, oils
including aqueous extracts of seeds have to be tested
on non targets insects including parasitoids before
being included in integrated pest management
programs currently being developed. Moreover
additional studies are required in other to develop
appropriated formulation and application method of R.
communis based pesticide.
ACKNOWLEDGEMENTS
The authors thank Dr TCHABI AttiAtti at the
“Université de Lomé” for his critical comments and
review of this paper. We also thank Mr. Batalia
Yao at the laboratory of the Ecole Supérieure
d’Agronomie (ESA)-Université de Lomé, Togo for
his technical assistance in oils extraction.
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on the diamondback moth
2909
Table 3: Effects of topical application of castor bean extracts and oil on adult DBM emergence, deformities, sex ratio and longevity
.
Adult longevity (days)
2
Treatments %
Emergence
1
%
Adults without
deformities
1
Sex ratio
(males/females) Male Female
Seed kernel extract (20%) 75.29 ± 2.41b 30.85 ± 1.09c 1.18 ± 0.08b 9.85 ± 0.98Ac 9.04 ± 1.01Ac
Root extract (20%) 79.84 ± 3.01b 53.49 ± 2.41b 1.04 ± 0.06c 11.08 ± 0.24Aa 10.82 ± 1.24Aa
Leaf extract (20%) 77.45 ± 1.20b 46.28 ± 1.98b 1.16 ± 0.13b 10.58 ± 1.08Ab 10.17 ± 0.98Ab
Oil (5%) 57.72 ± 1.57c 23.14 ± 2.78c 1.63 ± 0.15a 9.28 ± 0.86Ac 8.18 ± 1.04Ad
Oil (10%) -
3
-
3
-
3
-
3
-
3
Dursban (5%) 76.19 ± 1.26b 95.58 ± 2.45a 1.24 ± 0.09b 10.86 ± 0.98Ab 10.10 ± 0.85Ab
Control 1 (Emulsified water) 98,42 ± 1,08a 100.00 ± 0.00a 0.97 ± 0.05c 13.41 ± 0.95Aa 12.49 ± 1.06Aa
Control 2 (1% ethanol in water) 98.12 ± 1.58a 100.00 ± 0.00a 0.96. ± 0.41c 13.72 ± 0.83aA 12.72 ± 1.02Aa
Control 3 (untreated check) 98.87 ± 0.98a 100.00 ± 0.00a 0.98 ± 0.07c 13.25 ± 1.05Aa 12.48 ± 2.45Aa
df 7,16 7,16 7,16 7,16 7,16
F 206.31 1099 5.133 10.8 5.133
P < 0.0001 < 0.0001 0.003 < 0.0001 0.003
Means followed by the same lower case letter within a column and means adult longevity followed by the same upper case letter within line are not significantly different, (P <
0.05; Student-Newman-Keuls test).
1
Average of 90 pupae, 3 replications;
2
Average of 45 moths, 3 replications,
3
treated larvae died before pupation.
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on the diamondback moth
2910
Table 4: Effects of ingestion of castor bean extracts and oil on adult DBM emergence, deformities, sex ratio and longevity.
Adult longevity (days)
2
Treatments %
Emergence
1
%
Adults without
deformities
1
Sex ratio
(males/females) Male Female
Seed kernel extract (20%) 77.35 ± 1.28b 35.18 ± 1.13c 1.62 ± 0.19a 10.02 ± 1.09Ac 9.58 ± 1.52Ad
Root extract (20%) 87.14 ± 1.75b 56.27 ± 1.74b 1.35 ± 0.75a 12.74 ± 1.11Aa 11.96 ± 0.98Ab
Leaf extract (20%) 82.05 ± 2.13b 49.09 ± 1.09b 1.27 ± 0.09a 11.01 ± 1.13Ab 10.95 ± 1.02Ac
Oil (5%) 49.98 ± 0.98c 20.76 ± 1.97d 1.73 ± 0.45a 9.11 ± 0.98Ac 9.02 ± 1.55Ad
Oil (10%) -
3
-
3
-
3
-
3
-
3
Dursban (5%) 80.85 ± 2.91b 91.58 ± 3.01a 1.09 ± 0.26a 11.01 ± 1.21Ab 10.85 ± 0.96Ac
Control 1 (Emulsified water) 98.39 ± 1.54a 100.00 ± 0.00a 0.97 ± 0.74a 12.98 ± 1.09Aa 12.14 ± 1.68Aa
Control 2 (1% ethanol in water) 98.41 ± 1.22a 100.00 ± 0.00a 0.96 ± 0.81a 12.79 ± 1.10Aa 12.36 ± 1.39a
Control 3 (untreated check) 97.49 ± 1.90a 100.00 ± 0.00a 0.98 ± 0.09a 13.86 ± 1.08Aa 12.63 ± 1.62Aa
df 7,16 7,16 7,16 7,16 7,16
F 238.992 1380 1.048 6.745 2.834
P < 0.0001 < 0.0001 0.38 0.001 0.004
Means followed by the same lower case letter within a column and means adult longevity followed by the same upper case letter within linear not significantly different (P < 0.05;
Student-Newman-Keuls test).
1
Average of 90 pupae, 3 replications;
2
Average of 45 moths, 3 replications,
3
treated moth died before pupation.
Tounou et al. J. Appl. Biosci. 2011 Bio-insecticidal effects of plant extracts and oil emulsions of R. communis on
the diamondback moth
2911
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