Ceratophyllum demersum L. Extract as a Botanical Insecticide for Controlling the Maize Weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)

Abstract

Insecticidal activities of methanol and water extracts, obtained from in vitro propagation of Ceratophyllum demersum L. on maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) were laboratory studied. In vitro whole plant regeneration was obtained from nodal explants of C. demersum cultured on liquid Murashige and Skoog medium (MS) treated with various combinations of 6-Benzylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA). Different concentrations (2.5, 5, 10 and 20 mg/ml) of C. demersum extracts were tested. The highest mortality (100%) caused were determined by both extracts at the concentrations of 10 and 20 mg/ml after 96 h of exposure. LC50 of methanol and water extracts after 96 h were 1.720 and 1.015 mg/ml, respectively. It was detected that mortality was correlated with concentration and exposure time. These findings suggest that C. demersum extract could be considered as an effective biocontrol agent and could be used as an alternative to chemical insecticides.

Full text

Egyptian Journal of Biological Pest Control, 27(1), 2017, 11-15
Ceratophyllum demersum L. Extract as a Botanical Insecticide for Controlling the Maize
Weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)
Dogan1, M.; B. Emsen1; M. Aasim2* and E. Yildirim3
1Dept. of Biology, Kamil Ozdağ Faculty of Science, Karamanoglu Mehmetbey University, Karaman, Turkey.
2*Dept. of Biotechnology, Fac. of Science, Necmettin Erbakan Univ., Konya, Turkey, mshazim@gmail.com
3Dept. of Plant Protection, Faculty of Agriculture, Atatürk University, Erzurum, Turkey.
(Received: July 4, 2016 and Accepted: August 16, 2016)
ABSTRACT
Insecticidal activities of methanol and water extracts, obtained from in vitro propagation of Ceratophyllum demersum L.
on maize weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) were laboratory studied. In vitro whole
plant regeneration was obtained from nodal explants of C. demersum cultured on liquid Murashige and Skoog medium
(MS) treated with various combinations of 6-Benzylaminopurine (BAP) and 1-Naphthaleneacetic acid (NAA). Different
concentrations (2.5, 5, 10 and 20 mg/ml) of C. demersum extracts were tested. The highest mortality (100%) caused were
determined by both extracts at the concentrations of 10 and 20 mg/ml after 96 h of exposure. LC50 of methanol and water
extracts after 96 h were 1.720 and 1.015 mg/ml, respectively. It was detected that mortality was correlated with
concentration and exposure time. These findings suggest that C. demersum extract could be considered as an effective
biocontrol agent and could be used as an alternative to chemical insecticides.
Key words: Ceratophyllum demersum extract, Insecticidal activity, Maize weevil, Pest control.
INTRODUCTION
Maize weevil, Sitophilus zeamais Motschulsky
(Coleoptera: Curculionidae) is an important
economic stored grain pest of wheat, rice and corn.
The insect causes damage by feeding inside the grain
both as larvae and adults and resulting in quality and
weight loss of the product and ultimately low
germination rate (Yildirim, 2012). In order to control
these stored grain pests, the most preferable method
is chemical pesticide which results pest control in
very short time. However, as a result of intense and
unconscious using of pesticides, these chemicals in
their original form with their by-products can remain
in food, soil, water and air and ultimately exert
negative impacts on people and other non-target
organisms. It was determined that some of pesticides
had carcinogenic and even mutations builder effects
on the nervous system (Wan et al., 2015).
Considering the damages of these chemicals,
botanicals, natural or microbial insecticides provide
alternative source of safe biopesticides for pest
control. Furthermore, these biological insecticides
affect human life less in comparison with synthetic
pesticides on account of aforementioned property
(Yotavong et al., 2015). In recent years, many
researchers have studied the potency of plants based
insecticides and showed positive results (Emsen et
al., 2013; Liu et al., 2013 and Emsen et al., 2015).
Ceratophyllum demersum L., a perennial plant of
family Ceratophyllaceae (Arber, 2010), is an aquatic
medicinal plant. It has been widely used by people
since ancient times as medicine to treat various
ailments and diseases. In Indian medicine, it is used
for treating jaundice, scorpion bites and as an
antipyretic and antimalarial; whereas in Chinese
medicinal system, it is used for hemoptysis,
antidiarrhoeal and in wound healing (Bolotova,
2015). Fareed et al. (2008) reported that the aqueous
and organic solvents extracts of C. demersum have
antimicrobial effect against isolated strains of
bacteria and fungi. Additionally, it was determined
that aqueous extracts of C. demersum have inhibitory
activity against five species of water bloom
microalgae including Oscillatoria sp.,
Aphanizomenon flos-aquae, Chlamydomonas sp.,
Pandorina morum and Ankistrodesmus sp. (Sun et al.,
2012). Furthermore, inhibitory activity against
oxidative stress mechanisms of C. demersum has also
been reported (Karatas et al., 2015).
The present study aimed to measure toxicity
degree of different extracts obtained from C.
demersum propagated by tissue culture techniques in
the laboratory on the maize weevil, S. zeamais.
MATERIALS AND METHODS
Plant materials and culture conditions
C. demersum plants were obtained from
Department of Biology, Karamanoglu Mehmetbey
University, Karaman, Turkey and sterilized
accordingly to Karatas et al. (2014). Nodal explants
were cultured on liquid MS medium with 3% sucrose
and additionally supplement with different
combinations of 6-Benzylaminopurine (BAP)
(0.25, 0.50, 0.75, 1.00 and 1.25 mg/l) and 0.25 mg/l
1-Naphthaleneacetic acid (NAA) in Magenta GA7
vessels. The pH of the medium was adjusted to
5.6-5.8 using either 1N NaOH or 1N HCl prior to
autoclaving at 121°C and 118 kPa for 20 min. All the

12
cultures were maintained at 24°C and 16 h light
photoperiod (1500 lux) using white Light Emitting
Diodes (LED) lights. The data were recorded for
shoot regeneration after 8 weeks of culture. Eight
weeks later, the experiment was terminated and
statistical analyses were applied for shoot
regeneration frequency (%), mean number of shoots
per explant, and shoot length (cm).
Preparation of extracts
Randomly, selected 50 plants from each 6-
Benzylaminopurine (BAP) and 1-Naphthaleneacetic
acid (NAA) medium were taken and thereafter, a total
of 100 plants were used for each type of extraction.
Ten grams of the plants were dried for 7 days under
room conditions, powdered and then performed
extraction process with 250 ml methanol and water
solvents with Soxhlet extractor. The solvents in
extracts obtained were evaporated by rotary
evaporator. Crude extracts were solved in 5%
dimethyl sulfoxide (DMSO) and stock solutions were
obtained. The crude extracts were dissolved in
methanol and water solvents, existed in 5% DMSO.
Different concentrations from each sample were
prepared at 2.5, 5, 10 and 20 mg/ml.
Insect rearing
S. zeamais was collected from the storage houses
located in Tokat, Turkey. The insects were reared in
the laboratory at 25±1°C, 64±5% R.H. at the Dept. of
Plant Protection, Atatürk University, Turkey. Corn
grains were purchased from a local market and
sterilized by storing at -20°C. in order to kill any
previous infestation; corn grains were washed using
tap water and dried at 25°C.
Bioassay
Thirty-three corn grains and 33 adults of S.
zeamais were introduced in 100×20 mm glass Petri
dishes in triplicate. Amounts of solutions applied
were 2.5, 5, 10 and 20 mg/ml in each Petri dish. After
exposure, mortality of adults was determined at 24,
48 and 96 h duration. Grains were cut into two pieces
to let the insects visible inside the grains. The
completely inactive insects were considered dead.
Petri dishes applied with %5 DMSO solvent served
as control.
Statistical analyses
All the experiments were carried out in triplicate.
Differences among the variables were exhibited using
Duncan test and significance was declared with 95%
confidence intervals. Median lethal concentration
data (LC50) were determined using probit regression
analysis. Similarities and dissimilarities among the
LC50 values were tested via Hierarchical cluster
analysis (HCA) with Ward’s minimum variance
method. In order to determine relations among the
variables, Pearson’s bivariate correlation test was
used. Statistical Package for Social Sciences (SPSS,
version 21.0, IBM Corporation, Armonk, NY, USA)
was used for analyses.
RESULTS AND DISCUSSION
Nodal explants of C. demersum were cultured in
vitro. The whole plant regeneration on liquid
MS medium contained 0.25-1.25 mg/l BAP and
0.25 mg/l NAA. Shoot formations began to be
monitored on the culture medium at the end of 8 days
and multiple shoot regenerations began to be
markedly observed on MS medium after 4 weeks of
culture. Callus formation was not detected on nodal
explants of C. demersum in culture medium
supported the previous findings of Karatas et al.
(2014 and 2015) and Dogan et al. (2015). High
frequency axillary shoot proliferation and plant
regeneration were obtained from nodal explants, after
8 weeks of culture (Fig. 1).
Shoot regeneration frequency, shoots per explants
and mean shoot length ranged 77.77 - 100.00%,
64.67 - 92.76 and 3.08-3.53 cm, respectively (Table
1). Results revealed that maximum shoot regeneration
frequency (100%) was obtained on MS medium
containing 0.25-0.75 mg/l BAP + 0.25 mg/l NAA.
Whereas, maximum number of shoots per explants
(92.67) and mean shoot length (3.53 cm) were
obtained from medium containing 0.75 mg/l BAP +
0.25 mg/l NAA and 0.25 mg/l BAP + 0.25 mg/l
NAA, respectively. Results further revealed that
increased BAP concentration exerted negative effects
on shoot regeneration behavior. The results are in line
with those of Karatas et al. (2013) who reported
decreasing mean shoot length of Bacopa monnieri
depended on increasing BAP concentrations
irrespective of NAA in the culture medium.
LC50 determined for different exposure times
showed that water extract of C. demersum with lowest
LC50 value was the most efficient treatment against S.
zeamais recorded after 96 h of exposure (1.015
mg/ml). Contrarily, the highest LC50 value of
methanol extract was 24.900 mg/ml after 24 h.
According to the calculated LC50 values at 24, 48 and
96 h after exposure, insecticidal potency of extracts
was in the ascending order of methanol < water. Also,
it was observed that there was a significant difference
(p < 0.05) among the LC50 data (Table 2). Likewise,
decreasing LC50 findings depending on exposure time
in many biological insecticide studies performed on
S. zeamais was demonstrated (Yildirim et al., 2012;
Kordali et al., 2013 and Yildirim et al., 2013).
However, De Oliveira et al. (2012) recorded that

13
Fig. (1): In vitro whole plant regeneration from nodal
explants of C. demersum cultured on liquid culture
medium after 8 weeks.
Table (1): Effects of different concentrations of
BAP-NAA on in vitro shoot regeneration of
C. demersum from nodal explants
Growth
regulators
(mg/L)
Shoot
regeneration
Frequency (%)
Mean number
Of shoots per
explant
Shoot
length
(cm)
BAP
NAA
0.25
0.25
100.00a
77.00ab
3.53a
0.50
0.25
100.00a
86.33a
3.49a
0.75
0.25
100.00a
92.76a
3.34b
1.00
0.25
88.89ab
75.67ab
3.16c
1.25
0.25
77.77b
64.67b
3.08d
Means followed by different superscript letters in the
same column differ significantly at p < 0.01.
Fig. (2): Dendrogram built from LC50 of C. demersum
extracts against S. zeamais at different exposures.
Note: Methanol extract (M), water extract (W), at
different exposure time (A, B, C) are different
groups.
Table (2): LC50 values (mg/ml) of C. demersum extracts against S. zeamais adults at different exposure time
Treatment
Exposure time (h)
LC50 (limits)
Slope ± standard error (limits)
Methanol extract
24
24.900f (20.169 – 34.635)
2.545 ± 0.359 (1.842 – 3.429)
48
16.013e (13.176 – 20.885)
1.922 ± 0.231 (1.469 – 2.375)
96
1.720b (1.129 – 2.199)
2.517 ± 0.396 (1.741 – 3.293)
Water extract
24
14.542d (11.536 – 20.290)
1.476 ± 0.211 (1.062 – 1.889)
48
6.959c (5.894 – 8.217)
1.882 ± 0.212 (1.467 – 2.297)
96
1.015a (0.384 – 1.566)
2.127 ± 0.444 (1.256 – 2.998)
Values followed by different superscript letters in the same column differ significantly at p < 0.05.
Fig. (3): Percentage mortality of S. zeamais exposed to different concentrations of C. demersum extracts after
(a) 24 h (b) 48 h (c) 96 h. Each value is expressed as mean ± standard deviation (n = 3).

14
methanol extract of Vitex cymosa (Family:
Lamiaceae) showed high insecticidal effect against S.
zeamais. Earlier, Othira et al. (2009) reported that
hexane extract had high insecticidal activity
compared to water extract. Such different results for
extracts of various plants suggest that insecticidal
compounds in different plants differed according to
the plant species and the solvent diverse.
HCA was carried out by methanol (M) and water
(W) for exposure time (h) of 24, 48 and 96 h. LC50
data of methanol (M-24, M-48 and M-96) and water
(W-24, W-48 and W-96) extracts showed that the six
treatments can be divided into three groups (group A,
B and C). Group A was larger than those of groups B
and C. Accordingly, HCA, M-96, W-96 and W-48
were located within group A, M-48 and W-24 created
a separate group (group B). M-24 was single
treatment in group C and it had the most distant
relationship with the other groups (Fig. 2). Therefore,
it was indicated that M-24 was ineffective treatment.
The results of HCA revealed that M-96, W-96 and W-
48 under group A had the lowest HCA coefficient that
showed the highest insecticidal activity.
When different exposure times on the insects were
investigated, they showed that the most effective time
was 96 h. After 96 h of exposure, treatments with the
concentrations of 10 and 20 mg/ml of both extracts
had the highest insecticidal activities (> 98%) but
statistically insignificant. At this treatment, the
insecticidal activities (81.81%) of the lowest
concentration (5 mg/ml) of water and methanol
extracts, respectively, were insignificantly different
(Fig. 3c). The insecticidal activity was crucial in early
time for rapid insect control. Hence, the results after
24 h of exposure are important, it was detected that
there was no insect mortality in control group and
statistically there was insignificant difference
between control and treatments at the concentrations
of 2.5 and 5 mg/ml for methanol extract. The
maximum percentage mortality of S. zeamais after 24
h was determined at the treatment of the highest
concentration (20 mg/ml) of water extract (57.57%)
and this value was different from all other treatments
(Fig. 3a). Based on all S. zeamais treatments, it was
revealed that mortality rate was concentration and
exposure time dependent.
Table (3): Correlation between different variables and
C. demersum extracts for insecticidal activity
against S. zeamais
Treatment
Mortality -
Concentrationa
Mortality -
Exposure timea
Methanol extract
0.432b
0.870b
Water extract
0.502b
0.814b
aPearson correlation coefficient; bcorrelation is significant
at the 0.01 level.
Correlation coefficients between exposure times
and mortalities caused by methanol and water extract
were higher and recorded 0.870 and 0.814,
respectively. On the other hand, high positive
correlations between mortality and concentration
variables for both extracts were remarkable (Table 3).
Similarly, increasing insecticidal activity on S.
zeamais depending on concentration was reported by
Akinneye and Ogungbite (2013). They displayed that
some medicinal plants such as Zanthoxylum
zanthoxyloides, Aristolochia ringens and Garcinia
kola had 100% insecticidal effect against S. zeamais
within 72 h.
Although chemical insecticides are frequently
used as repellents at the present time, biological
products, plant origin, have safer repellent potential
for people and environment (Wan et al., 2015).
Residues caused by synthetic pesticides are
fundamental problems for the nature as they disrupt
the ecological balance. Therefore, these adverse
conditions may affect other organisms including
humans (Lacey et al., 2015). Plants used as
ecofriendly biological insecticides have many active
components and their metabolites do not create
problem on the environment and/or humans
(Veerakumar et al., 2014). Because of these reasons,
this exploratory study revealed that C. demersum
extract had promising perspectives for controlling
one of the storage insect pests, S. zeamais.
ACKNOWLEDGMENT
This study was supported by the Karamanoğlu
Mehmetbey University through the Scientific
Research Project commission (BAP) (Project no: 05-
M-15).
REFERENCES
Akinneye, J. O. and O., Ogungbite 2013. Insecticidal
activities of some medicinal plants against
Sitophilus zeamais (Motschulsky) (Coleoptera:
Curculionidae) on stored maize. Arch.
Phytopathol. Plant Prot., 46: 1206-1213.
Arber, A. 2010. Water Plants. A study of Aquatic
Angiosperms. Cambridge Univ Press New York,
US. 88 p.
Bolotova, Y. V. 2015. Aquatic plants of the Far East
of Russia: a review on their use in medicine,
pharmacological activity. Bangladesh J. Med.
Sci., 14: 9-13.
De Oliveira, T.A., B. Ronchi-Teles, C. R. V. Da
Fonseca, S. L. R. Da Silva, P. A. Santos and C. V.
Nunez 2012. Insecticidal activity of Vitex cymosa
(Lamiaceae) and Eschweilera pedicellata
(Lecythidaceae) extracts against Sitophilus
zeamais adults (Curculionidae). Emirates J. Food

15
Agric., 24: 49-56.
Dogan, M., M. Karatas and M. Aasim 2015. An
efficient in vitro plantlet regeneration of
Ceratophyllum demersum L., an important
medicinal aquatic plant. Fresenius Environ. Bull.,
24: 3499-3504.
Emsen, B., A. Aslan, E. Yildirim and S. Ercisli 2013.
Toxicity effects of some lichen species extracts
against the Colorado potato beetle, Leptinotarsa
decemlineata Say (Coleoptera: Chrysomelidae).
Egypt J. Biol. Pest Control, 23: 193-199.
Emsen, B., E. Yildirim and A. Aslan 2015.
Insecticidal activities of extracts of three lichen
species on Sitophilus granarius (L.) (Coleoptera:
Curculionidae). Plant Prot. Sci., 51: 156-161.
Fareed, M., A. Haroon and S. Rabeh 2008.
Antimicrobial activity of some macrophytes from
Lake manzalah (Egypt). Pakistan J. Biol. Sci., 11:
2454-2463.
Karatas, M., M. Aasim, M. Dogan and K. M. Khawar
2013. Adventitious shoot regeneration of the
medicinal aquatic plant water hyssop (Bacopa
monnieri L. Pennell.) using different internodes.
Arch. Biol. Sci., 65: 297-303.
Karatas, M., M. Aasim and M. Dogan 2014. Multiple
shoot regeneration of Ceratophyllum demersum L.
on agar solidified and liquid mediums. Fresenius
Environ. Bull., 23: 3-9.
Karatas, M., M. Dogan, B. Emsen and M. Aasim
2015. Determination of in vitro free radical
scavenging activities of various extracts from in
vitro propagated Ceratophyllum demersum L.
Fresenius Environ. Bull., 24: 2946–2952.
Kordali, S., B. Emsen and E. Yildirim 2013.
Fumigant toxicity of essential oils from fifteen
plant species against Sitophilus zeamais
Motschulsky (Coleoptera: Curculionidae). Egypt.
J. Biol. Pest Control, 23: 241-246.
Lacey, L., D. Grzywacz, D. Shapiro-Ilan, R. Frutos,
M. Brownbridge and M. Goettel 2015. Insect
pathogens as biological control agents: Back to the
future. J. Invertebr. Pathol., 132: 1-41.
Liu, Z. L., Q. Z. Liu, L. Zhou and G. Jiang 2013.
Essential oil composition and insecticidal activity
of Salvia umbratica flowering aerial parts from
China against Sitophilus zeamais. J. Essent. Oil.
Bear. Plants, 16: 672-678.
Othira J. O., L. A. Onek, L. A. Deng and E. Omolo
2009. Insecticidal potency of hyptis spicigera
Preparations against Sitophilus zeamais (L.) and
Tribolium castaneum (herbst) on stored maize
grains. African J. Agric. Res., 4: 187-192.
Sun, Y. Y., S. Z. Xu, J. Zhang and C. Wang 2012.
Growth inhibition of Ceratophyllum demersum on
five species of water bloom microalgae. Huanjing
Kexue yu J., 7: 1-7.
Veerakumar, K., M. Govindarajan, M. Rajeswary and
U. Muthukumaran 2014. Mosquito larvicidal
properties of silver nanoparticles synthesized
using Heliotropium indicum (Boraginaceae)
against Aedes aegypti, Anopheles stephensi, and
Culex quinquefasciatus (Diptera: Culicidae).
Parasitol. Res., 113: 2363-2373.
Wan, N. F., X. Y. Ji, J. X. Jiang, Y. M. Zhang,
J. H. Liang and B. Li 2015. An ecological
indicator to evaluate the effect of chemical
insecticide pollution management on complex
ecosystems. Ecological Indicators, Ecol. Indic.
53: 11-17.
Yildirim, E. 2012. Pests of stored product and their
control methods, Third edition; Atatürk University
Agricultural Faculty Press, Erzurum, 191: 123.
Yildirim, E., B. Emsen, A. Aslan, Y. Bulak and S.
Ercisli 2012. Insecticidal activity of lichens
against the maize weevil, Sitophilus zeamais
Motschulsky (Coleoptera: Curculionidae). Egypt.
J. Biol. Pest Control, 22: 151-156.
Yildirim, E., B. Emsen and S. Kordali 2013.
Insecticidal effects of monoterpenes on Sitophilus
zeamais Motschulsky (Coleoptera:
Curculionidae). J. Appl. Bot. Food Qual., 86: 198-
204.
Yotavong, P., B. Boonsoong, W. Pluempanupat, O.
Koul and V. Bullangpoti 2015. Effects of the
botanical insecticide thymol on biology of a
braconid, Cotesia plutellae (Kurdjumov),
parasitizing the Diamondback Moth, Plutella
xylostella L.. Int. J. Pest. Manag., 61: 171-178.