Journal of Entomology and Zoology Studies 2016; 4(1): 262-268
E-ISSN: 2320-7078
P-ISSN: 2349-6800
JEZS 2016; 4(1): 262-268
© 2016 JEZS
Received: 18-11-2015
Accepted: 20-12-2015
Mubashar Ahmad Malik
Department of Entomology,
University of Agriculture,
Faisalabad, Pakistan.
Mujahid Manzoor
Department of Entomology,
University of Agriculture,
Faisalabad, Pakistan.
Habib Ali
College of Plant Protection,
Fujian Agriculture and Forestry
University, Fuzhou-China.
Abrar Muhammad
College of Plant Protection,
Fujian Agriculture and Forestry
University, Fuzhou-China.
Saif ul Islam
College of Plant Protection,
Fujian Agriculture and Forestry
University, Fuzhou-China.
Muhammad Qasim
College of Plant Protection,
Fujian Agriculture and Forestry
University, Fuzhou-China.
Nazir Ahmad
Department of Plant Breeding
and Genetics, the University of
Agriculture Peshawar, Pakistan.
Atif Idrees
College of Plant Protection,
Fujian Agriculture and Forestry
University, Fuzhou-China.
Arif Muhammad
College of Plant Protection,
Fujian Agriculture and Forestry
University, Fuzhou-China.
Hafiz Sohaib Ahmed Saqib
College of Plant Protection,
Fujian Agriculture and Forestry
University, Fuzhou-China.
Correspondence
Habib Ali
College of Plant Protection,
Fujian Agriculture and Forestry
University, Fuzhou-China.
Evaluation of imidacloprid and entomopathogenic
fungi, Beauveria bassiana against the red palm weevil
Rhynchophorus ferrugineus (Coleoptera:
Curculionidae)
Mubashar Ahmad Malik, Mujahid Manzoor, Habib Ali, Abrar
Muhammad, Saif ul Islam, Muhammad Qasim, Nazir Ahmad, Atif Idrees,
Arif Muhammad, Hafiz Sohaib Ahmed Saqib
Abstract
The Red Palm Weevil is a serious problem of the date palm in Pakistan. The sole and combine
application effects of Beauveria bassiana (Balsamo) Vuillemin (Hyphomycetes) and sub-lethal dosages
of Imidacloprid against the second and fourth instar larvae of Rhynchophorus ferrugineus Olivier was
investigated in laboratory. Additionally, the in vitro hyphal growth of B. bassiana amended with three
different concentrations (50, 250, 500 µl L-1) of Confidor® 200 SL was also assessed. The results showed
significantly higher mortality for the second and fourth instar larvae of RPW when B. bassiana and
Imidacloprid applied together compared to their sole application. The highest mortality level (100%) for
the second instar was recorded after 15 days of treatment with B. bassiana (1×106 conidia/ml) and
Imidacloprid (1 µl L-1) in combination, while for the fourth instar larvae it was achieved after 20 days of
application. However, the presence of Imidacloprid did not significantly affect the hyphal growth of B.
bassiana on Sabouraud Dextrose Agar (SDA), and greater colony sizes compared to untreated control
were recorded in all cases. The maximum number of sporulation (173, 159.83 conidia ml-1) and
percentage (89.15, 79.4%) of mycosed R. ferrugineus cadavers was observed at lower dose rate (1×106
conidia/ml) of B. bassiana, exhibited the potential to be exploited for the control of RPW.
Keywords: Entomopathogenic fungi, Imidacloprid, Mortality, Mycosis, Sporulation.
1. Introduction
The invasive Red Palm Weevil (RPW) Rhynchophorus ferrugineus Olivier (Curculionidae:
Coleoptera), is considered as economically important and destructive pest insect of date palm
in Pakistan [46]. It was first reported in 1992 from Khairpur Sindh [3], from where the pest
spread to other date palm growing areas of Pakistan. At present, the pest has successfully
invaded many areas including Asia, Africa, Oceania, and Europe [18], and more recently it has
been reported from California and Australia [32, 36]. The female RPW lay their eggs singly in
the apertures made with their rostrum at the leaf base [35]. On hatching the larvae made their
way into the center of palm and make galleries by feeding inside the trunk, the mature larvae
made their cocoons from the chewed fiber and pupate at the leaf base [22]. The adult emerged
from the cocoon, resided on the same tree and reproduced till the death of host [13].
The existing methods of RPW management largely rely on the IPM strategies, which include:
phyto sanitation, use of conventional insecticide, pheromone traps and bio-control agents. The
chemical control method against RPW, include repeated fumigation, spraying and injecting of
synthetic insecticides into infected palms [19]. These approaches are not enough as under this
category the use of certain chemicals causes the development of resistance in the pest and
further have negative environmental and human impacts [1]. Neonicotinoids, comparatively
new group of synthetic insecticides, similar to natural product nicotine present in the insect
nervous system, agonists the nicotinic acetylcholine receptors (nAChR) [30]. The peculiar mode
of action and Chemorational ability of the neonicotinoid insecticides make them a better
choice in pest management, especially in case of concealed borers. Imidacloprid [1-(6-chloro3-pyridylmethyl)-2-nitroimino-imidazolidine] possess both systemic and contact mode of
action and is compatible with different application methods for example foliar application,
seed treatment [45], soil drench and stem application in different crops and trees [10].
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Journal of Entomology and Zoology Studies
Imidacloprid causes irreversible blockage of postsynaptic
nicotinergic acetylcholine receptor of the central nervous
system [34]. Laboratory and semi-field trials of Imidacloprid
(SL) against RPW has been reported by Cabello et al. [11] and
Kaakeh, [29] which strengthen the case for its further evaluation
against RPW having different geographical origin.
An additional and alternate tool for RPW control is
entomopathogenic fungi. Upon contact with the host,
entomopathogenic fungi penetrate the cuticle of the host,
germinate the spores, and overcome the host immune system.
The blastopores proliferate producing conidia in cadavers of
the host [51], hence the host can be infected either by direct
contact, horizontal transmission from infected cadavers to
healthy host or through the germination of appressoria from
dead cadavers [31, 38]. Entomopathogenic fungi are considered
unique due to the above mentioned characteristic among the
other biological control agents, especially in case of borer
insect. The eggs and other developmental stages of R.
ferrugineus, except the adult stage, occur in the tree trunk, and
entomopathogenic fungi is more likely to infect the adult stage
of the host insect [23], and thus serve as an inoculum for the
trunk inhabiting stages of RPW. The potential of hypocerealen
species Metarhizium brunneum (Petch) (Hypocreales:
Clavicipitaceae), Metarhizium anisopliae (Metsch.) Sorokin,
Beauveria bassiana (Balsamo) Vuillemin (Ascomycota:
Clavicipitaceae) as biocontrol agents against several cryptic
insect species has been investigated by various researcher [43,
40]
.
Several studies have been conducted to study the combined
effects of entomopathogenic microbes with sub-lethal doses of
synthetic insecticides, proved to augment the effectiveness of
entomopathogenic organisms [2], specifically against the
invasive pests, for e.g. longhorned beetle, Anoplophora
glabripennis (Motschulsky) (Cerambycidae: Coleoptera),
when Imidacloprid was used in combination with M.
brunneum. Chemical induces nutritional stresses, which
increases susceptibility of economically important pest against
their pathogens [7, 15, 21, 24]. Successful control against different
insect pest has increased the combine use of sub-lethal dosages
of Imidacloprid with other entomopathogenic microbes.
The present study was designed with the aim to examine the
sole and combine use of B. bassiana and Imidacloprid against
the second and fourth instar larvae of R. ferrugineus and to
study the post-mortal sporulation and mycosis.
2. Materials and methods
Test insect
The larvae of RPW used in the experiment were obtained from
Integrated Pest Management laboratory, Department of
Entomology, University of Agriculture, Faisalabad, Pakistan.
Both larvae and adult of R. ferrugineus were reared on clean,
fresh and uninfested sugarcane stems. The laboratory
conditions maintained at 26±2 oC, 70±5% RH, and a
photoperiod of 12:12 D: L hours for rearing purpose [29].
Insecticide and Fungal isolation
Imidacloprid commercial liquid formulation (Confider® 200
SL), obtained from Bayer Crop Sciences, Limited, Pakistan
which contain 200g/L active ingredients was used in the
experiment. The fungal strain B. bassiana strain used in the
experiment was originally isolated from the infected pupae of
RPW by the single spore method, modified by Choi et al. [12],
and later the fungus was identified based on the morphology
according to Barnett and Hunter [4]. Conidia was picked from
the surface of 14-days old well sporulating culture using a
sterilized scalpel. The14-days old well sporulating culture was
previously subculture and maintained on Potato Dextrose Agar
(PDA) and incubated at 26 °C and 75% RH. The harvested
conidia were then added in sterilized water containing 0.05%
Tween 80. After serial dilution, required conidial suspensions
of 1×106, 1×107 and 1×108 conidia ml-1 were prepared and
enumerated under hemocytometer. B. bassiana produced more
than 95% viable spores in a preliminary bioassay.
Radial growth test
The conical flasks containing Sabouraud Dextrose Agar (SDA)
media were autoclaved at 121 oC for 25-30 minutes, upon
cooling the three concentrations (50, 250, 500 µl L-1) of
Imidacloprid were added to these flasks. For control treatment
the same SDA media without any concentration of insecticide
was used. After gently shaking the flasks for 2-3 minutes, the
media was poured into Petri plates (9 cm), about 15ml
amended media was transferred into each Petri plate. Eleven
Petri plates were used for each concentration. To check the
effect on radial growth, three mm diameter cores of B.
bassiana from fresh unsporulated fungal cultures were picked
and placed in the center of individual Petri plate. Radial
colonies growth was measured by using two cardinal
diameters in every two days up to 15 days [50]. The whole
experiment was replicated three times.
Bioassay
Seven treatments were tested in the bioassays each for B.
bassiana alone (1×106, 1×107, 1×108 conidia ml-1),
Imidacloprid alone (1 µl L-1) and Imidacloprid with the higher,
intermediate and lower dose of B. bassiana, respectively. To
treat the second and fourth instar larvae of R. ferrugineus, a
batch of 10 larvae for each instar were dipped directly in B.
bassiana conidial suspension of for 60s [14], and were air dried
for 10 minutes in sterilized Petri plate (2.5 cm diameter) lined
with moist filter paper [33]. The treated larvae were provided
with vertically cut sugarcane pieces (1×4 cm), previously
dipped in the specific concentration of Imidacloprid (1 µl L-1).
In case of alone treatments, one batch (n=10) dipped in fungal
suspensions was air dried and then fed on fresh untreated
sugarcane pieces, while another fresh batch (n=10) was only
fed on Imidacloprid treated sugarcane pieces to represent the
fungal alone and Imidacloprid alone treatments, respectively.
For the control group, the larvae were dipped in distilled water
having 0.01% Tween 80. In each group, larval mortality rate
was calculated after 5, 10, 15 and 20 days of treatment. The
experiment was repeated for three times with six replicates for
each treatment.
Mycosis and Sporulation
To measure the sporulation and mycosis data, dead larvae of
R. ferrugineus were collected every day after each mortality
count and placed on an autoclaved Petri plate which was
immediately transferred into refrigerator at 5-6 oC. For surface
sterilization, these cadavers were washed with a 75% ethanol
solution for 2 minutes, rinsed with deionized water and then
the surface sterilized dead larvae were placed on Sabouraud
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Journal of Entomology and Zoology Studies
Dextrose Agar (SDA) and incubated for 9-10 days at 25±2 oC
and 75% RH. The number of larvae showing external fungal
growth was measured directly under the stereo microscope.
Sporulation was calculated by stirring mycosed insects in a
beaker containing 0.05% Tween 80. After thorough stirring of
the solution for 10 minutes, hemocytometer and compound
microscope were used to count the number of conidia in the
solution [48].
Statistical analysis
The data was subjected to statistical analysis, mortality means
were corrected by Abbott’s formula. However, throughout the
experiment, control mortality was very low, therefore
mortality data was excluded from the analysis, and mortality
data was analyzed using two-way analysis of variance
(ANOVA) having treatment and interval as the main factors
while mortality was the response variable for each larval
instar. The means were compared by using Tukey’s test at
significance level of α= 0.05%, while the entire analysis was
carried out using Minitab 13.2.
3. Results
Radial growth test
In in vitro trials, vegetative growth of B. bassiana on
Sabouraud dextrose agar (SDA) media was not affected
significantly by the different concentration of Imidacloprid and
greater colony sizes were witnessed on all three different dose
rate of Imidacloprid as compared to control treatment (Fig.1).
(D3: F3, 47=7.44; P<0.01, D5 F3, 47=3.30; P<0.01, D7 F3,
47=12.2; P<0.01, D9 F3, 47=14.1; P<0.01, D11 F3, 47=10.7;
P<0.01, D13 F3, 47=25.7; P<0.01, D15 F3, 47=16.3; P<0.01).
Fig 1: Radial growth rate (mm/days ±SE) of B. bassiana on SDA amended with 50, 250 and 500 µl/L imidacloprid (Imd) (within each day
means followed by the same letter are not significantly different
Larval mortality of R. ferrugineus
For the second instar larvae of R. ferrugineus, the mortality
was significantly affected by the treatment, interval (F6,41
=18.4; P< 0.01; F6,41=20; P< 0.01; F6,41 =22; P< 0.01; F6,41
=26.6; P< 0.01) and their associated interaction treatment ×
interval F18,167 =2.72; P< 0.01). Initially at day 5, highest larval
mortality was achieved at the lowest dose rate of B. bassiana
3.33% and at the single alone dose of Imidacloprid 37.19%.
During the experiment the highest mortality (100%) rate was
recorded when (1 × 106 conidia ml-1) of B. bassiana was
applied in combination with 1ppm of Imidacloprid after 20
days (Table 1).
Larval mortality of the fourth instar larvae of RPW was also
significantly affected the main effect (F6,41 =18.4; P< 0.01;
F6,41 =20; P< 0.01; F6,41 =22; P< 0.01; F6,41 =26.6; P< 0.01)
and their associated interaction (F3,167 =2.28; P< 001). Among
the four used alone treatment, the highest larval mortality
17.67% was observed for single and alone dose rate of
imidacloprid than that of applying B. bassiana 2.50%.
Similarly after 20 days, mixed application of B. bassiana (1 ×
106 conidia ml-1) and Imidacloprid (1ppm) gave significantly
higher mortality 82.14% (Table 2).
Mycosis and sporulation
Treatments significantly affected the mycosis (2nd instar: F6, 41
=21.1, P< 0.01; 4th instar: F6, 41 =21.9, P< 0.01 and sporulation
(2nd instar F6, 41 =44.2; P< 0.01; 4th instar F6, 41 =47.7, P< 0.01)
in the cadavers of R. ferrugineus. Maximum mycosis (89.15,
79.4%) and sporulation (173, 159.83 conidia ml-1) was
recorded where B. bassiana (1×106 conidia/ml) was applied at
its lower dose rate against the 2nd and 4th instars larvae of R.
ferrugineus, respectively. However reduced rate of mycosis
(10.83, 5%) and sporulation (28, 13 conidia ml-1) was
observed for treatments where B. bassiana was combined with
Imidacloprid (Fig. 3).
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Journal of Entomology and Zoology Studies
Fig 2: Mycosis (% SE) in cadavers of R. ferrugineus treated with B. bassiana alone and combined with imidacloprid (Bb1:1×106, Bb21×107:
Bb3: 1×108conidia/ ml; Imd: 1µl/L); means with the same letters are not significantly different; Tukey-Kramer test at 5% significance
Fig 3: Sporulation (conidia/ml) on cadavers of R. ferrugineus treated with B. bassiana alone and combined with imidacloprid (Bb1: 1×106 Bb2
1×107 Bb3: 1×108 conidia/ml; Imd: 1 µl/L); means with the same letters are not significantly different; Tukey-Kramer test at 5% significance
Table 1: Mean mortality (±SE) of 2nd instar larvae of R. ferrugineus larvae after different interval with one dose rate of Imd (1 ppm), three- dose
rates of B. bassiana (Bb1, 1×106; Bb2, 1×107; Bb3, 1×108) within each treatment followed by the same letter are not significantly different; HSD
test at P= 5%
Treatment
Bb1
Bb2
Bb3
Imd
Bb1+Imd
Bb2+Imd
Bb3+Imd
Mortality(±SE)
Days 5
3.33±1.66e
5.83±2.38de
11.00±1.57cde
16.93±1.67bcd
21.88±3.22bc
28.72±4.50ab
37.19±3.65a
Days 10
10.96±3.94e
18.50±5.07de
32.93±5.34cde
39.69±4.61bcd
48.20±4.95bc
61.66±7.02ab
74.47±3.83a
Days 15
23.64±5.08d
39.73±4.97d
46. 49±5.54bc
71.09±7.36cd
72.80±7.80b
80.48±5.10ab
100±0.00a
Days 20
54.16±4.41d
63.46±4.53cd
77.80±6.08bc
84.07±3.93ab
100±0.00a
100±0.00a
100±0.00a
Table 2: Mean mortality (±SE) of 4th instar larvae of R. ferrugineus larvae after different interval with one dose rate of Imd (1 µl/L), three- dose
rates of B. bassiana (Bb1, 1×106; Bb2, 1×107; Bb3, 1×108) within each treatment followed by the same letter are not significantly different; HSD
test at P= 5%
Treatments
Bb1
Bb2
Bb3
Imd
Bb1+Imd
Bb2+Imd
Bb3+Imd
Mortality(±SE)
Days 5
2.50±1.70c
4.16±2.00c
8.37±2.77bc
12.58±5.11bc
17.67±5.09abc
25.30±5.58ab
32.89±4.44a
Days 10
9.21±2.99E
16.00±4.14de
21.93±4.72de
32.10±4.67cd
44.03±4.94bc
57.50±6.48ab
68.55±3.22a
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Days 15
20.26±4.62d
31.27±5.15cd
42.28±3.62c
65.17±2.60b
67.71±5.57ab
73.59±6.27ab
85.57±1.62a
Days 20
46.53±4.01e
55±4.9de
68.64±3.26cd
79.51±7.37bc
82.14±3.89abc
94.07±2.74ab
100.00±0.00a
Journal of Entomology and Zoology Studies
4. Discussion
The present study suggests that the simultaneous use of a
chemical insecticide enhance the pathogenicity of
entomopathogenic fungi against R. ferrugineus. It is already
established that the synthetic insecticides impose physiological
changes or chemical stresses in insects, rendered them more
vulnerable to hyphomycetes, subsequently fungal spore
penetrate easily into cuticle of insect and successful
penetration results extensive vegetative growth of fungi which
drain the nutrition and ultimately cause death [8, 27, 47, 5, 20, 25-26].
Prior to evaluate the integrated effect of Imidacloprid and B.
bassiana against test insect species, the compatibility test of
both compounds revealed no inhibitory effect of Imidacloprid
on the radial growth of B. bassiana. During a similar study,
Jaramillo et al. [28] found that the conidial or hyphal growth of
M. anisopliae increased positively in Imidacloprid treated and
blank formulation group compared to control untreated group.
Higher colony sizes were observed in M. anisopliae, when
treated with amended neonicotinoids insecticides in a
laboratory experiment [37]. Contrary to this, a significantly
reduced vegetative growth of B. bassiana has been reported by
Anderson et al. [2] with five different non neonicotinoid
insecticides. Likewise, Saenz-de-Cabezón Irrigaray et al. [44]
reported inhibitory effect of triflumuron on the mycelial
growth of B. bassiana.
The mixed application of synthetic insecticides with
entomopathogenic organisms is not only the way to enhance
their insecticidal effect but this is also an alternate approach to
reduce resistance development in different insects as in
combinations they synergize the effect of each other thus
allowing to be used at lower concentrations [6]. Imidacloprid
causes irreversible blockage of postsynaptic nicotinergic
acetylcholine receptor of the central nervous system [34] and
proved to be effective against various sucking insect pests
from different orders include: Diptera, Coleoptera, and
Lepidoptera, and [7].
In our bioassays, significantly higher mortalities of RPW
larvae were observed at combined use of B. bassiana with a
low concentration of Imidacloprid as compared to their sole
applications. Several factors like poor nutrition, overcrowding,
weakened immune system and chemical stress predisposed the
insects to entomopathogenic diseases [27]. The synergistic
phenomenon first reported by Easwaramoorthy et al. [16],
where two insecticides augmented the efficacy of Verticillium
lecanii used against coffee green scale Coccus viridis Green
(Coccidae: Hemiptera). Later on, the combined application of
B. bassiana with sub-lethal dose of abamectin, triflumuron and
carbaryl insecticides caused maximum mortality of Colorado
potato beetle [2]. Another successful use of the synergistic
effect of Imidacloprid and two entomopathogens against
sugar-cane rootstock borer weevil Diaprepes abbreviatus
(Cuculionidae: Coleoptera) [39], and adult guava weevil
Conotrachelus psidii Marshall (Curculionidae: Coleoptera) [9].
The similar findings in present study could be explained by the
fact that sub-lethal dose of Imidacloprid may act as a
behavioral modifier or physiological stressor that reduce the
mobility of test insects thus not allowing them to remove the
attached conidia from their skin. Thus, insecticide intoxication
augmented the pathogenicity of fungi leading to easy conidial
attachment, cuticle penetration, spore germination, or
devastating the immune system of host [21].
Sub-lethal concentration of Imidacloprid not only enhanced
the effectiveness of B. bassiana but also curtailed the amount
of fungal inoculums required to cause high level of larval
mortality of R. ferrugineus. In this experiment, significantly
higher mortality was observed in the combined application of
B. bassiana with imidacloprid against second instar as
compared to fourth after 20 days which are in congruent with
the results of Jaramillo et al. [28] who observed similar result
when M. anisopliae and imidacloprid applied against the
subterranean burrower bug Cyrtomenus bergi Froeschner
(Cydnidae: Hemiptera).
The infection of entomopathogenic fungi cab be disseminated
into the healthy population through the post-mortal sporulation
and mycosis in the dead ones [42]. In the current bioassays,
maximum number of sporulation and mycosis in cadavers of
R. ferrugineus was recorded at the lowest and sole
concentration of B. bassiana as compared to all other
treatments. Similar trend in mycosis and sporulation were
reported by Riasat et al. [41] for the cadavers of R. dominica
(Bostrychidae: Coleoptera) and Tefera and Pringle [48] for
Chilo partellus (F.) (Pyralidae: Lepidoptera), respectively. The
possible reasons of maximum mycosis and sporulation at low
conidial concentration as compared to high concentration
could be due to the fact that the rapid death of the specimen
occurred when treated with high concentration and thus just a
few could be available for sporulation. Moreover, the high
dose rate of fungus also cause the conidial self-inhibition
scenario [48], Contrary to this, Vandenberg [49] found higher rate
of sporulation and mycosed percentage in the cadaver’s of leaf
cutting bee Megachile rotundata (F.) (Hymenoptera:
Megachilidae) at intermediate concentration of B. bassiana
instead of its lower and higher dose rate.
5. Conclusions
To our knowledge, this might be the first study in which the
combine effect of entomopathogenic fungi and Imidacloprid
has been tested against R. ferrugineus. These laboratory trials,
exhibited the potential of this integrated approach for the
successful IPM of R. ferrugineus and it can be exploited for
the control of other insect species of date palms in
combination with certain other reduced risk insecticides.
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