Biological control of larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) with entomopathogenic fungi - Beauveria bassiana (Balsamo) vaillemin (Hypocreales: Cordycipitaceae)

Biological control of larger grain borer, Prostephanus truncatus (Horn)(Coleoptera: Bostrichidae) with entomopathogenic fungi - Beauveria bassiana (Balsamo)
vaillemin (Hypocreales: Cordycipitaceae)
IJEN
Biological control of larger grain borer, Prostephanus
truncatus (Horn) (Coleoptera: Bostrichidae) with
entomopathogenic fungi - Beauveria bassiana
(Balsamo) vaillemin (Hypocreales: Cordycipitaceae)
Osipitan AA1*
, Popoola AO2
, Afolabi CG3
, Oke OA4
1*, 2, 3
Department of Crop Protection, College of Plant Science and Crop Production, Federal University of Agriculture, P.
M. B. 2240, Abeokuta, Ogun State, Nigeria.
4
Department of Biological Science, College of Natural Science, Federal University of Agriculture, P.M.B. 2240,
Abeokuta, Ogun State, Nigeria.
*Corresponding author e-mail: osipitan1@yahoo.com, Tel: (+234)8033930581
The use of synthetic insecticide has been faced with challenges of resistance among other
drawbacks. This has necessitated the search for bio-pesticide that are environmentally friendly,
non-toxic to humans and have a residual effect. This study evaluated the entomopathogenic
fungi, Beauveria bassiana for biological control of larger grain borer (LGB) Prostephanus
truncatus in maize grains. Pathogenicity examination of dead adult LGB in maize grains treated
with conidia of B. bassiana was done to confirm the source of LGB mortality in B. bassiana
treated maize grains. Adult dead LGB were subjected to high humidity and observed for the
growth of white mould (Muscadine disease), which was cultured on Potato Dextrose Agar and
identified. Eighty six percent of the dead insects from treated maize grains showed fungal
growth B. bassiana. Mortality of LGB generally increased with the concentration and the
exposure time of the treatments. The “weight of grain dust”, “percentage of grain damaged” and
“percentage of grain weight loss” were significantly (p<0.05) higher in the untreated maize
kernels. Beauvaria bassiana formulation was effective in controlling LGB and is recommended
for maize storage. Further studies should be conducted to test the formulation under farmer
situations in order to deal with practical challenges.
Key words: Beauveria bassiana, fungal growth, pathogenicity, potato dextrose agar, mortality
INTRODUCTION
Maize is a major staple food crop in Africa and it
contributes significantly to the agricultural sector; a
substantial part of the outputs however, is lost to insect
pests on the field and in storage. This constitutes a
major constraint to food security and income generation
in Sub-Saharan Africa (Abebe et al., 2009; Osipitan et al.,
2011).
Larger grain borer (LGB) – (Prostephanus truncatus) is
a native of Mexico, Central America and exotic to Africa.
The insect has since been introduced into Africa through
Tanzania in 1981 (McFarlane, 1988; Pike et al., 1992;
Boxall, 2002), where it is currently a more destructive
pest of stored maize and cassava than in its native
Central America (Dick et al., 1988). LGB is currently
International Journal of Entomology and Nematology
Vol. 2(1), pp. 002-008, February, 2015. © www.premierpublishers.org. ISSN: 2326-7262
Research Article

Popoola et al. 002
established in most parts of Africa threatening maize
production due to its aggressive nature and extensive
damage it causes within a short period of time. The insect
represents the main storage pest in maize stock with
destructive effects over long periods of time (Maboudou
et al., 2004). Storage losses in LGB-infested maize vary
between 15% and 30% depending on regions (ADA,
2010).
Entomopathogenic fungi were among the first organisms
to be used for the biological control of pests and more
than 700 species from around 90 genera are
pathogenic to insects (Florez, 2002). They are
generally safe in terms of low risks as compared to
chemical pesticide and bear a considerable potential for
the control of different stored product pests (Anonymous,
2000; Cox et al., 2004). Beauveria bassiana is one of the
mostly used entomopathogenic fungi because of its
proven efficiency. It has been used to control stored
grains pests such as Acanthoscelides obtectus,
Rhyzopertha dominica, Sitophillus oryzae, S. granarius
S. zeamays, Tribolium castaneum and other pests of
economic importance (Padin et al., 2002; Smith et al.,
2006; Shams et al., 2011; Mahdneshin et al., 2011). It is
a common soil borne saprophyte fungus that occurs
worldwide and attacks range of both immature and adult
insects such as silkworms, whiteflies, aphids,
grasshopper or lady beetles. Commercial formulations of
the fungus are available and registered by the U.S.
Environmental Protection Agency (EPA) for a wide range
of insect control applications (Lord, 2001).
In the last 50 years, synthetic chemicals pesticides have
been the mainstay of insect pest control. However,
insecticide resistance, pest resurgence and concern over
environmental impact of agricultural inputs have led to
search for alternative biologically-based control measure
that is relatively safer, cost effective and
environmental/ecologically friendly (Francisco et al.,
2012). Gerg (1992) opined that pathogens play important
roles in the population dynamics of many insect species.
Insect pathogens such as B. bassiana if properly
harnessed could serve as an environmentally friendly
alternative to chemical insecticides (Pawar and Borikar,
2005). This study therefore, evaluates an isolate of B.
bassiana for pathogenicity on larger grain borer (P.
truncatus) in maize grains.
MATERIALS AND METHODS
Assessment of the isolate of Beauveria bassiana for
pathogenicity to Prostephanus truncatus
Source of Beauveria bassiana and larger grain borer
(LGB)
Commercially formulated conidia of B. bassiana strain
GHA (Botanigard 22 WP) was sourced from Lawn and
Garden Product Inc., Fresna Califonia, United States of
America. Adult LGB were obtained from the
Entomological Laboratory of the Department of Crop
Protection, Federal University of Agriculture, Ogun State,
Abeokuta, Nigeria.
Mortality Bioassay
The isolate of B. bassiana was used at the rate of 0.00,
0.35, 0.70, 1.05 and 1.40g per 40 ml of water per 100 g
maize kernels. One hundred grams (100g) samples of
the maize grains were measured into 500 cm
3
glass jars
covered with netted lid that provide sufficient aeration.
Ten ml each of the treatments was introduced into the
kernels with the aid of micro syringe, mixed thoroughly
and air dried in the laboratory. Twenty adults of P.
truncatus of 1-5 days were introduced into each of the
glass jars. The control was 100 g maize grains in 500 cm
3
glass jars covered with netted lid, infested with P.
truncatus of 1-5 days, but not treated with conidia of B.
bassiana. The treatments and control were replicated
four times and arranged on work tables in the laboratory
in a Completely Randomized Design. The mortality of the
introduced LGB was recorded at 5 days interval for 15
days to determine the effectiveness of the treatments
with exposure time
. The treated maize grains were left for two months
to assess the residual effects of the treatments on at
least two filial generations (progenies) of the introduced
LGB. At two months post-treatment of the maize grains,
the insects were sieved out of the grains and separated
into dead and living. The grains were separated into
damaged and undamaged and each category counted
and weighed. The number of dead LGB and weight of
grain dust (g) were also recorded. Percentage grain
damage and weight loss were calculated as follows:
(i) % Grain damage= Weight of damaged grains x 100%
Total weight of grains
(ii) % Grain weight loss=Initial weight- Final weight x 100%
Initial weight
Pathogenicity Examination of Dead Insects
Ten dead LGB from B. bassiana treated maize grains
were washed in 70% ethanol, rinsed three times in sterile
distilled water to remove any surface contaminants and
kept separately in Petri dishes (Adel and Chelav, 2013).
These plates were then incubated in a plastic box with
high relative humidity (approximately 100%) to observe
the growth of fungus (Khashaveh, et al., 2011; Guarana
et al., 2012; Adel and Chelav, 2013). The same treatment
was done to ten dead insects from the untreated maize
kernels. The white mould from the cadavers was isolated

Int. J. Entomol. Nematol. 003
Table 1. Weight of grain dust, percentage grain damage and grain weight loss of maize kernels treated
with Beauveria bassiana
Concentration
(g/40ml water)
Weight of Grain Dust (g) % Grain Damage % Grain Weight Loss
1.4 4.90±2.16d
22.30±2.26e
6.78±0.35d
1.05 8.10±0.55c
35.80±1.36d
10.70±1.32c
0.7 12.50±0.90b
48.80±1.4c
14.80±0.65b
0.35 13.22±0.66
b
63.10±1.15
b
16.37±1.13
b
Control 18.10±0.29a 77.00±1.49
a
20.25±0.32
a
Means within the same row with the same letter are not significantly different according to Duncan’s
New Multiple Range Test (n = 20, P ≤ 0.05)
on Potato Dextrose Agar (PDA) medium with antibiotics
(Streptomycin) to limit the growth of bacteria. Subsequent
subculturing was grown on PDA. The fungal growth was
identified under the microscope with the assistance of a
pathologist.
Statistical Analysis
Mortality in the control was corrected by using Abbott’s
(1925) formula. Data on insect mortality and percentage
mortality were normalized using arcsine transformation.
Data collected were subjected to analysis of variance
(ANOVA) and significant means were separated by
Duncan’s New Multiple Range Test P ≤ 0.05. Probit
analysis was used to estimate both LC50 and LC95 of
the isolates with 95% Confidence Limits.
RESULTS
Mortality of LGB in maize kernels treated with
Beauveria bassiana at five, ten and fifteen days after
treatment
Mortality of LGB generally increased with concentration
and exposure time of the treatments. At 0.35 g/40ml H2O,
10 insects died at 5 days After Application Treatment
(AAT), 44.38 died at 10 days AAT and 47.60 at 15 days
AAT. At 0.70 g/40ml H2O, 27.57 insects died at 5 days
AAT, 47.07 died at 10 days AAT and 52.40 died at 15
days AAT. At 1.05 g/40ml H2O, 33.55 insects died at 5
days AAT, 48.42 died at 10 days AAT and 65.17 died at
15 days AAT. At 1.4 g/40ml H2O, 41.18 insects died at 5
days AAT, 94.80 died at 10 and 15 days AAT. None of
the LGB in the untreated maize kernels which serve as
control died at 5, 10 and 15 days (Fig.1).
Percentage grain damage and weight loss of maize
kernels treated with Beauveria bassiana.
There were significant differences (P < 0.05) in the
percentage of the grains damaged and weight loss of
maize kernels treated with different concentration of B.
bassiana (Table 1). The Percent Grain Damaged (PGD)
and Percent Grain Weight Loss (PGWL) were high in the
control. PGD and PGWL varied directly with the
concentration of the treatments. The PGD (63.10) in
maize treated with 0.35g of the treatment was
significantly (P < 0.05) higher than the PGD (22.30) in
maize kernels treated with 1.4 g (Table 1). Similarly, the
PGWL (16.37) in maize kernels treated with lowest
conidia mixture (0.35 g/40ml H2O) of B. bassiana was
significantly (P < 0.05) higher than the PGWL in maize
kernels treated with higher conidia mixture (1.4 g /40ml
H2O) of B. bassiana (Table 1).
Weight of grain dust in LGB-infested maize grains
treated with B. bassiana
The Weight of Grain Dust (WGD) in maize grains treated
with of 0.35 g/40ml H2O conidia mixture was significantly
(P < 0.05) higher than the WGD from maize grains
treated with higher concentration (1.4 g /40ml H2O) of
conidia mixture (Table 1). Significantly (P < 0.05) higher
grain dust (18.10 g) was obtained from untreated maize
grains compared to all treated maize grains (Table 1).

Popoola et al. 004
Figure 1. Mortality of LGB in maize grains treated with Beauveria bassiana (n = 20, P ≤ 0.05)
Table 2. Mortality and reproductive success of LGB in Beauveria bassiana-treated maize kernels
Concentration
g/40ml water
Total number
of LGB
Number of
Living LGB
Number of
Dead LGB
% of dead
LGB in total
Grain
Dust (g)
1.4 20.06±0.25
c
0.06±0.00
b
20.00±1.25
c
99.70±0.16
d
4.90±0.16
d
1.05 17.80±0.14
b
4.30±0.85
b
13.50±1.22
b
75.84±0.16
c
8.10±0.55
c
0.7 16.55±0.35b
5.30±0.94b
11.25±0.35b
67.98±0.16b
12.50±0.90b
0.35 17.00±0.44
b
6.75±1.49
b
10.25±0.48
b
60.29±0.16
b
13.22±0.66
b
Control 69.00±1.22
a
67.50±6.23
a
1.50±0.23
a
2.17±0.16
a
18.10±0.29
a
Means within the same row with the same letter are not significantly different according to Duncan’s New Multiple Range Test at (n =
20, P ≤ 0.05)
Mortality and reproductive success of LGB in maize
kernels treated with Beauveria bassiana at sixty days
after application of treatments
The mean number of dead and living LGB at sixty days
after application of treatments is shown on Table 2. The
total (dead and living) number (69.00) of the insect in the
untreated maize kernels was significantly (P < 0.05)
higher than the number in all the treated maize grains.
The total number of LGB in maize grains treated with 1.4
g/40ml H2O of conidia mixture was significantly (P < 0.05)
higher than the number of the insect in maize grains
treated with other conidia mixtures (Table 2).
The mean number (68.00) of living LGB was significantly
(P < 0.05) higher in the untreated maize grains compared
to the treated ones. The number of the insect in other
treated maize grains were not significantly (P > 0.05)
different from each other. At the highest conidia mixture
(1.4 g /40ml H2O), the mean number of living LGB was
0.06 and it was 6.75 at the lowest (0.35 g/40ml H2O)
conidia mixture (Table 2).
Significantly (P < 0.05) higher number (20.00) of LGB
died in maize grains treated with conidia mixture of 1.4 g
/40ml H2O compared to other treated and untreated
maize grains. Similarly, significantly (P < 0.05) higher
percent (99.70) of the insect died in the maize grains
treated with the conidia mixture at highest rate of 1.4 g
/40ml H2O. The mean number (1.50) and percent (2.17)
of dead LGB in the untreated maize grains (control) was
significantly (P < 0.05) lower compared to the number in
all the treated maize grains (Table 2).
0
20
40
60
80
100
120
0 0.35 0.7 1.05 1.4
5days
10days
15day
bc
ab
a
a
b b
b
a
c
bc
a
b
Concentration of Beauveria bassiana (g/40 ml H2O) hhhH2)
water)

Plate 1. Dead LGB with the white muscadine disease
Plate 2. Growth of Beauveria bassiana hyphae from dead LGB
Pathogenicity examination of the dead LGB and
identification of fungal growth
In the pathogenicity examination of the dead insects,
86.0% of the cadavers subjected to the examination grew
the white moulds (muscadine disease) associated with
Beauveria bassiana (Plate 1). Growth of B. bassiana
hyphae from culture of moulds from dead LGB examined
microscopically is shown in Plate 2.
DISCUSSION
In this study, the spores of Beauveria bassiana strain
GHA caused 86.0 % mortality of LGB in treated maize
kernels. This is similar to the results of Bourassa et al.
(2001) which reported that B. bassiana IMI330194 led to
100 % mortality of P. truncatus larvae. Similarly, Hussein
et al. (2013) in a trial of the insecticidal efficacy of
Trichoderma album on Rhyzopertha dominica reported

Popoola et al. 006
that T. album caused 20% mortality within seven days
post spraying at the lowest concentration and 100%
mortality at the highest concentration of the spores. The
authors reported that the spores of the fungi germinated
on the host cuticle, penetrated them and spread through
the body. Hluchi and Samsinakova (1989) reported that
the formulation of entomopathogenic fungi Boverosil®
caused 90% mortality of insect at 5.92 x 10
8
conidia/ml.
Similarly, Héraux et al. (2005) reported that lytic enzymes
secreted by the fungus may likely play a role in the
process of damage. Askary et al. (1998) in a laboratory
bioassays of B. bassiana against several life stages of
the pine beauty moth, Panolis flammea reported high
mortality of the fifth instar larvae and spraying the spores
of the pathogen against the field populations of the
grasshopper, Melanoplus sanguinipes resulted in high
rates of population decline. Weiguo et al. (2005) reported
that entomopathogenic fungi produce proteases,
chitinases and lipases which can degrade insect cuticle.
In the pathogenicity examination of the dead LGB, 86.0%
of the cadavers showed symptoms of infection
suggesting that most of the insects died as a result of
infection by B. bassiana. Mul et al. (2009) and Kaoud
(2010) suggested that after killing the insect the fungus
could grow out of the insect cadaver and produce more
spores thus increasing the chance for other individuals to
be killed. Butt et al. (2001) and Uma Devi et al. (2008)
indicated that the fungal isolates particularly B. bassiana
are found naturally in soils, parasitizing insects, killing or
disabling as it has rapid germination and sporulation, with
a high virulence and good discharge of conidia which
makes it an efficient control agent. Lingappa et al. (2005)
reported that fungal pathogen particularly, Beauveria
bassiana, Metarhizium anisopliae, Verticillium lecanii and
Nomuraea rileyi have been found to be promising in the
control of several agricultural pests.
The results of this study indicated higher efficacy of the
biopesticide and mortality of LGB at higher concentration
suggesting that the mortality was directly related to dose.
This is in agreement with the findings of Caston and
Makaka (2008) which indicated that an increase in the
concentration of spores generally increases the mortality
and might generate a faster result. Malarvannan et al.
(2010) studied the effect of B. bassiana on Spodoptera
litura at four different concentrations (2.4 × 107
, 2.4 × 106
,
2.4 × 105
, 2.4 × 104
conidia/ml) and reported that the least
pupation (43.33%) was observed in larvae treated with
the highest spore concentration (2.4 × 107
) of the fungi
and the healthy moth emergence was least in (2.4 × 10
4
)
spore concentration of the treatment, while the fecundity
was completely arrested in the highest.
CONCLUSION
This study concluded that the entomopathogenic fungus,
B. bassiana was effective in the control of larger grain
borer in stored maize grains. A long term use of this
formulation of entomopathogenic fungi is therefore
recommended for maize storage. However, further
investigation should be conducted to identify other
isolates of entomopathogenic fungi that have potential as
biopesticide against LGB and other important storage
insect pests. These studies were conducted under
laboratory environments which differ from the actual
farmer environments. Further studies should therefore be
conducted to test the formulation under farmer situations
in order to deal with practical challenges.
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Accepted 20 January, 2015
Citation: Popoola AO, Osipitan AA, Afolabi CG, AA, Oke
OA (2015). Biological control of larger grain borer,
Prostephanus truncatus (Horn)(Coleoptera: Bostrichidae)
with entomopathogenic fungi - Beauveria bassiana
(Balsamo) vaillemin (Hypocreales: Cordycipitaceae).
International Journal of Entomology and Nematology,
2(1): 002-008.
Copyright: © 2015 Osipitan et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium,
provided the original author and source are cited.

Biological control of larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) with entomopathogenic fungi - Beauveria bassiana (Balsamo) vaillemin (Hypocreales: Cordycipitaceae)

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Biological control of larger grain borer, Prostephanus truncatus (Horn) (Coleoptera: Bostrichidae) with entomopathogenic fungi - Beauveria bassiana (Balsamo) vaillemin (Hypocreales: Cordycipitaceae)