Laboratory Evaluation of Entomopathogenic Fungi Metarhizium anisophilae
and Beauveria bassiana Against Termite, Macrotermes (Isoptera: Termitidae)
Termites, Macrotermes are major agricultural and domestic problem in Ethiopia
causing serious damage with loss up to 100%. The use of synthetic termitecids
was the most commonly used prevention measure to reduce the termites attack. However,
these synthetic termiticides were known to be very harmful to the environment
and non-target organisms. Therefore, efficacy of entompathogenic fungi, Metarhizium
anisopliae (isolates PPRC-2 and MM) and Beauveria bassiana (isolates
PPRC-56 and 9609) were evaluated against Macrotermes. The isolates were
obtained from Ambo Plant Protection Research Center, Ethiopia. For each isolate
concentrations of 1x105 to 1x109 conidia mL-1
were prepared and used as treatments. Untreated and standard (Diazinon 60% EC)
checks were used for comparison. The treatments were laid out in Completely Randomized
Design (CRD) and replicated thrice. The fungal isolates were evaluated by direct
spraying of spore suspensions on worker Macrotermes spp. The result of
the study revealed that all fungal isolates used were able to infect and cause
mortality at all concentrations. The percent mortality of Macrotermes varied
from 60 to 100% for M. anisopliae isolate MM at 1x105 to M.
anisopliae isolate PPRC-2 at 1x109, respectively. Similarly, the
percentage mortality of Macrotermes varied from 25-95% for B. bassiana
isolate 9609 at low concentration and isolate PPRC-56 at highest concentration,
respectively. The isolates had LT50 ranging from 7.74 days in M.
anisopliae isolate PPRC-2 to 8.80 days in B. bassiana isolate 9609.
The concentration response with the isolate PPRC-2 showed the lowest LC50
of 3.21x105 conidia mL-1 followed by isolates MM, PPRC-56
and 9609 with LC50 of 3.82x105, 4.39x105 and
5.08x105 conidia mL-1, respectively. In conclusions, the
present study suggests that the use of entompopathogenic fungi, M. anisopilae
and B. bassiana, at higher concentrations for seven days is an eco-friendly
effective mycoinsecticides that causes more than 95% mortality of Macrotermes.
Received: December 05, 2012;
Accepted: March 11, 2013;
Published: June 21, 2013
Termites belong to the insect order Isoptera and have long been recognized
as important agricultural and domestic pests (Owusu et
al., 2008; Samb et al., 2011). Among
all termites, the largest termite species are the Macrotermes spp. known
to be fungus growing and mound building. Macrotermes builds large epigial
nests (mounds) from where they forage outwards to distances up to 50 m in galleries
(Osipitan and Oseyemi, 2012). Macrotermes are
economically important insect pests causing serious damage up to 100% loss to
agricultural crops and various domestic products (Abdurahman,
1990; UNFAO, 2000; Sekamatte,
2001; Nyeko et al., 2010). Damage by these
termite species to agricultural and commercial products also provides entry
for secondary infection by pathogen especially Asperagilous, which cause
indirect yield loss and contamination of products with aflatoxins (Osipitan
and Oseyemi, 2012).
Management of termites has largely relied on broad spectrum and persistent
organo chlorine insecticides in the world (Logan et al.,
1990). Nowadays, most of the persistent insecticides were banned or withdrawn
from the market for human health and environmental reasons. Also, synthetic
insecticides are not affordable by poor farmers of Africa including Ethiopia.
Thus, there were serious limitations and increasing legal restrictions associated
with the application of persistent and deleterious insecticides; because of
which the search for environmentally benign alternative methods of termite management
has been intensified by entomologists. Among the diverse potential alternatives
available for termite management, the use of entomopathogenic fungi is getting
momentum (Michael, 2005).
Entomopathogenic fungi, Beauveria bassiana (Basamo) and Metarhizium
anisophilae (Metchnikoff) are effective in the management of different species
of termite (Milner et al., 1998; Milner,
2003) which may be used in different methods among which direct exposure,
soil barrier and in baits system were able to achieve good control in termite
colony. The genus Metarhizium, Beauveria and Paecilomyces are
fungal pathogens of insects that have shown great promise in commercial development.
M. anisopliae, with worldwide distribution has been isolated from more
than 200 insect species across seven orders and has shown great potential as
bio-control agents. Strains of Metarhizium differ in their host range,
necessitating selection of the most virulent against a target insect (Zimmermann,
M. anisopliae has an advantage over B. bassiana in microbial
management of termite due to their social behavior and high production of fungal
biomass (Sun et al., 2002). Pik-Kheng
et al. (2009) reported that isolates of M. anisopliae were
pathogenic against subterranean termite, Coptotermes curvignathus, causing
100% mortality at 1x107 conidia mL-1 within 3 days post-inoculation.
According to David et al. (2010), a positive
relationship between virulence and repellency effect of different isolates of
the fungus M. anisopliae on M. michaelseni was determined. Further,
they compared the volatile profiles of two isolates of M. anisopliae,
on the same species of termite and found that the volatiles of each isolate
The entomopathogenic fungus, M. anisophilae, has more host ranges and
is widely used as bio-pesticide agents on several types of insect pests which
include onion thrips, storage cowpea, white grub, cattle ticks and different
species of termites such as Reticulitermes spp., Rhinotermitidae,
Coptotermes formosanus and Odontotermes formosanus (Maniania
et al., 2003; Sun et al., 2003; Wang
and Powell, 2004; Cherry et al., 2005; Samson
et al., 2005; Bahiense et al., 2006;
Dong et al., 2009). Many strains of M. ansisopliae
have been isolated from termites and are reported as effective myco-insecticides
(Sun et al., 2003; Wright
et al., 2005) for the management of subterranean termites. Entomopathogenic
fungi isolate, Aspergillus terreus, spore suspension at different concentration
was tested on different stage of tick, Hyalomma anatolicum anatolicum
and has shown promising result as bio-pesticides (Suliman
and Mohammed, 2012).
Biological control of different agricultural and domestic pests has been reported
as promising option in the current and future state of pest management (Bittencourt,
2000; Chandler et al., 2000; Kaaya,
2003; Sharma et al., 2011; Jaramillo
and Borgemeister, 2006). Entomopathogenic fungi, M. anisophilae,
pathogenicity has been studied and found to cause mortality on different stage
of insect pests such as filth fly parasitoid, Spalangia Cameroni, Malaria
mosquito, Anopheles Gambia (Nielsen et al.,
2004; Scholte et al., 2006). Thus new management
approach in Macrotermes is deemed of prime necessity in Ethiopia. However,
the report on the use of entomopathogenic fungi for the management of termite
in Ethiopia is very meager despite the economic importance of Macrotermes
as insect pest. Therefore, the current study was aimed at determining the efficacy
of some available isolates of entomopathogenic fungi (B. bassiana and
M. anisophilae) at different concentrations for the management of Macroterms.
MATERIALS AND METHODS
Description of the experimental area: The experiment was conducted in
Entomology and Plant Pathology Laboratory, Jimma University, College of Agriculture
and Veterinary Medicine (JUCAVM) which is located at 354 km Southwest of Addis
Ababa, Ethiopia. JUCAVM is at geographical coordinate of 7°42 N latitude
and 36°50 E longitude with an altitude of 1710 masl. The temperature
ranges from 11.8°C (minimum) to 26.8°C (Maximum) with maximum relative
humidity and mean rainfall of 91% and 1500 mm per annum, respectively (Abera
et al., 2011).
Termite collections and establishment for test: Termite, Macrotermes
spp., population were collected from two main sites, namely Saye Kebele of Mana
and near Agaro town of Agro Woredas. At these two localities, termite mounds
were dug up using spade and soil containing termites were put on plastic sheets.
Termites were collected from the plastic sheets using camel hair brush and placed
in plastic boxes (polyethylene plastic box) (2217x7 cm3) as described
by Gitonga et al. (1995). Wooden plants (termite
infested materials) were added to the plastic boxes as feed for the termites.
Then the top parts of the plastic boxes were covered with a mesh cloth that
allows air ventilation in and out easily but preventing the escape of the termites.
Moistened wad of cottons were placed in the plastic boxes to maintain the required
moisture level (more than 60%) for the survival of termites. The boxes carrying
the termites were transported to JUCAVM, Entomology and Plant Pathology Laboratory
and placed in a cool and dark area until needed for the experiments. Periodically,
dry wooden materials were provided to the termites population and the
plastic boxes were inspected for maintenance of the required moisture level.
Source of entomopathogenic fungi isolates: Two already cultured species of entomopathogenic fungi (M. anisopilae and B. bassiana) and two isolates of each species, viz., MM, PPRC-2 (M. anisopliae) and 96O9 and PPRC-56 (B. bassiana) used in this experiment were obtained from Ethiopian Institutes of Agricultural Research, Ambo Plant Protection Research Center.
Preparation of spore suspension: To prepare fungal spore suspension,
the conidia were harvested by flooding each plate with 10 mL of 0.05% Tween
80 (sigma) as a wetting agent in sterile distilled water. The conidial suspensions
were prepared by mixing the solution using a magnetic stirrer for 5 min and
the suspensions were filtered through sterile muslin cloth to eliminate the
coagulated medium. The spore suspension of 1x105, 1x106,
1x107, 1x108 and 1x109 per mL of distilled
water were prepared for each isolates by using haemocytometer as described by
Experimental design and treatments: There were 22 treatments (Table 1) replicated thrice and laid-out in Completely Randomized Design (CRD). In this experiment, there were two controls (the untreated and the standard controls, viz., sterile water and 0.21% a.i. Diazinon, respectively) for comparison. Each isolate was tested at five concentrations (1x105, 1x106, 1x107, 1x108 and 1x109 conidia mL). The experiments were conducted under laboratory conditions at 25±2°C temperature and 60% RH in dark places.
Median Lethal concentration (LC50) and time (LT50):
Median lethal time (LT50) and Median lethal concentration (LC50)
were determined for each concentration by taking in to account the time and
concentration required at which the inoculums of fungus caused 50% of the mortality
on worker termite population.
|| Entomopathogenic fungi isolates investigated in the present
|APPRC: Ambo Plant Protection Research Center
Lethal time (LT50) and lethal concentration (LC50) required
to achieve 50% mortality per replicate was obtained from probit analysis.
Mortality of termite due to fungal isolates: To test the efficacy of
each of the fungal isolates on termites, twenty worker termites were placed
in a Petri dish on a filter paper and sprayed with the spore solutions as described
by Julia and Lina (2010). To determined how many termites
were dead without being infested with entomopathogenic fungi, a control group
were sprayed with only sterile distilled water as a negative control. The efficacy
was evaluated on a daily basis for 15 days, by counting dead termites which
were later converted to percentage mortality. Petri dishes containing the treated
termites were maintained at 25±2°C in the dark. Mortality data were
corrected for the corresponding control mortality by the formula:
where, CM is corrected mortality, T is mortality in treated insects and C is
mortality in untreated insects (Abbott, 1925).
Data analysis: The data recorded for different response variables in
the study were analyzed statistically by using one-way analysis of variance
model in SAS version 9.2 Software packages. Based on significant differences
of treatments, mean separation was done using Turkeys studentized (HSD)
test. Mortality rate were corrected using Abbot (1925)
formula. United State Environmental Protection Agency (USEPA) probit analysis
version 1.5 was used for analyzing median lethal time and median lethal concentrations
for each isolates at different concentrations (Finney, 1971).
All recorded variables were analyzed according to the following statistical
where; Yij is any response, μ: is the general mean effect, Ti is the ith treatment effect and Eij is the experimental error.
Percentage mortality of worker termites: Percentage mortality of worker
termites was calculated for the different concentrations of the four isolates
and showed increasing mortality with increasing spore concentration. Cumulative
mortality of worker termite over exposure period (one to seven days) was significantly
different (p<0.05) for the fungi isolates (Fig. 1).
||Cumulative Percent mortality of termite treated with M.
anisopliae (MM and PPRC-2) and B. bassiana (9609 and PPRC-56)
||Percent Mortality of Macroterms treated with different
fungal isolates at 1x109 conidia mL-1 (highest concentration)
On the first day of exposure, maximum mortality (100%) was registered from
standard check, while the untreated control had zero mortality. These were significantly
different from all concentration of the isolates. Among the isolates, maximum
mortality was observed from PPRC-2 isolates (19 to 100%) followed by MM isolates
(14 to 100%) on day one up to day seven after treatment. Minimum mortality was
registered from 9609 isolates (4 to 100%) followed by isolate PPRC-56 (11 to
100%) within the same period of exposure. Generally, as time progressed, cumulative
mortality for all isolates increased attaining 100% mortality on the seventh
day after application of the spore concentrations.
At the highest concentration, 1x109 conidial mL-1, all B. bassiana and M. anisopliae isolates gave complete reduction in population of worker termites resulting to 100% mortality after sixth day of exposure (Fig. 2). The initial data for pathogenicity of all the four isolates indicated that all of them were virulent, even one day after exposure causing significant mortality (up to 20%) when compared with the untreated control (zero mortality). The mortality percentage for all the isolates gradually increased and on sixth day after exposure all of the isolates gave 100% mortality similar to the standard control.
Mean percentage mortality of the worker termites due to the different isolates
of the entomopathogenic fungi at different concentrations was significantly
different over time (Table 2). After one day of exposure 100%
and zero mortality was registered from standard and negative controls, respectively.
Among the isolates, maximum and significant mortality (23.33%) was registered
from PPRC-2 at highest concentration. However, this concentration was followed
by and non-significantly different from PPRC-2 isolates (21.67%) at 1x108
conidia mL-1, PPRC-2 isolates (20.00%) at 1x107 conidia
mL-1, PPRC-2 isolate (16.67%) at 1x106 conidia mL-1
and MM isolates (18.33%) at 1x109 conidia mL-1. Minimum
mortality (1.67%) was recorded from 9609 isolates at lowest concentration followed
by 9609 isolate (3.33%) at 1x106 conidia mL-1, (5%) at
1x107 conidia mL-1, (6.67% ) at 1x108conidia
mL-1, (8.33%) at 1x109 conidia mL-1 and PPRC-56
isolate (5%) at 1x105 conidia mL-1 and these were on par
with each others. This indicates that M. anisopliae isolate PPRC-2 at
higher concentration is the most pathogenic fungi against termite followed with
M. anisopliae isolate MM. On the second days after exposure, maximum
mortality was also registered from standard check (100%). There was no mortality
registered from untreated control (0%). From the isolates, maximum and significant
mortality (53.33%) was registered from PPRC-2 at higher concentration followed
by PPRC-2 (50%) at 1x108conidia mL-1, PPRC-2 (46.64%)
at 1x107 conidia mL-1 and MM (46.67%) at 1x109
conidia mL-1 which were non-significantly different among each other.
Significantly minimum mortality of 3.33% and 8.33% were recorded from 9609 isolates
at 1x105 and 1x106 conidia mL-1, respectively
which were also on par with the negative control.
On day three, highest and significant mortality (100%) was registered from standard check (Diazinon). No mortality was registered (0%) from control (Water). Highest and non-significant mortality of 81.67% was recorded from the isolates PPRC-2 at higher concentration followed by PPRC-2 at 1x108 conidial mL-1 (78.33%), MM isolate at 1x109 conidia mL-1 (73.33%) and PPRC-2 at 1x107 conidial mL-1 (71.67%) in that order. However, lower and significant mortality, 13.33 and 21.67%, were obtained from 9609 isolates at 1x105 and 1x106 conidial mL-1, respectively.
Similarly, on day four, standard check gave significantly higher worker termites
mortality (100%) while no mortality (0%) was obtained from negative control.
|| Mean percent mortality of termite treated with B. bassiana
and M. anisopliae isolate at different concentrations over time
|Means followed by the same letter within a column are not
significantly different (Turkeys Studentized range test, p<0.05)
Among the isolates, significant and higher mortality (100%) were recorded from
the isolate PPRC-2 at 1x109conidia mL-1, 1x108conidia
mL-1 and MM isolates at 1x109 conidia mL-1 followed
by PPRC-2 (96.67%) at 1x107conidia mL-1, PPRC-56 (95.00%)
at 1x109 conidia mL-1, PPRC-2 (93.33%) at 1x106
conidia mL-1 and MM isolate (93.33%) at 1x108 conidia
mL-1. There were no significant differences among these concentrations
of the respective isolates.
After fifth day of exposure, highest and significant mortality, 100%, of workers termite was registered from standard check; isolates PPRC-2 at 1x107 to 1x109 conidial mL-1, MM at 1x107 to 1x109 conidial mL-1, PPRC-56 at 1x109 conidial mL-1 and 9609 isolate at 1x109 conidial mL-1. Minimum significant mortality (3.33%) was recorded from untreated control. The mortality in the negative control treatment might be because of ageing. From the isolates, minimum significant mortality (45.00%) was registered from 9609 isolate at 1x105 conidial mL-1.
Six days after exposure, minimum and significantly lower percentage mortality was 6.67% from negative control treatment while the other treatments showed non-significant mortality among each other. From the isolates, 9609 isolates registered 68.33% at 1x105 conidial mL-1 and 83.33% at 1x106 conidial mL-1 where as isolate MM scored 98.33% mortality at 1x105 conidial mL-1. However, the rest isolates and standard checks, Diazinon were not significant different from each other with 100% mortality.
The trend on percent morality of worker termite gradually increased over exposure period and attained maximum mortality of 100% from sixth day on wards similar to standard check (Diazinon). In addition, it can be noticed that as concentration of the isolates increased, efficacy also increased on specific day after exposure.
Median lethal time (LT50) for fungal isolates: The median lethal time (LT50), time taken for the death of 50% of worker termite due to different isolates at different concentration (1x105 to 1x109conidia mL-1) were found to be different. From the isolates, M. anisopliae isolate PPRC-2 has shown the shortest median lethal time (7.74 days) at the highest concentration to observe 50% death of the target insect pests. On the other hand, median lethal time noted for the death of 50% of termite treated with B. bassiana isolates 9609 was the longest (8.80 days) at lowest concentration (1x105) (Table 3). Thus, the isolates of each entomopathogenic fungi were found to be promising in terms of the time taken as a management option against termite.
Median lethal concentration (LC50) for each fungal isolates:
Results of the probit analysis obtained from applying known conidial suspensions
of the two fungi isolates have shown that the LC50 for each isolate
||Median lethal time (LT50) of B. bassiana
(9609 and PPRC-56) and M. anisopliae (MM and PPRC-2) isolates against
termite on day seven after treatment
|ais the single hypothesis one-sided p-value of
the association between upper and lower limit (based on Fishers Exact
||Median lethal concentration (LC50) of selected
fungal isolates against termite, Macrotermes treated with aqueous
conidial suspensions of different isolates of B.bassiana (9609 and
PPRC-56) and M. anisopliae (MM and PPRC-2) on day seven
|CL: Confidence limits, X2-Chi-Square
Among the two isolates of each M. anisopliae and B. bassiana
tested; M. anisopliae isolate PPRC-2 had the least LC50 value
(2.35x109 conidia mL-1) followed by PPRC-2 (2.44x108conidia
mL-1) and MM (2.48x109conidia mL-1). While,
the median lethal concentration to killed 50% of termite treated with B.
bassiana isolates 9609 took the highest lethal concentration (5.08x105
conidia mL-1) followed by 9609 isolates (4.55x106 conidia
mL-1), MM (4.40x105 conidia mL-1), MM (4.39x106
conidia mL-1) and MM (4.02x107conidia mL-1)
Spore concentrations of two species of entomopathogenic fungi (M. anosopliae
(isolates MM and PPRC-2) and B. bassiana (9609 and PPRC-56) were applied
on worker termite to determine fungi isolate(s) with high efficacy against Macrotermes
under laboratory conditions. Both fungi isolates were found to be pathogenic
to Macrotermes. However, there was a variation in their virulence against
Macrotermes. The two isolates of B. bassiana were significantly
less effective when compared with that of M. anosopliae isolates in terms
of virulence. Among the isolates, PPRC-2, were found more virulent to the Macrotermes
than the other isolates. Virulence due to M. anisopliae isolates on Macrotermes
was not significantly different as compared to that of standard check after
fifth days of the exposure period both causing 100% mortality. This indicates
that from all the entomopathogenic fungi, M. anisopliae PPRC-2 isolate
was the best entomopathogen for the control of Macrotermes. The next
best entomopathogen was MM, followed by PPRC-56 and 9609 from B. bassiana
in that order. These findings are in conformity with earlier reports (Singha
et al., 2006; Ahmed et al., 2009;
Dong et al., 2009; Pik-Kheng
et al., 2009) who have shown a similar pattern of activity with isolates
of these two entomopathogenic fungi against subterranean termite Coptotermes
curvignathus. Both the fungal species were reported to produce an enzyme
exoproteases with insecticidal activity.
The highest concentration (1x109conidia mL-1) of the fungal isolates M. anisopliae (PPRC-2 and MM) had nearly 100% mortality similar to the synthetic chemical (Diazinon) from day four on wards. On average, all the fungal isolates showed a high level of mortality, ranging from 25 to 75% (9609) and 45 to 95% (PPRC-56) for the isolate of B. bassian while 60 to 100% (MM) and 75 to 100% (PPRC-2) for M. anisopliae isolate when compared to 0.21% Diazinon with 100% mortality over time.
Currently the understanding of how effective the fungal isolates on Macrotermes
was very limited due to less experiments, but many reports are available
showing that the isolates cause a high mortality on other insects (Kannan
et al., 2008). In this study it has been shown that all the fungal
isolates are effective against termite and achieved appreciable mortality. An
increase in the concentration of spores generally increased the mortality and
generated a faster mortality.
After termites were infested with fungus spores, the mortality rate was lowest
on the first two days and increased rapidly on the subsequent days. Percent
mortality of termites depends on concentration of conidia and isolates of fungi.
Termites that have been treated with conidia of M. anisopliae initiate
to die within two days after inoculation. After six to seven days white mycelium
had developed and green conidia were appeared around the insect cadavers. The
reason for this is that fungus spores required the amount of time to thrive
and sprout their mycelia into the termites (Krutmuanga and
Mekchay, 2005; Brogden et al., 2005) opined
that mortality increased after the second day of exposure because specific toxin,
Destruxin, from the extracts penetrated swiftly into the termites hemocoel.
In this study, the calculated values of LT50 for the isolates suggest
that, in general, the M. anisopliae isolates PPRC-2 (7.74 to 8.19 days)
and MM (7.95 to 8.54 days) elicited mortality quickly than did the B. bassiana
isolates 9609 (8.26 to 8.80 days) and PPRC-56 (8.11 to 8.59 days). This observation
conforms to the earlier reports (Singha et al.,
2006; Ahmed et al., 2009; Dong
et al., 2009; Pik-Kheng, et al., 2009;
Cherry et al., 2005). The median lethal time taken
for fifty percent death of target insect (LT50) was more in the current
result may be due to different factors such as difference in physiological combinations,
methods of application of spore suspension and the amount used, the age of the
insects used plus the condition where the experiments were conducted and other
characteristics of entomopathogenic fungi. Also according to Todorova
et al. (2002) and Alizadeh et al. (2007),
lethal time (LT50) depends on the spore suspension and with the increase
in spore suspension there is a decrease in time taken. Adane
et al. (1996) reported the lowest times of 2.74 and 5.54 day for
B. bassiana on bruchid and maize weevil, respectively.
The median lethal concentration (LC50) of different fungal isolates
against termite, Macrotermes depend upon the fungal species or its isolate.
Earlier studies confirmed that conidia of V. lecanii were highly pathogenic
against aphids (Vu et al., 2007). The susceptibility
of target insect to fungal infection is concentration dependent (Liu
et al., 2002; Wright et al., 2005).
Ansari et al. (2004) also reported that mortality
of insect pests due entomopathogenic fungi depends on the concentration of conidial
suspension, time of exposure and temperature. Entomopathogenic fungi M.
anisopliae isolates had low LC50 values (3.21x105
to 3.82x105) compared to B. bassiana isolates (4.39x105
to 5.08x105) which is in line with the findings of Singha
et al. (2011).
Based on this finding, we suggest that M. anisopliae and B. bassiana as a useful option for the management of Macrotermes spp. However, further investigations are strongly recommended to be carried out on the possibility of field application as well as finding other isolates of Entomopathogenic fungi that have potential as bio-termiticidal activities against such termite.
We greatly acknowledge Ambo plant protection research center, Ethiopia for providing the test material, Entomopathogenic fungi (M. anisopliae and B. bassiana isolates). We are also indebted to thank Jimma University College of Agriculture and Veterinary Medicine for providing laboratory space and facilities as well as financial support to conduct the study.
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