Pakistan J. Zool., vol. 43(3), pp. 543-548, 2011.
Virulence of Entomopathogenic Bacteria Xenorhabdus bovienii and
Photorhabdus luminescens Against Galleria mellonella Larvae
Ali Murad Rahoo, Tariq Mukhtar,* Simon R. Gowen and Barbara Pembroke
School of Agriculture, Policy and Development, University of Reading, Reading RG6 6AR, UK
Abstract.- Keeping in view the serious health and environmental apprehensions associated with the use of
pesticides, entomopathogenic symbiotic bacteria have the potential to supersede pesticides for the management of
various pests. Lab experiments were conducted to test the toxicity of two bacteria Xenorhabdus bovienii and
Photorhabdus luminescens at different bacterial concentrations against Galleria mellonella larvae and influence of
different abiotic factors viz.: substrates, temperatures and moisture levels were ascertained on the efficacy of these
bacteria. P. luminescens and X. bovienii caused the maximum mortality (99 and 90%, respectively) at a concentration
of 4 x 107 cells/ml. Mortality caused by P. luminescens was significantly higher than that of X. bovienii. Highest
mortality was observed on sand as compared to filter paper. A temperature of 30 oC and a moisture level of 20 % were
found optimum for the maximum mortality.
Key words: Entomopathogenic bacteria, Xenorhabdus bovienii, Photorhabdus luminescens, Galleria mellonella
INTRODUCTION
Despite health and environmental hazards
associated with the use of pesticides and
development of insect resistance to insecticides,
pesticides are still mainly relied on for pest control.
Due to rising concerns on the use of these
pesticides, alternative control strategies are being
sought. Entomopathogenic nematodes and the
bacteria having symbiotic relationship with them are
gaining considerable attention for the biological
control of insect pests. Xenorhabdus and
Photorhabdus spp. belonging to Enterobacteriaceae
family (Thomas and Poinar, 1979) have symbiotic
associations
with
the
nematodes
of
Steinernematidae and Heterorhabditidae families,
respectively. This bacteria-nematodes association is
highly pernicious to many insect species. These
bacteria are vectored by the infective juveniles
while being retained in the intestine. After
penetration into the host, the symbiotic bacteria are
released into the haemocoele of the latter from the
gut of the infective juveniles. In the host body, rapid
proliferation of bacteria and subsequent secretion of
several metabolic compounds bring about the
_____________________________
*
Corresponding author. Department of Plant Pathology, Pir
Mehr Ali Shah Arid Agriculture University, Rawalpindi,
Pakistan. E-mail: drtmukhtar@uaar.edu.pk
0030-9923/2011/0003-0543 $ 8.00/0
Copyright 2011 Zoological Society of Pakistan.
establishment of breeding ground both for
nematodes and bacteria and ultimately the insect is
killed within 24 to 48 hours (Bowen et al., 1998;
Khandelwal et al., 2004). The bacteria grow rapidly
in the haemolymph which serve as a nutrient source
for the infective juveniles. The infective juveniles
then undergo two to three generations and produce
new infective juveniles which then leave the insect
carcass to find new hosts. (Thomas and Poinar,
1979). Several toxins secreted by bacteria and
nematodes are responsible for bringing about the
mortality of the insect host.
These entomopathogenic nematodes and their
symbiotic bacteria are environmentally benign and
produce some of the protinacious toxic metabolites
that can be incorporated and expressed in crop
plants or other microorganisms (Raichon et al.,
1994). These bio-control agents can also be applied
in inundative and inoculative forms. These entities
can cause rapid mortality to a wide range of insect
pests akin to majority of chemical pesticides
(Morris, 1985). Although the efficacy of these
entomopathogenic symbiotic bacteria has been
successfully tested against some insects (AbdelRazek and Gowen, 2002; Bussaman et al., 2006,
2009) yet there is dearth of information regarding
the biotic and abiotic factors influencing the
efficacy of these bacteria. The studies reported in
this paper aimed at the evaluation of the infectivity
and toxicity of two bacteria Xenorhabdus bovienii
and Photorhabdus luminescence symbiotically
544
A.M. RAHOO ET AL.
associated
with
Steinernema
feltiae
and
Heterorhabditis bacteriophora respectively at
different bacterial cell concentrations and the
determination of some of the key factors
(temperature, substrate and moisture) influencing
the infectivity of these bacteria against Galleria
mellonella (L).
MATERIALS AND METHODS
Isolation of symbiotic bacteria
X. bovienii and P. luminescens, symbiotic
bacteria, used in the experiments were isolated from
two entomopathogenic nematode species viz. S.
feltiae and H. bacteriophora following the
techniques described by Akhurst (1983). The
nematodes used, were already preserved in the Plant
Protection Lab of Department of Agriculture,
University of Reading, UK and subcultured on last
instar larvae of G. mellonella (Dutky et al., 1964).
White traps were used for harvesting infective
juveniles (White, 1929). The infective juveniles of
S. feltiae were kept in deionized distilled water at a
temperature of 5-7oC and those of H. bacteriophora
at 15oC.
Six G. mellonella last instar larvae were
placed in Petri dishes lined with two layers of filter
papers and inoculated with approximately 500
infective juveniles each of S. feltiae and H.
bacteriophora in 1 ml of distilled water. The Petri
plates were then sealed with Nescofilm and
incubated at 25oC. After three days the cadavers of
G. mellonella were surface sterilized for 5 minutes
in 70 % alcohol and left to dry in a laminar flow
chamber. Larvae were split opened with sterilized
needles and scissors and a drop of the exuding
haemolymph was streaked onto NBTA plates
(nutrient agar (BDH) =37 g, Bromothymol blue
(Raymond) = 25 mg, 2, 3, 5- triphenyl-tetrazolium
Chloride (1 %) (BDH) = 4 ml and distilled water =
1000 ml) with a sterilized inoculating loop. The agar
plates were sealed with Nescofilm and incubated at
25oC for 24 hrs in the dark. Single colonies of each
bacterium were transferred onto fresh NBTA plates.
The process was continued until colonies of uniform
size and morphology were obtained. The
pathogenicity of the isolates was confirmed by
inoculating G. mellonella larvae with the bacterial
cells. The haemolymph of the dead infected larvae
was streaked onto NBTA plates and incubated at
25oC for 3 days. The bacteria were confirmed by the
formation of their characteristic colonies.
Preparation of bacterial suspensions
A single pure colony of each of X. bovienii
and P. luminescens was added to sterilized (15psi at
121oC for 30 mins) nutrient broth No. 2 (30 g/ 1000
ml) and placed in a shaking incubator at 150 rpm for
24 hrs at 28oC in dark. The optical density of the
broth containing bacteria was measured using
spectrophotometer adjusted to 600 nm wave length.
The concentration of bacterial cells in broth was
determined by making dilutions of the broth, plating
onto NBTA plates and counting colony forming
units (cfus) after three days. The concentration of
broth suspension was adjusted to 4 x 107 cells / ml.
To obtain bacteria in water, the broth suspension
was centrifuged at 4100 rpm for 20 mins in 250 ml
centrifuge tubes. A pellet of bacterial cells was
formed at the bottom of centrifuge tubes. The
supernatant was drained off and sterilized tap water
was added and repeated the same procedure for
three times to obtain bacteria in water. The optical
density of the suspension was measured and
concentration of bacterial cells was determined as
mentioned above and adjusted to 4 x 107 cells / ml.
Further concentrations (4 x 106, 4 x 105, 4 x 104, 4 x
103, and 4 x 102) were made by adding requisite
amount of sterilized broth or water and 3% Tween
80 as an emulsifier.
Effect of different bacterial cells concentrations on
the mortality of G. mellonella larvae
The objective of this experiment was to
assess the mortality of G. mellonella larvae with
different concentrations (4 x 107, 4 x 106, 4 x 105, 4
x 104, 4 x 103, and 4 x 102 cells/ml) of X. bovienii
and P. luminescens and evaluate the best mortality
concentration for each bacterium in broth and water.
The required concentrations for each bacterium
were prepared in broth and water and 3% Tween-80
was added. Hundred gram of fine sterilized sand
was put into 9 cm diameter Petri-dishes and the
moisture contents were adjusted to 10 % by adding
EFFECT OF BACTERIA AGAINST GALLERIA MELLONELLA LARVAE
requisite amount of sterilized distilled water. Ten G.
mellonella larvae of similar size, age and colour
were placed in each Petri-dish. The larvae were then
sprayed with 2 ml of each bacterial suspension by a
hand sprayer. Each treatment was replicated thrice.
All Petri-dishes were covered, sealed with Para film
and incubated at 25ºC. Mortality in each
concentration was recorded daily up to fourth day.
Death of larvae due to bacterial infection was
confirmed as described earlier.
Efficacy of bacterial suspension on G. mellonella
larvae on filter paper and sand substrates
The objective of this experiment was to test
the efficacy of best cell concentration (4x 107) of X.
bovienii and P. luminescens on the filter paper and
sand substrates against the larvae of G. mellonella.
Fresh suspension of X. bovienii and P. luminescens
in broth and water were prepared as described
earlier. The concentrations were adjusted to 4x 107
cells per ml and 3% Tween-80 was mixed in all
suspensions. There were three replications for each
treatment. Broth and water served as controls. Ten
G. mellonella larvae were separately placed in Petri
dishes containing double Whatman filter papers and
100 g fine sterilized sand. The larvae on filter papers
and sand were then sprayed with 2 ml from each
bacterial suspension with a hand sprayer. The Petri
dishes were sealed with parafilm and incubated at
25ºC. The mortality of G. mellonella was assessed
after 3 and 6 days. The mortality of larvae due to
bacteria was confirmed as described earlier.
Effect of temperature on the pathogenicity of
bacteria
The objective of this experiment was to test
the pathogenicity of X. bovienii and P. luminescens
cells in broth and water at three different
temperatures against G. mellonella on sand. The
procedure described in the experiment two was
followed. G. mellonella larvae were incubated at
20ºC, 25ºC and 30ºC and mortalities were recorded
as mentioned in the experiment two.
Effect of different moisture levels on the mortality of
G. mellonella larvae treated with bacteria
The purpose of this experiment was to
ascertain the mortality of G. mellonella larvae at
545
different moisture regimes (10 %, 15 % and 20 %).
Petri dishes containing 100 g fine sterilized sand
were adjusted to 10%, 15% and 20% moisture
contents and the procedure described in experiment
two was followed.
Statistical analysis
Corrected mortalities of G. mellonella in all
the experiments were calculated by using Abbott’s
formula (1925).
All the experiments were repeated thrice.
Since there were no discrepancies in the mean
corrected mortalities of all the corresponding
treatments of the repeated experiments, the data of
the three trials were amalgamated before statistical
analysis.
All the data were subjected to analysis of
variance by Genstat 12th edition, version
12.1.0.3278 (2009). Means were separated by
Duncan’s Multiple Range Test. A significant level
of P ≤ 0.05 was used in statistical analyses.
RESULTS
Effect of bacterial cells concentrations on the
mortality of G. mellonella larvae
The effects of bacterial suspensions on G.
mellonella mortality were variable. The mortality
caused by P. luminescens was significantly greater
than that of X. bovienii both in broth and water (F =
2.46, df = 1, 46, P < 0.001). Concentrations also had
significant effects (F = 3.01, df, 5, 46, P = 0.0201)
on the mortality of G. mellonella larvae, being the
maximum (89.5 %) in the highest concentration.
The interaction among bacteria, suspensions and
concentrations was also significant (F = 3.17, df = 5,
46, P = 0.015). The maximum individual mortality
(99 %) was observed in case of P. luminescens in
broth followed by X. bovienii in broth (90%) at the
highest concentration of bacterial cells/ml (4x107).
The minimum individual mortality (15 % and 10 %)
in broth was observed at the lowest concentrations
of bacterial cells (4x102) of P. luminescens and X.
bovienii respectively. However, P. luminescens
caused the maximum mortality of 80% in water
followed by X. bovienii in water (70%) at the
highest concentration. The minimum mortality (8 %
and 12%) was recorded in X. bovienii and P.
546
A.M. RAHOO ET AL.
luminescens in water respectively. The mortality
caused by P. luminescens was significantly higher
as compared with X. bovienii. Similarly the
mortality observed in broth was significantly higher
than water. The minimum mortalities were observed
at the lowest concentrations of both the suspensions
(Fig. 1). There was an increase in the mortality with
an increase in the concentration. These relationships
are shown by equations given below:
YPlb = 16.771 x -0.8667
YXbl = 16.486 x -6.8667
YPlw = 14.086 x -5.4667
YXbw = 12.657 x -6.8
R2= 0.9983
R2= 0.9973
R2= 0.9929
R2= 0.9958
mortality compared to bacteria in water. The
mortality on filter paper was significantly lower
than that on sand (F = 5.93, df = 1, 30, P = 0.029) as
shown in Figure 2.
100
80
60
40
20
0
3 Days
F. paper
Photo in broth
Photo in broth
Xeno in broth
Photo in water
6 Days
Xeno in broth
3 Days
6 Days
Sand
Photo in water
Xeno in water
Xeno in water
120
% Mortality
100
Fig. 2. Effect of X. bovienii and P.
luminescens on mortality of G. mellonella
larvae on filter paper and sand substrates. (LSD
= 3.185)
80
60
40
20
0
C1
C2
C3
C4
Concentrations
C5
C6
Fig.1. Effect of different concentrations of
P. luminescens and X. bovienii on the mortality
of G. mellonella. (LSD = 9.056)
C1= 4 x 102, C2 = 4 x 103, C3 = 4 x 104, C4 = 4
x 105, C5 = 4 x 106 and C6 = 4 x 107
Efficacy of bacterial on G. mellonella larvae when
treated on filter paper and sand substrates
The effects of bacterial suspensions on the
mortalities of G. mellonella when tested on filter
paper and sand substrates varied significantly (F =
10.18, df = 1, 30, P < 0.001). Significantly greater
mortality of G. mellonella was obtained in sand
compared to filter paper. Days also had a significant
effect on the mortality (F = 208.36, df = 1, 30, P <
0.001) . The mortality after 6 days was significantly
greater than that after 3 days. The maximum
individual mortality (98.33%) of G. mellonella
larvae was caused by P. luminescens followed by X.
bovienii (91.67%) in broth on sand after 6 days.
The maximum individual mortality caused
respectively by P. luminescens and X. bovienii in
water was found to be 88 % and 81 % in sand after
six days. The bacteria in broth resulted in higher
Effect of temperature on the pathogenicity of
bacteria against G. mellonella larvae
Temperature was found to have a significant
effect on the mortality of G. mellonella larvae (F =
158.50, df, 2, 46, P < 0.001). The highest mortality
was recorded at 30oC followed by 25oC while the
mortality at 20oC was found to be the minimum.
The maximum individual mortality (98 %) of G.
mellonella larvae was recorded in case of P.
luminescens at 30 ºC followed by X. bovienii (95 %)
in broth after 6 days. The mortality caused either by
P. luminescens or X. Bovienii at 25oC in broth after
6 days was the same. Bacteria in broth were found
to be more effective than those in water (Fig. 3).
Effect of moisture levels on the mortality of G.
mellonella larvae
The effect of moisture levels on the mortality
of G. mellonella larvae was also found to be highly
significant (F = 38.62, df = 2, 48, P < 0.001). The
maximum individual mortality (92 %) was achieved
at 20% level of moisture in case of P. luminescens
in broth after 6 days followed by X. bovienii (80 %).
The mortality after 6 days was significantly greater
than that after 3 days. The mortality in water was
significantly lower as compared to broth. There was
EFFECT OF BACTERIA AGAINST GALLERIA MELLONELLA LARVAE
a significant difference in the mortality of larvae
when treated with P. luminescens and X. bovienii
both in water and broth (Fig. 4).
% Mortality
100
80
60
40
20
0
3 Days
6 Days
3 Days
20
6 Days
3 Days
25
6 Days
30
Temperature (Celsius)
Photo in broth
Xeno in broth
Photo in water
Xeno in water
% Mortality
Fig. 3. Effect of X. bovienii and P.
luminescens on mortality of G. mellonella
larvae at three temperature regimes. (LSD =
1.376)
100
80
60
40
20
0
3 Days
6 Days
3 Days
10%
6 Days
3 Days
15%
6 Days
20%
Moisture levels
Photo in broth
Xeno in broth
Photo in water
Xeno in water
Fig.4. Effect of X. bovienii and P.
luminescens on the mortality of G. mellonella
larvae at three moisture levels. (LSD = 1.642)
DISCUSSION
Entomopathogenic bacteria, P. luminescens
and X. bovienii, have been reported to be greatly
pestilent to several insect pests (Dowds and Peters,
2002). Ansari et al. (2003) demonstrated the
virulence of P. luminescens and X. bovienii against
Hoplia phylanthus and G. mellonella comparable
with other bacterial species. In the present studies
the effects of these two bacteria were found to be
highly significant (P<0.001). The mortality caused
by P. luminescens was significantly greater than that
547
of X. bovienii. Effectiveness of these bacteria has
been tested by different workers for the control of
insects in several lab as well field experiments.
Abdel-Razek (2003) reported that X. nematophilus
and P. luminescens respectively caused 40 and 60 %
mortalities of pupae of diamondback moth (Plutella
xylostella). It has also been proved that culture of P.
luminescens (Strain GPS 12) caused 76 % mortality
of female Luciaphorus sp. within 2 days after
bacterial treatment (Bussaman et al., 2006) and
those of X. nematophila (XI strain) and P.
luminescens (Imported strain) resulted in 85 and 83
% mortalities of L. perniciousus respectively within
72 h after application. X. nematophila (XI strain)
also reduced the fecundity of the mushroom mite
(Bussaman et al., 2009). The deleterious effects of
these bacteria have been imputed to several
enzymes and toxins, which as suggested by Au et al.
(2004), caused destruction of the humoral immune
system of insects by inflicting damage upon the
haemocytes and inhibiting phygocytosis. The oral
toxicity of these bacterial metabolites to insects has
recently been revealed (Bowen et al., 1998; ffrenchConstant and Bowen, 2000). Partially purified
mixtures of these complexes have shown virulence
against many insect pests belonging to Coleoptera,
Dictyoptera and Lepidoptera orders (Bowen et al.,
1998). Several proteins have been purified from
bacterial supernatants, their insecticidal properties
described and the genes encoding them, cloned
(Bowen et al., 1998; ffrench-Constant and Bowen,
2000; ffrench-Constant et al., 2002; Yang et al.,
2009). The toxicity of these proteins has been
ascertained against a variety of insects. It has also
been proved that less than five bacterial cells per
larva can cause more than 50% mortality (ffrenchConstant et al., 2002).
On sand substrate the highest mortality might
be attributed to the abrasions caused on the cuticle
of G. mellonella during its movement that facilitated
the penetration of bacteria in the body as compared
to filter paper. Temperature and moisture also
influenced the mortality, being the maximum at
30oC and 20 % moisture level. This might be due
the fact that G. mellonella can survive better in hot
climate and is more active at higher temperatures.
The most favourable temperature for normal and
healthy development and activity of G. mellonella
548
A.M. RAHOO ET AL.
was found to be 28oC (Jyothi and Reddy, 1996;
Yogesh et al., 2009). The findings of Yang and Li
(1988) also corroborated our results who proved that
the symbiotic bacteria can grow at 18-35oC with the
optimum temperature of 30oC. The results of our
experiments suggest that P. luminescens and X.
bovienii have a great potential for controlling pests
and understanding the key factors for their effective
use in biological control programmes.
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