Egyptian Journal of Biological Pest Control, 24(1), 2014, 247-253
Efficacy of Entomopathogenic Nematodes and Fungi as Biological Control Agents
against the Cotton Leaf Worm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)
Souad A. Shairra and Gehan M. Noah
Biological Control Dept., Plant Protection Res. Institute Agricultural Res. Center, Giza, Egypt.
(Received: May 25, 2014 and Accepted: June 25, 2014)
ABSTRACT
Pathogenicity of the two entomopathogenic nematodes, Heterorhabditis bacteriophora Poinar (HP88 strain) and
Steinernema riobrave and two fungi, Metarhizium anisopliae and Beauveria bassiana as well the combined effect of
both against 3rd instar larvae of the cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) was studied
under laboratory conditions. Data revealed that the tested nematodes differed in their efficacy against 3 rd instar larvae of
S. littoralis. The LD50 values for S. riobrave reached 438.8 and 180.1/ IJs ml/100g diet indicated the most potent
nematodes at 48 and 72 h post treatment, respectively. On the contrary, LC50 reached 584.5 and199.2/ IJs ml/100 g diet,
for H. bacteriophora, respectively. The LC50 values of fungus, B. bassiana revealed least mortality percentage (7.84 x
1011spores ml-1), while M. anisopolie was the most potent (2.89x 108spores ml-1) after 7 days of treatment. Probit
regression lines for the combination of the tested concentration of fungi and nematodes (B. bassiana+ HP88) and (M.
ansoplae + S. riobrave) against 3rd instar of S. littoralis showed that highest larval mortality increased with increasing
fungal spores and/ or nematodes juvenile's concentrations.
Key words: Spodoptera littoralis, entomopathogenic nematodes, entomopathogenic fungi, Efficacy.
INTRODUCTION
Spodoptera littoralis (Boisd.) (Lepidoptera:
Noctuidae) is one of the most notorious chewing
insect pests that causes heavy losses in cotton, thus,
deprives the farmers from getting high yield.
Insecticides of synthetic origin have been used to
manage insect pests for more than 50 years (Charnley
and Collins, 2007). However, due to adverse effects
of insecticides on environment, their rational use is
being advocated. Development of an effective control
method against the cotton leaf worm has been
urgently needed since it does serious damage to many
important agricultural crops in Egypt. There is a
serious interest in the use of microbial insecticides for
biological control of insect pests, as alternatives to
chemical control, since they neither leave toxic
chemical residues in the environment nor do they
induce resistance in their insect hosts (Evans, 1999).
And hence, the public awareness and concern for
environmental quality, has led to more focused
attention on research aiming at developing biological
agents (Hidalgo et al., 1998 and Shairra, 2010). A
promising strategy with good potential to control
insect pests and at the same time, to minimize adverse
effects of chemical insecticides is the use of
entomopathogenic microbial agents such as
nematodes and fungi.
The entomopathogenic nematodes (EPNs) belong
to
both
families
Steinernematidae
and
Heterorhabditidae associated with their symbiotic
bacteria
Xenorhabdus,
and
Photorhabdus,
respectively, have been used commercially as
biocontrol agents of economic insect pests (Gaugler,
2002). After penetration of the infective juveniles
(IJs) into the selected host insects, they release their
symbiotic bacteria into the insect hemocoel where
they grow causing septicemia and/ or toxemia leading
to host death (Georgis, 1992).
Biological control methods offer alternative
choices to use the unsuccessful pollutant chemicals.
Among biological control agents, EPNs and the
entomopathogenic fungi (EPFs) seemed to be the
most appropriate weapon for controlling this serious
pest in soil (Ibrahim and Shairra, 2011). EPFs are
classified as fungi that infect, invade, and eventually
kill their host insects (Singkaravanit et al., 2010).
Because of low availability and cultivation
difficulties, there have been only a few studies on the
characterization of the products of EPFs. It is
estimated that over 800 fungal species belong to more
than 90 genera have been described as pathogenic
against different insect species (Thackar, 2002) but
only a dozen of EPFs species are available for pest
management at grower level (Hajek and St. Leger,
1994). Fungal biological control agents have
demonstrated efficacy against a wide range of insect
pests including Spodoptera species (Purwar and
Sachan, 2005; Lin et al., 2007; Asi et al., 2013;
Gabarty et al, 2014 and Husnain et al., 2014).
The present study aimed to evaluate the infectivity
of different isolates of two nematode species, two
fungi species and combined effect of both against
larvae of S. littoralis under laboratory conditions.
MATERIALS AND METHODS
Host insect:
Spodoptera littoralis larvae were reared on castor
leaves, (Ricinus communis) according to Ibrahim
(1974(. Newly molted 3rd instar larvae were utilized
248
in all experiments.
Nematode species:
Imported nematode species, Steinernema
riobrave; Heterorhabditis bacteriophora Poinar
(HP88 strain), supplied by Dr. El-Sadawy, National
Research Center, Giza, Egypt were used in
experimentation. For culturing, last instar larvae of
both the greater wax moth, Galleria mellonella L. and
S. littoralis were used as insect hosts according to
Shamseldean (1994). Infective juveniles (Ijs) were
harvested with the White's traps (White, 1927). The
Ijs were kept under sterile conditions at 20±2°C and
used in all experiments.
Fungus species:
Tow
entomopathogenic
fungi
isolates,
Metarhizium anisopliae and Beauveria bassiana,
imported by Gaara Establishment, Import and Export
Company. The Manufacture Company was M/S.T.
Stares Company Limit- India. The fungi were kept at
4°C as stock culture. Conidia developed at 25°C from
the stock culture were used for the bioassays.
Susceptibility of S. littoralis larvae to the nematode
species
Third instar larvae, in five groups, were chilled at
4°C for 5 min. About 18 larvae were then infected
with S. riobrave and H. bacteriophora Poinar (HP88)
strains at each dose of the suspensions (16, 32, 64,
128 or 256 IJs /larva / ml distilled water). Plastic cups
(5 x 6 x 6 cm), each contained 120 g of dried beach
sand added with larvae diet (100g leaf of
R. communis) and moistened with 1ml of distilled
water containing the IJs of the nematodes. R.H. of
the soil was (80%) throughout the bioassay. The
host insects were kept in the cups which covered and
placed in an incubator at 27+2oC. Control experiment
was conducted by placing the host larvae on
sand moistened with 1ml distilled sterilized water.
Number of dead larvae in each cup was recorded
at 24, 48, 72, 96 & 120 hours post infection. Each
dose was repeated 3 times for each nematode
isolate or strain. Values of LC50 and Slope were
calculated.
Dose-mortality effect of EPFs against 3rd instar
larvae of S. littoralis
Third instar larvae of S. littoralis were sprayed
individually by different spore concentrations
(1x104, 1x106 and 1x108 conidia ml-1) of the fungal
isolates, M. anisopliae and B. bassiana. For the
control treatment, larvae were sprayed with
0.1% Tween 80 solution. Each treatment, had a batch
of 18 larvae, was replicated three times. Insect
mortality was recorded daily up to ten days.
The median lethal concentrations (LC50) were
determined.
Combined effect of entomopathogenic fungi and
nematodes on S. littoralis larvae
Individuals in three groups of 3rd instar larvae of S.
littoralis were placed in a Petri dish of 9 cm diameter,
lined with a filter paper disk. Larvae of the first group
were fed for 2 days on castor leaves contaminated
with M. anisopliae or B. bassiana, suspensions
(1x108, 1x106, 1x104 spores/ml-1), respectively.
Second group larvae were co-infected with the
nematodes S. riobrave or H. bacteriophora Poinar
(HP88 strain) suspensions (104 +64; 106+32 and
108+16, spores/ IJs / larva / ml distilled water),
respectively. Larvae of the third group were fed
normally and served as control. Control and
experimental larvae were anesthetized on ice for 5
min before used under laboratory temperature ranged
between 25 and 27±2°C. Number of cadavers was
recorded at 3,7 and 10 days post-infection.
Accumulative mortality percentages of the host
individuals were calculated. Each treatment was
repeated three times.
Statistical analysis
Percentages of mortalities were corrected
according to Abbott's formula (Abbott, 1925). The
data were then subjected to probit analysis through
software Computer program to obtain the LC50 and
slope values. Or using a software package “Ldp-line”
a copyright by Ehab Bakr, Plant Protection Res.
Institute, Giza, Egypt and Finney (1971). Analysis of
variance (ANOVA) was conducted on all data using
“COSTAT”, computer program software. Means
were compared by Duncan’s multiple range test
(Duncan, 1955).
RESULTS AND DISCUSSION
Susceptibility of S. littoralis larvae to nematodes
Results in Table (1) show the corrected mortality
percentages of S. littoralis larvae after feeding on
castor leaves treated with different concentrations of
nematodes Ijs (Ijs/ml/100 g diet). Obtained data
clearly revealed that the tested nematodes differed in
their efficacies against 3rd instar larvae of S. littoralis.
Results indicated that the mortality rates increased
with increasing the concentration and period after
treatment. Corrected mortality percentages after
24 and 48 hours of treatment with nematodes,
H. bacteriophora (HP88) and S. riborave ranged
between 4.49-15.07% for both and ranged between
4.49-35.24 and 4.49-39.11% at the lowest (16
ijs/ml/100g diet) and the highest doses (256
ijs/ml/100g diet) concentrations, respectively (Table,
1). While the corresponding figures after 72; 96 and
120 hours of treatment with the respective nematodes
ranged between (7.17-54.88 and 6.30-58.79%);
(14.71-72.10 and 7.97-71.02%) and (20.73-86.32 and
249
Table (1): Corrected mortality % of S. littoralis larvae treated with H. bacteriophora (HP 88) & S. riborav at
successive days post treatment under laboratory conditions
Nematode strain / concentration
( Ijs/ml/ 100g diet )
16
H. bacterio-phora
32
(HP88)
64
128
256
16
32
S. riborave
64
128
256
24h
4.49
6.29
8.62
11.53
15.07
4.49
6.29
8.62
11.53
15.07
Corrected mortality (%) after
48h
72h
96h
4.94
7.17
14.71
9.13
18.56
26.11
15.51
28.93
40.85
24.30
41.44
57.03
35.24
54.88
72.10
4.47
6.30
7.97
8.96
13.75
17.96
16.17
25.66
33.48
26.37
41.46
52.54
39.11
58.79
71.02
120h
20.73
36.77
55.55
73.14
86.32
31.19
40.93
51.26
61.51
71.02
Table (2): LC50 (ml/100 g diet) and slope values for H. bacteriophora (HP88) and S. riborav against 3rd instar
larvae of S. littoralis at confidence limits (95%)
Treatment
H. bacteriophora (HP88)
S. riborave
H. bacteriophora (HP88)
S. riborave
H. bacteriophora (HP88)
S. riborave
H. bacteriophora (HP88)
S. riborave
H. bacteriophora (HP88)
S. riborave
LC50 Confidence limits (95%)
24h after treatment
19271.86 (1974.96 - 2.89 E+09)
19271.86 (1974.96 - 2.89 E+09)
48h after treatment
584.47 (345.22 - 1523.03)
438.78 (195.86 - 13950.09)
72h after treatment
199.18 (147.88 - 303.80)
180.11 (143.13 - 244.70)
96h after treatment
94.76 (77.55 - 119.18)
116.99 (97.85 -114.30)
120h after treatment
52.27 (43.50-62.15)
58.85 (42.57-80.04)
Slope
0.551
0.551
1.056
1.181
1.126
1.455
1.358
1.629
1.587
0.867
Fig. (1): Probit regression lines of combination of the tested concentrations of fungi and nematodes (B.
bassiana+ HP88) against 3rd instar of S. littoralis.
250
Fig. (2): Probit regression lines of combination of the tested concentration of fungi and nematodes (M.
ansoplae + S. riobrave) against 3rd instar of S. littoralis.
Table (3): Efficacy (corrected mortality %) of the
fungi, B. bassiana and M. ansoplae against 3rd
instar larvae of S. littoralis at different days post
treatment
Fungi Concentration
( spores/ml-1/100g diet)
104
B. bassiana
106
108
104
M. ansoplae
106
108
Corrected mortality (%) after
3-days 7-days 10-days
4.27
8.93
9.95
21.75
19.80
41.44
6.15
11.97
19.37
10.67
25.81
49.80
17.18
45.15
80.36
31.19-71.02%) at the lowest (16ijs/ml/100g diet) and
the highest doses (256ijs/ ml/100g diet)
concentrations, respectively. It could be noted that
mortality percentages were distributed in first two
intervals of treatments (i.e. about 50% of the recorded
mortality occurred within 72 hours). On the other
hand, higher concentrations of nematodes caused an
acute effect while latent effect was observed in the
case of lower ones. Statistical analysis of the
accumulative percentage mortalities of S. littoralis
larvae revealed a significant increase by increasing
the doses in a comparable manner for the tested
nematode isolates (Table 1). Data showed that death
of the insect larvae started within 2-3 days after
application with the nematode, H. bacteriophora
(HP88) at the concentrations 16; 32; 64, 120 and 256,
respectively and increased within 3-5 days after
application. Obtained results agree with those of
Shairra (2007) who found a positive relationship
between doses and host mortality, mainly due to the
numbers of infecting nematodes. However, the
defense reactions against the nematodes and their
associated bacteria may play an important role. ElBishry et al. (2002) demonstrated that nematode dose,
infective juvenile age, exposure period, host species,
host size, larval diet and starvation were the factors
affected penetration of three isolates
Heterorhabdits (AS1, RM1 and HP88).
of
Ricci et al. (1996) reported that complete
mortality was achieved when the insects were
exposed to S. riboravis or S. feltiae for only 2h and
the number of individuals that penetrated into the
cadavers increased gradually as the time of exposure
lengthened. As shown in table (1), statistical analysis
revealed no differences in LC50 values for both
nematode species. The LC50 values showed S.
riobrave as the most potent nematode after 48 & 72h
post treatment, as LC50 reached 438.779 and 180.113/
IJs ml/100g diet, respectively. On the contrary, for H.
bacteriophora the respective values were 584.4752
and 199.182/ IJs ml/100 g diet. H. bacteriophora had
the lowest LC50 value (94.764 and 52.267/IJs ml/100
g diet) (Table 2). While, S. riobrave recorded a
moderate effect against S. littoralis, with LC50
reached 116.990 and 58.850, after 96 and 120h,
respectively. Infectivity of Heterorhabditis to its
insect hosts considerably differs depending on the
nematode species and insect host. Also, the range in
number of invading nematodes varied.
These results may imply that variable insect
susceptibility to heterorhabditids is not due to
numbers of infecting nematodes but it is mainly due
to the defense reactions against the nematodes
and their associated bacteria. Shairra (2000)
and Shamseldean et al., 2008 demonstrated that
in case of G. mellonella, S. littoralis or S. litura,
for example, 100 %mortality was achieved with
10, 200 and 1000 IJs/caterpillar, respectively 48h
after exposure to the tested nematodes. In the present
study, the two tested nematode species grew faster in
S. littoralis larvae. Low susceptibility of insect pest to
the nematode isolates observed may be attributed to
physical or behavioral host reaction which prevents
IJs from penetrating the target host. This finding
agrees with El-Bishry et al., )2002) and Ibrahim and
251
Table (4): LC50 (spores/ml-1/100 g diet) and slope values of B. bassiana & M. ansoplae against 3rd instar larvae
of S. littoralis at confidence limits (95%)
Treatment
B. bassiana
M. ansoplae
B. bassiana
M. ansoplae
B. bassiana
M. ansoplae
LC50 Confidence limits (95%)
3days after treatment
14
2.330E x 10 (4.819E x 1010-1.000E x 1038)
7-days after treatment
7.839E x 1011 (6.571E x 109-1.830E x 1019)
2.897E x 108 (4.763E x 107-9.023E x 109)
10-days after treatment
5.835E x 108 (9.057E x 107-2.079E x 1010)
1.027E x 106 (4.384E x 105-2.410E x 106)
Shairra, (2011).
Susceptibility of S. littoralis larvae to fungi
Corrected mortality percentages, 3 days after
treatment with the fungi, ranged between (0 - 6.15 %)
at the lowest (5x104 spores/ml-1/100g diet) and (017.18%) at the highest (5x108 spores/ml-1/100g diet)
concentrations for B. bassiana and M. ansoplae,,
respectively (Table 3).
Corrected mortality percentages increased after
7 days of treatment and ranged between 4.27-11.97
and 19.80-45.15% at the lowest (5x104 spores/ml1
/100g diet) and the highest (5x108 spores/ml-1/100g
diet) concentrations of fungi, B. bassiana & M.
ansoplae, respectively (Table 3). Results indicated
that mortality rates of both fungi increased with
increasing the concentration and the period after
treatment. Corrected mortality percentages, 10 days
after treatment with fungi, ranged between 8.93-19.37
and 41.44-80.36%, at the lowest (5x104 spores/ml1
/100g diet) and the highest (5x108 spores/ml-1/100g
diet) concentrations of fungi, B. bassiana & M.
ansoplae, respectively. Virulence of EPFs varied
from species to species and strain to strain against S.
littoralis (Lin et al., 2007). Susceptibility of the insect
to EPFs decreases with advancement in age of larvae
of the target host (Purwar and Sachan, 2005).
Cumulative mortality indicated that the strains of M.
anisoplaie and B. bassiana affected the 3rd instar
larvae of S. littoralis. B. bassiana revealed least
mortality percentage while M. anisopolie showed the
highest mortality rate against S. littoralis larvae.
These results agree with those of Godonou et al.
(2009), and Husnain et al. (2014). About 50% of the
recorded mortality recorded within the 7 days at
tested M. anisoplaie concentrations, except the lowest
one, while in case of B. bassiana, the most of
mortality occurred after 10 days following treatment
(Table 3).
Dose-mortality effect of tested fungi against larvae
of S. littoralis
Third instar larvae of S. littoralis were susceptible
Slope
0.149
0.218
0.264
0.282
0.430
to all EPF isolates used in the bioassay in a dose
dependent manner (Table 4). Mortality caused by
each fungus was low at lower spores’ concentrations.
It increased with increase of spores’ concentration.
The highest mortality was induced by M. anisopliae,
followed by B. bassiana. The median lethal
concentration (LC50) value for 3rd instar larvae was
(LC50 = 0; 7.839E+11& 5.835E+08 conidia ml-1) with
B. bassiana at 3; 7 and 10 days post treatment (Table
4). On the other hand, the fungus M. anisopliae
showed (LC50= 2.330E+14; 2.897E+08 and
1.027E+06 conidia ml-1) at 3; 7 & 10-days after
treatment. M. anisopliae was the most potent fungi
after 7 days of treatment (LC50 reached 2.897E+08
conidia ml-1). On the contrary, B. bassiana fungi
(LC50= 7.839E+11 conidia ml-1) was the least
effective fungi (Table 4). Ten days after treatment, M.
anisopliae became the most effective fungus, as it had
the lowest LC50 value (1.027E+06 conidia ml-1). This
might be due to defense mechanisms of target insect.
It is well documented that older instars of the cotton
leaf worm are able to tolerate toxic effect of this
fungus.
EPFs that infect insects have received
considerable attention by scientists for their potential
use in biological control of pests. Some pathogenic
fungi have restricted host ranges while others have a
wide host range, e.g., B. bassiana and M. anisopliae.
Many researchers have focused on the selection of
virulent strains for target pests and their development
as biological control agents (Angel-Sahagum et al.,
2005 and Godonou et al., 2009). Similarly, Anand
and Tiwary (2009) observed highest morality of 2nd
instar larvae of S. litura at the highest spores’
concentration of fungal isolates. The growth indicated
by white spores of B. bassiana on the dead Agrotis
ipsilon larvae treated with the LC50 (2 x 108spore/ml)
after 7, 10, 13 and 16 days. The mycelium started to
grow after 7 days from death of infected larvae, and
then the insect cadaver was covered by mycelium
after 10 days. The formation and discharge of spores
were found after 13 and 16 days; respectively
(Gabarty et al., 2014).
252
Combined effect of fungi strains and nematodes
isolates on larvae of S. littoralis
The combined effects of fungi strains based on
B. bassiana or M. anisopliae and entomopathogenic
nematode, H. bacteriophora (HP88) and S. riborave
differed than the applications of each pathogen alone
(Figs. 1 and 2). In the present work, most
combinations showed increased of host mortality.
Combined applications of entomopathogens and
sublethel dosages of synthetic insecticides or other
biological control agents have been proposed as a
strategy to improve the efficacy of microbial control
agents (Anderson et al., 1989). Several studies have
reported additive or synergistic effects from the
combination of entomopathogens, mostly fungi, with
insecticides (Ericsson et al., 2007). Rao et al., (1990)
reported that more attention should be paid to the use
of bioinsecticides based on bacteria, fungi, and
viruses. These groups have unique modes of action
(Thompson et al., 1999) and their properties may
differ considerably from the conventional agents with
which growers are familiar. In the present study, it
was observed that highest larval mortality of
S. littoralis increased with increase in fungi spores
and nematodes infective juveniles’ concentrations
and time elapsed after treatment. The interaction
between fungi and nematodes may allow reducing
chemical application rates. Additionally, the
nematodes may become established and begin to offer
a long term reduction in the larval populations (Klein
and Georgis, 1992). This study gives additional
support to the importance of combinations between
the entomopathogenic fungi and nematodes for the
control of insect pests.
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