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Egyptian Journal of Biological Pest Control, 26(2), 2016, 229-236

Toxicological Studies and Field Applications of a New Bacillus thuringiensis Isolate (Bt1)
and Two Chemical Pesticides on Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae)
Fifi, M. Reda1 ; W. A. Hassanein1 ; E. A. H. Sherief2 and Shahera, Moabed2
1 Dept.

Botany and Microbiology, Fac. of Science, Zagazig Univ., 44519, Zagazig, Egypt.
afmo67@yahoo.com Fifi.reda133@yahoo.com
2 Plant Protection Research Institute, Sharkia Branch, Zagazig, Egypt.
(Received: February 3, 2016 and Accepted: March 20, 2016)

ABSTRACT
A total of 35 bacterial cultures were isolated from dead and living larvae of cotton leafworm Spodoptera littoralis (Boisd.)
(Lepidoptera: Noctuidae) and soil samples. Only one bacterial isolate (Bt1) exhibited the highest pathogenicity against
the pest. Identity of one of the most potent isolates suspected and encoded as Bt1 was confirmed by amplifying its 16S
rRNA gene. Partial nucleotide sequence of the amplified 16S rRNA gene of the tested strain was submitted in GenBan k
with accession number KU646830. Pathogenicity of Bt1 strain was tested against 2nd and 4th instars of S. littoralis larvae
at different concentrations (2x109 , 2x108 , 2x107 and 2x106 CFU/ ml). Based on the LC50 value, the 2nd instar larvae were
more susceptible than the 4th one. It recorded 1.9x109 CFU/ ml and 4.1x1011 CFU/ ml for 2nd and 4th larval instars,
respectively, after seven days. Latent effect of Bt1 strain was recorded on some S. littoralis biological aspects as pupation,
pupal weight and adult emergency that decreased, in addition to some failure in pupation and emergence of deformed
adults. In vivo, screening of Bt1 strain culture filtrates against 2nd and 4th larval instars of S. littoralis cleared that filtrat e
including protease had significant effects on larval mortality. Purified protease gave single homogenous band of 68 kDa
by SDS- PAGE. Also, its optimum pH and temperature were 7.0 and 35o C, respectively, with wide pH and thermal
stabilities. Furthermore, purified protease showed high activity against 2nd and 4th instars S. littoralis larvae than crude
protease. A field application was conducted to compare the efficacy of the recommended pesticides (Challenger Super
and New benzuron) and Bt1 culture against the 2nd and 4th instars S. littoralis larvae. Results showed that Challenger Super
was the most effective insecticide and caused 89.61, 93.6 and 88.68% reduction in S. littoralis population after 3, 7 and
10 days post treatment, respectively. However, Bt1 strain was the least toxic effect where the mean reduction was 40.5%.

Key words: Bacillus thuringiensis, Spodoptera littoralis, Pesticides, Toxicity, Proteases, Field study.
INTRODUCTION
Over the years, the widespread use of pesticides
has led to several problems as pesticide resistant
insects, a reduction in beneficial insect populations,
moreover, the harmful effects to humans and
environment (Haq et al., 2004). These problems
invited researchers to create and develop several
strategies to control insects using both synthetic and
natural molecules. Microbial pesticides are natural
disease-causing microorganisms such as viruses,
bacteria, protozoa, and fungi that infect or intoxicate
specific pest groups (Khetan, 2001).
The cotton leafworm, Spodoptera littoralis
(Boisd) is one of the most important major pests of
cotton plants (Gossypium hirsutum L.) as well corn
(Zea mays L.) and sugar beet (Beta vulgaris L.).
Larvae are polyphagous causing important economic
losses in 112 host plants on many field and vegetable
crops (Fouad et al., 2011). In addition to the chemical
control as traditional pesticides, numerous studies
were carried out on the possible microbial control
agents against S. littoralis, including insect viruses,
fungi, and bacteria (Masetti et al., 2008).
In particular, Bacillus thuringiensis (Bt) is a grampositive, spore-forming bacterium that produces
parasporal inclusions during the sporulation phase.
These inclusions are composed of proteins (Cry

proteins) or -endotoxins, which are highly toxic to a


wide variety of insect pests and some invertebrates
(Vilas-Bas et al., 2007). In general, toxins insert
themselves into midgut epithelial cells, where lead to
pore-forming toxins that lyse the cells and
subsequently causing insect death (De Maagd et al.,
2003). Beside that solubilized protoxins are activated
by midgut proteases and bound with the receptors of
the epithelial cells (Pigott and Ellar, 2007). Due to
their high specificity and safety to most non-target
organisms or to environment, in general, Bt proteins
are preferred and widely used as biopesticides in
agriculture pest management (Van Frankenhuyzen,
2009). B. thuringiensis showed high virulence against
several lepidopteran larvae (Salama and Foda, 1982).
Commercial Bt products consist of a mixture of
spores and crystals that produced in large fermentors
and applied as foliar sprays, much like synthetic
insecticides (Sanchis et al., 1999). In addition, some
of the commercial formulation of Bt recorded
significant biochemical and physiological effect on
treated larvae (Kamel et al., 2010). Therefore,
biological control agents, either microorganisms or
microbial products, have long been drawing attention
as alternatives to chemical pesticides (Mahfouz and
Abou El-Ela, 2011).
Cry toxins Bt produces additional virulence
factors including phospholipase C (Martin et al.,
2010), proteases (Infante et al., 2010) and hemolysins

230
(Nisnevitch et al., 2010). The effect of environmental
conditions on the production of proteolytic enzyme
could play an important role in the induction or
repression of the enzyme by specific compounds
(Wang et al., 2008). Enzymes are gradually replacing
the use of harsh chemicals in various industrial
processes (Malathu et al., 2008). Proteases, which
hydrolyze peptide bonds of proteins are also known
as peptidyl-peptide hydrolase, constitute 60-65% of
the global enzyme (Genckal and Tari, 2006). In
addition to microbial origin, proteases are produced
from plants and animal organs and microorganisms
(Sevinc and Demirkan, 2011).
This study aimed to: i) isolate and identify the
bacterial isolated from S. littoralis larvae and soil
samples in Egypt, ii) bioassays of the bacterial strains
against S. littoralis larvae, iii) study the latent effect
of selected strains on several biological aspects of S.
littoralis larvae, iv) optimize, purify and characterize
the biopesticide agents produced by selected strain,
and v) enable the use of selected Bt1 strain as
biological control agent against the insects in the
field.
MATERIALS AND METHODS
Sampling
Soil samples were collected from different
localities in Sharkia Governorate, Egypt according to
the method described by Johnson et al. (1959). Also,
S. littoralis larvae were obtained from the Cotton
leafworm Research Department, Plant Protection
Research Institute, Sharkia Branch, Zagazig, Egypt.
Isolation of bacteria
Spread plate technique of (Wollum,1982) was
used for isolation of bacteria from dead and living S.
littoralis larvae and soil samples. This was done by
subsequent steps: sample homogenization, followed
by serial dilution and then pour plating on nutrient
agar (Goettel and Inglis, 1997). Isolates were
distinguished based on colony color and morphology.
Pure cultures of the bacterial colonies were prepared
and stocked in 20% glycerol.
Preparation of bacteria
Total bacterial isolates were inoculated separately
into nutrient broth media and incubated at 30o C
overnight. After incubation, the bacterial density was
measured at optical density (OD 600 ) and adjusted to
approximately 2x109 CFU/ml (Moar et al., 1995).
Twenty milliliters of these cultures were centrifuged
at 3000 rpm for 10 min. Pellets were re-suspended
separately in 5 ml of phosphate buffer solution and
also filtrates were used in bioassay testes.
Laboratory bioassays
Laboratory sensitive strain of S. littoralis was
obtained from a well-established culture maintained

at Plant Protection Research Institute, Agriculture


Research Center, Giza, Egypt. This strain was reared
over ten months away from any insecticidal
treatments according to (El-Defrawi et al., 1964).
Generally, leafdipping technique (Abo El-Ghar
et al., 1994) was applied in both preliminary and
toxicity tests. Toxicological and biological responses
of S. littoralis larvae to both treatments of culture
filtrates and pellets of each isolate as well as the final
purified product (protease) were studied. Also, four
different concentrations of (2x109 , 2x108 , 2x107 and
2x106 CFU/ml) from Bt1 strain were used in
concentration response test. Ten of newly molted 2nd
and 4th instars of S. littoralis larvae were placed in a
clean jar. Fresh castor bean leaves, as diet, were
individually dipped for (10 sec.) in 10 ml of each
tested concentration (filtrates, pellets and crude &
purified enzyme). Four replicates were used for each
treatment. After a feeding period of 48 hrs on treated
leaves, survived larvae were transferred to other clean
jars, and supplied daily with fresh untreated clean
castor leaves. Similar number of larvae was supplied
with untreated castor leaves considered as the control
group. Mortality percentages was recorded daily then
corrected by Abbotts formula (Abbott, 1925).
Determination of the biological aspects was recorded
the latent effect as: pupation, pupal weight and adult
emergence, in addition to study the failure of pupation
and deformed adults.
Identification of the selected isolates
The selected bacterial isolates were preliminary
identified according to Bergeys Manual of
Systematic Bacteriology (Holt et al., 1994 and Todar,
2005).
16S rRNA gene sequencing
Total DNA was extracted from tested
bacterial isolate according to (Sambrook and
Russel, 2001). The gene coding for 16S rRNA was
amplified from each tested isolate by PCR
with universal primers (forward primer [F27]
5'-AGAGTTTGATCCTGGCTCAG-3' and reverse
primer [R1492] 5'-GGTTACCTTGTTACGACTT3') (Chnbey et al., 2000). Molecular size of
amplified fragments should have molecular size
approximately 1500 bp. The amplification conditions
were as follows: 94C for 10 min and 35 cycles of
denaturation at 95C for 30 s, annealing/extension at
56C for 1 min, 72C for 1 min and an extension at
72C for 10 min. Presence and yield of specific PCR
products (16S rRNA gene) were monitored by
running 1% agarose gels. Then the PCR product was
cleaned up by using GeneJET PCR Purification Kit
(Fermentas, Germany). Amplied DNA fragments
were partially sequenced at GATC Biotech AG
(Konstanz, Germany) using ABI 3730xl DNA
sequence, using forward primer (F27). The 16S

231
rDNA sequence, which has been determined in the
present study, was deposited at NCBI web server
(www.ncbi.nlm.nih.gov). Sequence analysis and
comparison to published sequences made using the
Basic Local Alignment Search Tool (BLAST)
program
(http://www.ncbi.nlm.nih.gov/blast)
(Altschul et al., 1997).
Protease production and purification
Bt1 was cultivated on nutrient broth medium with
0.2% gelatin (Ammar et al., 1991). Production of
enzyme was tested at different incubation periods (38 days), different temperatures (25-55C), different
pH-values (pH 4 -12) under shaking and static
conditions and in presence of different carbon and
nitrogen sources (Kaur et al., 2001). Protease activity
(U/ml) of the cell-free filtrate was determined using
the method of (Thangam and Rajkumar, 2000).
Protein content was estimated by the method of
(Lowry et al., 1951). All purification of Bt1 protease
was carried out at 4C. The culture supernatant was
first mixed with 70% ammonium sulphate, with
constant and continuously stirred (Phibbs and
Bernlohr, 1971). The precipitate was then separated
by centrifugation at 6000 rpm for 20 min, and the
protein was then re-suspended in small amount of
sodium phosphate buffer (pH 8.0). As mentioned by
Ammar (1975) the re-suspended pellets were applied
to a Sephadex G100 column then Sephadex G50 column
which pre-equilibrated with the same buffer.
SDS-PAGE Analysis
Molecular weight of Bt1 protease was determined
using SDS-PAGE according to Laemmli (1970).
Page Ruler Unstained Protein Ladder, Ferment as
marker was used.
Biochemical properties of Bt1 purified protease
Optimum temperatures of the tested enzymes were
determined after incubation the reaction mixture
(enzyme and substrate) at different temperatures (3070C). The enzyme activity was determined for each
temperature as described previously. Thermal
stability of tested enzymes was studied after preincubation of the enzyme at various temperatures (3080C), using 0.1M phosphate buffer (pH 8.0) for
different time (30-150 min). Relative enzymatic
activities were determined after the incubation of
reaction mixture at 37C for 30 min.
Field experiment
The experiment was conducted at a field
cultivated with sugar-beet (Beta vulgaris) infested
with S. littoralis at Manshiat Abaza, Elramal, Fakos,
Sharkia Governorate, Egypt in September 2015. Four
spray treatments were carried out. Separate sprayer
was used for each treatment by washing it carefully
with distilled water until the pH of washing water
became neutral before adding the next material.

Treatment (1): Spray with water (control).


Treatment (2): Spray with bacterial culture of Bt1.
Treatment (3): New benzuron (IGR) 48% SC, 125
cm3 / fedden.
Treatment (4): Challenger super (chemical
insecticide) 24% SC, 50 cm3/ 100 liter of water.
The experiment where set at a randomized block
design an area of about 12 Karate was divided into 4
plots (each plot of 3 karate). The cotton leafworm, S.
littoralis was counted and recorded in plot before
spraying and then counted and recorded after
spraying (1, 3, 7, 10 days). Reduction value of each
treatment was calculated using the equation of
Henderson and Tilton (1955) to compare the
difference between the effect of Bt and the
recommended chemical or biological pesticides.
Statistical analysis
Obtained results of mortality and biological
parameters were subjected to analysis of variance to
toxic and latent effect parameters, using a Costat
computer program Cohort Software (Costat statistical
software 2005).
RESULTS AND DISCUSSION
Isolation, identification and screening of bacterial
isolates
A total of 35 bacterial cultures were isolated from
dead and living larvae of S. littoralis and soil samples.
Bacterial isolates were screened for their insecticidal
activities against S. littoralis larvae. After preliminary
bioassays, only bacterial isolate Bt1 exhibited highest
pathogenicity against S. littoralis larvae. So, Bt1
isolate was selected and preliminary identified as Bt1
depending on morphological and biochemical
characteristics described in Bergey's Manual of
Systematic Bacteriology (Holt et al., 1994). PCR
amplification of 16S rRNA gene confirmed the
identification of the selected potent strain as Bt1.
Partial nucleotide sequence of amplified gene was
submitted to GenBank (http://www.ncbi.nlm.nih.
gov/GenBank/update.htm) under accession number
of KU646830. Yilmaz et al. (2013) stated that Bt with
different varieties is the most commonly used
entomopathogenic bacterium against many insect
species belonging to certain orders. Similar to our
findings, Osman et al. (2007) revealed that the pellets
of some Streptomyces isolates were more active
against S. littoralis than culture filtrates. Some Bt
insecticidal proteins are produced and secreted into
the culture medium during vegetative growth
(Abdelkafi- Mesrati et al., 2011).
Concentration response of Bt1 strain on 2 nd or 4 th
instars of S. littoralis larvae
Table (1) shows that 2nd and 4th larval instars
differed in their response to infection with tested

232
Table (1): Accumulative morality percentages of 2nd or 4th instar of Spodoptera littoralis larvae treated with
selected bacterial spores using leaf dipping technique under laboratory conditions
Concentration
CFU / ml
2x109
2x108
2x107
2x106
2x109
2x108
2x107
2x106

Treated larval
instars
nd

4th

Accumulate mortality %
Days post treatment
3
5
7
9
11
22.5
40
52.5
60
72.5
15
32.5
45
50
57.5
10
25
35
42.5
47.5
7.5
17.5
25
27.5
32.5
12.5
20
25
10
15
17.5
Pupal Stage
5
7.5
15
5
5
5

Mean mortality
49.5 a
40 b
32 c
22 d
-----

M eans columns followed by the same letter are not significantly different at p=5%

Table (2): Values of LC50 of 2nd or 4th instars of Spodoptera littoralis larvae treated with the selected
bacteria using leaf dipping technique under laboratory conditions
Days post
treatment
3
5
7
9
11

Treated larval
instars
2nd
4th
2nd
4th
2nd
4th
2nd
4th
2nd
4th

LC50
( CFU / ml )
5.8x10 11
1.2x1012
3.4x1010
1.6x1012
1.9x109
4.1x1011
3.4x108
0
7.9x107
0

Confidence Limits LC50 ( CFU / ml )


Lower
Upper
5.2x1011
6.3x1011
1.13x1012
1.4x1012
2.73x107
3.9x1010
12
1.25x10
1.9x1012
9
1.6x10
2.01x109
11
3.66x10
4.5x1011
8
2.98x10
3.7x109
0
0
7.5x107
8.26x107
0
0

Slope
0.4
0.26
0.25
0.27
0.28
0.27
0.3
0
0.36
0

Table (3): Latent effect of 2nd or 4th instars of Spodoptera littoralis larvae treated with Bt1 strain using leaf
dipping technique under laboratory conditions
Concentration
of tested strain
(CFU/ml)
2x109
2x108
2x107
2x106
Control

Larval instars
in days
2nd
4th
13.38
7.95
12.05
7.13
11.76
6.85
11.62
6.59
11.22
6.53

%
Pupation
2nd
4th
12
60
12.6
70
15.5
70
25
73.5
95
97.5

% Failure of
pupation
2nd
4th
91
40
87.6
30
85
30
75
27.4
5
2.6

Weight of
pupa (mg)
2nd
4th
256.5
265
244.16 271.5
272.64 276.42
278.29 289.37
281.3 304.11

% Adult
emergence
2nd
4th
50
55
60
60.8
66.5
64.3
70
72.5
95
97.6

% Deformed
Adult
2nd
4th
50
45.9
40
39.5
33.5
35.7
30
27.8
5
2.4

Table (4): Effect of crude and purified protease on larval, pupal and adult stages of Spodoptera littoralis
Enzyme
treatment
Crude
Purified
Control

larval instars
treated
2nd
4th
2nd
4th
2nd
4th

% of larval mortality

%
36
45b
21
26.25b
50
62.5a
39
48.75a
2
2.5
1
1.25

% of pupal mortality

%
9
20.45a
4
6.77b
7
23.33a
5
12.19a
1
1.3
0
0

% of adult
emergence
79.54a
89.83a
76.66a
87.8a
100
100

% Deformed
adult
5.70b
3.63b
8.68a
5.55a
0
0

M eans in columns followed by the same letter are not significantly different at P 5% according to Duncan's multiple range test
(Duncan, 1955).

Table (5): Effect of Bt1 culture, New benzuron, Challenger on Spodoptera littoralis larvae under field
conditions
Treatments
Bt1 strain
Newbenzuron
Challenger

After 3 days
36.35c
79.23b
89.61a

Reduction %
After 7 days
38.81b
91.50a
93.69a

M eans columns followed by the same letter are not significantly different at p=5%

After 10 days
46.33b
86.81a
88.68a

Mean of
reduction
40.50b
85.85a
90.66a

233
Bt1 strain. Cumulative mortality percentages were
increased as the time elapsed after treatment
increased. Maximum mortality of 72.5% against 2nd
instar larvae was obtained on day11 after application.
Meanwhile, the highest tested concentration of 2x109
CFU/ml gave 25% mortality against the 4th instar
larvae on the 7th day post treatment. These results are
in agreement with those of Mabrouk et al. (1995) who
reported that the pathogenicity of Bt isolates against
S. littoralis increased with increasing the time elapsed
after treatment. After 6 days of treatment, the
insecticidal activity of some isolates was higher than
that obtained after 4 days of treatment. There were
clear differences in the virulent activity among
different isolates. Ozkan-Cakici et al. (2014) reported
that Bt subsp. kurstaki (MnD) and Bt subsp. kurstaki
(BnBt) were the most effective, causing 100%
mortalities within 10 days after treatments.
LC50 values were determined from the plotted
regression lines for the tested Bt against the 2nd and
4th instars of S. littoralis larvae (Table 2). On basis
of the LC50 values, 2nd instar larvae of S. littoralis
(1.9x109 CFU/ ml) was more susceptible than the 4th
ones (4.1x1011 CFU/ ml) after seven days of
treatment. Similarly, EL-Sheikh et al. (2013) studied
the susceptibility of the 2nd instar larvae of S. littoralis
towards Bt and Teflubenzuron compounds. The LC50
of Teflubenzuron and Bt showed considerable effects
against the 2nd instar larvae of S. littoralis, particularly
in case of Teflubenzuron which had drastic toxic
effects comparing to Bt virulent.
Latent effect of Bt1 strain on S. littoralis
Data in table (3) showed an increasing in duration
of 2nd and 4th larval instars as comparing to control
within all tested Bt1 concentrations. Duration of 2nd
instar treated with (2x109 CFU/ml) of tested strain
was 13.38 days and 11.22 days in control, while in 4th
instar, it was 7.95 and 36.30 days in treatment and
control, respectively. On the other hand, larval
duration of the larvae treated with other
concentrations was near that of control. Pupation %
and pupal weight decreased by increasing Bt1 strain
concentrations. Also, the malformation % increased
by increasing the concentration of Bt1 strain in both
tested instar larvae. Maximum malformations were
50 and 45.9% induced by the high concentration of
(2x109 CFU/ml), while the minimum effects were
recorded (30 and 27.8%) at the lowest concentration
of (2x105 CFU/ml) for both tested instars,
respectively. From these results, it was obvious that
2nd instar larvae of S. littoralis were more sensitive
than 4th instar larvae in all aspects. AlOtaibi (2013)
reported that Bt delta-endotoxins are safe biological
insecticidal protein. The first commercialized Bt
insecticidal formulations were composed of sporecrystal preparations derived from wild-type strains.

Moreover, George and Crickmore (2012) reported


that Bt and its insecticidal toxins have been used in
agronomical pest control for decades. The mechanism
of Bt toxin action on insect pest involves specific
molecular interactions which makes Bt a popular
choice for pest control. Its specificity of action
reduces the concern of adverse effects on non-target
species, a concern which remains with chemical
insecticides.
Optimization of Bt1 protease production
In the present investigation, maximum protease
production was achieved when Bt1 strain was
cultivated in nutrient broth medium (pH 7) at 35C
for 2 days in presence of 1% casein and 2% maltose.
These results were almost similar to Ibrahim et al.
(2015) who reported that the optimum temperature
and pH for halotolerant alkaliphilic alkaline protease
producing Bacillus sp. NPST-AK15 were 40C and
pH 11, respectively. Also, Ahmetoglu et al. (2015)
who stated that the optimum temperature, pH and
incubation period for protease production by Bacillus
sp. KG5 were 40-45C, 7.0 and 24 h, respectively. It
was determined that the best nitrogen sources were
yeast extract and urea, while the best carbon sources
were lactose and galactose.
Purification and characterization of Bt1 protease
The protease enzyme was purified as an
extracellular enzyme from the liquid culture of Bt1
growing under the optimal culture conditions, by 70%
(NH4 )2 SO4 , dialysis, Sephadex G100 and finally
Sephadex G50 . The homogeneity and molecular mass
of purified protease enzyme appeared as a single
protein band of 68 kDa (Fig. 1). Similarly, Jain et al.
(2012) detected approximately the same molecular
mass (71 kDa) of protease produced from Bacillus sp.
SM2014. Meanwhile, Ahmetoglu et al. (2015) stated
that the molecular weight of purified Bacillus sp.
KG5 protease was found to be approximately 48 kDa.

Fig. (1): SDS- PAGE of purified protease from


Bacillus thuringiensis Bt1 Lane M (Broad-way
Daul prestained protein marker), Lane 1, the
purified protease enzyme.

234
Concerning the biochemical properties of the
purified protease, maximum protease activity was at
neutral pH 7 and 35C; with wide pH and thermal
stabilities. These results agree with those of Zambare
et al. (2011) who detected that the protease of P.
aeruginosa MCM B-327 exhibited wide pH and
thermal stabilities. Thermal and pH stability of the
enzyme may be related to that the enzyme maintains
its secondary and tertiary structure under stress but
lowering of enzyme activity at high temperature or
pH (above the maximum range) may be due to
enzyme aggregation, incorrect structure formation,
hydrolysis of S-S bonds, deamination and
denaturation (Ahern and Klibanov, 1987). Also, Abd
El-latif (2015) cleared that the puried serine protease
inhibitors from two cowpea cultivars were stable at
below 60C and were active at wide range of pH from
2 to 12.
In vivo effects of crude and purified Bt1 protease
on S. littoralis larvae
The crude and purified Bt1 proteases had
significant effects on larval mortality, pupation and
pupal weight of S. littoralis compared to control
(Table 4). The 2nd instar larvae were the most
susceptible to both enzymes. Mortalities of larvae
were 62.5and 48.75%, while those of pupae recorded
23.33 and 12.19% after the treatment of 2nd and 4th
larval instars, respectively. Obtained data cleared that
purified enzyme treatment was the most effective
against biological aspects compared to crude enzyme.
In this connection, Abd El-latif (2015) observed that
the puried serine protease inhibitors from two
cowpea were found to have deep and negative effects
on the mean larval mortality and larval weight of S.
littoralis.
Effects of Bt1 culture and insecticides on S.
littoralis larvae under field conditions
Data in table (5) summarize the effectiveness of
tested pesticides against S. littoralis under field
conditions. Reduction percentages of larval
population were measured till 10 days post treatment.
Data indicated that there was highly significant
difference between the reduction percentages for the
different treatments at 3 days, 7 days or 10 days. Rate
of decline increased with time. Challenger Super was
the most effective pesticide causing 89.61% decline
in S. littoralis population after 3 days of treatment, but
it recorded 93.69% and 88.68% after 7 and 10 days of
treatment, respectively, followed by New benzuron.
Meanwhile, Bt1 strain had the least toxic effect where
the mean reduction was 40.55%. This result agrees
with that of Abd-Elwahed et al. (2011) studied the
potency of certain commercial bio-insecticides where
Protecto (B. thuringiensis var. kurstaki) proved to be
virulent on the 2nd instar S. littoralis larvae. The tested
bioagents reduced the larval duration, percentages of
larval pupation as well as adult emergence of S.

littoralis treated at the 2nd instar larvae. Moreover,


Said et al. (2012) appeared that Virust was the most
effective compound as it recording 63.64% reduction
in the initial kill after five days, followed by Agerin
and Brofect that gave 51.61, 46.38% reduction.
While, Protecto was the least efficient bioinsecticide
as it gave 27.14% reduction. Ebeid et al. (2015) who
studied the effect of Egyptian Conyza and Challenger
36% SC insecticide against 4th instar larvae of S.
littoralis.
In conclusion, the present study suggests a new
biopesticide agent from culture and purified protease
(M. wt 68kDa) of potent local bacterial strain Bt1
KU646830 against S. littoralis. In the field
application, although the chemical insecticides
Challenger Super and New benzuron were more
effective against S. littoralis as compared to the high
concentration of Bt1 treatments at a long period of
time.
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