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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 (
 Bt
1) and Two Chemical Pesticides on
Spodoptera littoralis
(Boisd.) (Lepidoptera: Noctuidae)
 
Fifi, M. Reda
1
; W. A. Hassanein
1
; E. A. H. Sherief 
2
 and Shahera, Moabed
2
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 (
 Bt 
1) exhibited the highest pathogenicity against the pest. Identity of one of the most potent isolates suspected and encoded as
 Bt 
1 was confirmed by amplifying its 16S rRNA gene. Partial nucleotide sequence of the amplified 16S rRNA gene of the tested strain was submitted in GenBank with accession number KU646830. Pathogenicity of
 Bt 
1 strain was tested against 2
nd 
 and 4
th
instars of
S. littoralis
 larvae at different concentrations (2x10
9
, 2x10
8
, 2x10
7
 and 2x10
6
CFU/ml). Based on the LC
50
 value, the 2
nd 
 instar larvae were more susceptible than the 4
th
 one. It recorded 1.9x10
9
CFU/ml and 4.1x10
11
CFU/ml for 2
nd 
 and 4
th
 larval instars, respectively, after seven days. Latent effect of
 Bt 
1 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
 Bt 
1 strain culture filtrates against 2
nd
and 4
th
larval instars of
S. littoralis
 cleared that filtrate 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 35
o
C, respectively, with wide pH and thermal stabilities. Furthermore, purified protease showed high activity against 2
nd
and 4
th
 
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
 Bt 
1 culture against the 2
nd 
 and 4
th
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,
 Bt 
1 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 gram- positive, spore-forming bacterium that produces  parasporal inclusions during the sporulation phase. These inclusions are composed of proteins (Cry
 proteins) or δ
-endotoxins, which are highly toxic to a wide variety of insect pests and some invertebrates (Vilas-Bôas
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
.
 littoralis
 larvae and soil samples in Egypt, ii) bioassays of the bacterial strains against
.
 littoralis
 larvae, iii) study the latent effect of selected strains on several biological aspects of
.
littoralis
 larvae, iv) optimize, purify and characterize the biopesticide agents produced by selected strain, and v) enable the use of selected
 Bt 
1 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,
.
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
.
 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 30
o
C overnight. After incubation, the bacterial density was measured at optical density (OD
600
) and adjusted to approximately 2x10
9
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, leaf 
 – 
dipping 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 (2x10
9
, 2x10
8
, 2x10
7
 and 2x10
6
 CFU/ml) from
 Bt 
1 strain were used in concentration response test. Ten of newly molted 2
nd
and 4
th
 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 Abbott’s f 
ormula (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 Bergey’s 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'-GGTTACCTTGTTACGACTT-3')
(
Chénbey
 et al
., 2000
)
. Molecular size of amplified fragments should have molecular size approximately 1500 bp. The amplification conditions were as follows: 94°C for 10 min and 35 cycles of denaturation at 95°C for 30 s, annealing/extension at 56°C for 1 min, 72°C for 1 min and an extension at 72°C 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
). Amplified 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
 Bt 
1 was cultivated on nutrient broth medium with 0.2% gelatin (Ammar
et al
., 1991). Production of enzyme was tested at different incubation periods (3-8 days), different temperatures (25-55
C), 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
 Bt 
1 protease was carried out at 4°C. 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 G
100
 column then Sephadex G
50
 column which pre-equilibrated with the same buffer.
SDS-PAGE Analysis
 
Molecular weight of
 Bt 
1 protease was determined using SDS-PAGE according to Laemmli (1970). Page Ruler Unstained Protein Ladder, Ferment as marker was used.
Biochemical properties of
 Bt
1 purified protease
Optimum temperatures of the tested enzymes were determined after incubation the reaction mixture (enzyme and substrate) at different temperatures (30-70
C). The enzyme activity was determined for each temperature as described previously. Thermal stability of tested enzymes was studied after pre-incubation of the enzyme at various temperatures (30-80
C), 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 37
C 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
 Bt 
1.
 
Treatment (3): New benzuron (IGR) 48% SC, 125 cm
3
/ 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
 Bt 
1 exhibited highest  pathogenicity against
S. littoralis
larvae. So,
 Bt 
1 isolate was selected and preliminary identified as
 Bt 
1 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
 Bt 
1. 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
 Bt
1 strain on 2
nd
 or 4
th
 instars o
 S. littoralis
larvae
Table (1) shows that 2
nd 
 and 4
th
 larval instars differed in their response to infection with tested

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