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Egyptian Journal of Biological Pest Control, 21(2), 2011, 143-150
Characterization of Local Bacillus thuringiensis Isolates and Their Toxicity
to Ephestia kuehniella (Zeller) and Plodia interpunctella (Hubner) Larvae
Ugur Azizoglul *; Semih Ydmaz ; Abdurrahman Ayvaz ;
Salih Karaborkl* * ** and Mikail Akbulut***
*Erciyes University, Graduate School of Natural and Applied Sciences, Kayseri, Turkey
**Erciyes University, Seyrani Agricultural Faculty, Department of Agricultural Biotechnology, Kayseri, Turkey
**Erciyes University, Faculty of Science, Department of Biology, Kayseri, Turkey
****Osmaniye Korkut Ata University, Faculty of Arts and Sciences, Department of Biology, Osmaniye, Turkey
'Corresponding author: UgurAzizoglu, Erciyes University, Graduate School of Natural and Applied Sciences, 38039,
Kayseri, Turkey, E-mail: azizogluugur@hotmail.com, Tel.: +90 352 437 49 01/33068, Fax: +90 352 437 49 33
(Received: August 12, 2011 and Accepted: September 28, 2011)
ABSTRACT
Putative Bacillus thuringiensis isolates were obtained from soils of various agricultural fields. Of all the tested isolates
U14.1, U14.2 and U14.5 were found to be positive for cry] C. Besides, U6.6, U14.1, U14.4 and U14.5 were determined
to contain cly1B. These local isolates produced bi-pyramidal, spherical and cubical crystal proteins. U14.1, U14.4 and
U14.5 isolates exhibited similar protein banding patterns with Btk producing around 45, 70 and 130 kDa proteins.
However, U14.2 and U6.6 produced only 60 kDa major protein band. Some of the isolates also produced bands less
than 45 kDa showing the presence of some other low molecular weight proteins. The results of this study demonstrated
that isolates of Bt obtained from various agricultural fields may display toxicity against Ephestia kuehniella (Zeller) and
Plodia interpunctella (Hubner) larvae. As a result, spore-crystal mixture of these isolates may play an important role in
reducing the damage to stored products caused by these important pest insects.
Key words: Bacillus thuringiensis, Cry protein, lethal concentration, toxicity, Ephestia kuehniella
(Zeller), Plodia interpunctella (Hubner).
INTRODUCTION
Mediterranean flour moth, Ephestia kuehniella
(Zeller 1879, Lepidoptera: Pyralidae) and Indian
meal moth Plodia interpunctella (Hubner 1813,
Lepidoptera: Pyralidae) are important insect pests
that infest awide range of stored products
(Rees, 2003; Simmons and Nelson, 1975). Larvae of
these pests decrease both quality and quantity of
stored products through feeding, webbing, and fecal
matter (Hansen and Jensen, 2002; Johnson et al.,
1997).
The control of these pests in storage systems
mainly depends on fumigants such as methyl
bromide or phosphine. However, methyl bromide
has been banned in many countries since 2004
because of its ozone depleting properties (Hansen
and Jensen, 2002). Phosphine also causes serious
problems and in some countries insect control
failures have been reported in field situations
(Taylor, 1989; Collins et al., 2002). Furthermore, the
use of chemical pesticides has led to many other
problems, including environmental pollution and
human health hazards, such as cancer and several
immune system disorders (Bravo et al., 2011). Many
alternatives have been tested to replace these
fumigants for stored product and quarantine uses.
There is an urgent need to develop safe alternatives
that have the potential to replace the toxic fumigants,
yet are effective, economical and convenient to use
(Ayvaz and Karaborklii, 2008).
Bacillus thuringiensis (Bt) is known to be an
important microbial entomopathogen for the
biological control of many agricultural insect pests
and disease vectors (Santos et al., 2010). Bt is
characterized by its production of different
insecticidal toxic proteins in para-sporal crystals
during sporulation (Rowe and Margaritis, 1987;
Crickmore et al., 1998; Valicente et al., 2010). It is
mainly a soil-dwelling organism, but one that can act
as an opportunistic pathogen under appropriate
conditions (Raymond et al., 2010). Crystal proteins
of Bt are toxic to a wide variety of lepidopteran,
dipteran and coleopteran insects that causing serious
damage to economically important crops (Vidyarthi
et al., 2002). In fact, each habitat may contain
a novel B. thuringiensis strain awaiting discovery
which has a toxic effect on a target insect group
(Baig and Mehnaz, 2010). After ingestion of
Bt-treated diet, the crystal protein split up into
smaller toxic peptide fractions by certain proteolytic
enzymes in the mid-gut juice of susceptible larvae,
which processed to active toxins binding to the
brush border membrane receptors before insertion
into the membrane. This proteolytic activation is a
crucial step in the mode of action of the Cry proteins
(Dammak et al., 2010).
In the current study, toxicity of several local
Bt isolates against the Mediterranean flour moth,
E. kuehniella and the Indian meal moth,
P. interpunctella larvae were evaluated under
laboratory conditions.
144
MATERIALS AND METHODS
Isolation of Bacillus thuringiensis
Bt isolates were obtained from soil samples of
various agricultural fields (Table 1), by the method
of Travers et al., (1987). One g of soil sample was
added to 20 ml of Luria Bertani Broth buffered with
0.25 M sodium acetate (pH 6.8), and incubated for 4
h at 30 C then, centrifuged at 200 rpm. One ml of
sample was then heated at 80 °C for 5-10 min. Then
50 t1 aliquot was spread on nutrient agar in each
Petri dish and incubated overnight at 30 C. Bt
subsp. kurstaki (Instituto de Biotecnologia,
Universidad Nacional Autonoma de Mexico) was
used as reference strain.
Table (1): Bt isolates and their GPS Location
Bt
isolates GPS Location Agri c.
Field
U6.6 38°51'15.37"K; 350 19'17.47"D, 1082m Squash
U14.1 38°30'53.82"K; 36°29'59.12"D, 1708m Wheat
U14.2 38°30'40.93"K; 36°30'50.12"D, 1611m Beet
U14.4 38°30'49.22"K; 36°30'43.76"D, 1626m Apple
U14.5 38°30'11.43"K; 36°31'28.88"D, 1584m Potato
Polymerase chain reaction (PCR)
Molecular characterization of the isolates was
performed by PCR analysis using the primers:
cty2 (5'-TAAAGAAAGTGGGGAGTCTT-'3,
5'-AACTCCATCGTTATTTGTAG-'3)
ctyIC (5'-AAAGATCTGGAACACCTTT-'3
5'-CAAACTCTAAATCCTTTCAC-3'),
cly1B (5'-CTTCATCACGATGGAGTAA-3',
5'-CATAATTTGGTCGTTCTGTT-3')
crylAa/Ad 5'-TTATACTTGGTTTCAGGCCC-3',
5 ' -TTGGAGCTCTCAAGGTGTAA -3 ' )
Each reaction contained the reagents at afinal
concentration as 2.3 mM MgC12, I X taq buffer, 0.2
mM dNTP mix, 0.3 pmol primers (each), 0.5 U taq
DNA polymerase, and 30-100 ng template DNA.
The PCR amplification was performed under the
following conditions: Initial denaturation at 95 C
for 2min, followed by 34 °cycles at 95 °C for 1 min,
48-50 °C for 1 min, 72 C for Imin, and a final
extension step at 72 C for 5 min and the conditions
with the primers used were similar (Bravo et al.,
1998).
Spore-crystal mixture, freeze drying and electron
microscopy
Bt isolates were grown in 150 ml T3 medium (3 g
triptone, 2 g triptose, 1.5 g yeast extract, 0.005 g
MnCl2, 6 g NaH2PO4, 7.1 g Na2HPO4) and incubated
for 7 days at 30 °C to induce spore formation
(Travers et al., 1987). Suspensions were centrifuged
at 4 'C and 15.000xg for 10 min to harvest spore-
crystal mixtures. Pellets were washed twice and
centrifuged at 15.000xg for 10 min in 20 ml sterile
dH20.
Bt spore-crystal mixtures were freeze dried using
Labconco-Welch freeze-drier according to the
manufacturer's instruction and were stored at 4 C
until further use. Spore-crystal samples were spread
on a microscope slide and fixed after air dry at room
temperature. Samples were sputter coated with lOnm
Au/Pd using a SC7620 Mini-sputter coater and
viewed using aLE0440 scanning electron
microscope at 20kV beam current.
Plasmid DNA extraction
Plasmid DNA isolation was performed using the
methods of Jensen et al., (1995) and Porcar et al.,
(1999) with some modifications. The bacteria were
grown in 5 ml LB broth for 14h with continuous
shaking at 30 °C and 200 rpm. 4 ml of cells was
Relleted and resuspended, using 100 p.1 of TE buffer
(40 mM Tris-HCI, 2 mM EDTA, pH 7.9). Cells
were then lysed in 200 p.1 of lysing solution
(3% SDS, 15% sucrose, 50 mM Tris-hydroxide,
pH 12.5). The lysate was incubated at 60 °C for
30 min, and 2111 of proteinase K (20 mg/ml) was
added. The solution was inverted several times and
incubated at 41°C for 90 min. Thereafter. 1 ml of
phenol: chloroform:isoamyl alcohol (25:24:1) were
added to the solution and the tube was inverted
40 times. After centrifugation at 6.000xg for 7 min,
the upper aqueous layer was transferred to a clean
tube and 500 ul of chloroform: isoamyl alcohol
(24:1) was added and centrifuged again at 6000xg
for 7 min. Upper aqueous layer was subjected to
electrophoresis in 0.5% agarose gel (with 1X TBE
buffer and img/m1 ethidium bromide) at 65V and
5 °C for 8 h.
Protein electrophoresis
SDS-PAGE was "conducted as described by
Valicente et al., (2010) with some modifications.
The lyophilized spore crystal mixtures were
resuspended in 1 ml 0.01% Triton X-100 solution.
This step was repeated three times. Pellets composed
of a mixture of spore-crystal were solubilized in
500 ul buffer solution (0.01% Triton, l0 mM NaC1
and 50 mM Tris-HC1, pH 8.0), and one aliquot of
100 pi was withdrawn after this step. The mixture
was centrifuged at 14.000 rpm for 5 min and the
pellet was resuspended in 500 ul of sodium
bicarbonate buffer (50 mM sodium bicarbonate and
10 mM b-mercaptoethanol, pH 10.5) and maintained
at 37 °C for 3 h under continuous shaking. Samples
were then centrifuged at 14.000 rpm for 10 min, and
the supernatant were transferred to a new tube. The
remaining pellets were resuspended in 250 pi. of
0.1 M Tris, pH 8.0. Equal amounts of supernatant
and resuspended pellet were sampled and equal
volume of sample buffer (0.0625 M Tris, 2.3% SDS,
10% glycerol, 5% 13-mercaptoethanol and 0.1%
bromophenol blue, pH 6.8) was added. The mixture
was maintained for 5-10 min in boiling water.
Sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) was performed using 12%
running and 5% stacking gels. The molecular mass
of proteins was determined with SM0431 protein
molecular weight marker (Fermentas) and Btk HD1
strain was used as a reference. The gel was stained
with 0.4% comassie brilliant blue 8250 described by
Temizkan and Arda (2004).
Bioassay
E. kuehniella and P. interpunctella larvae were
reared on their artificial diet at 27±1 °C with a
photoperiod of 14:10 (L: D) h and 60±5% RH in a
rearing cabinet (Ayvaz et al., 2010). Freeze-dried
spore-crystal mixture was suspended in sterile
distilled water at 250, 1000 and 1500 pg ml"'
concentrations. One gram of nut was ground and
soaked into 1 ml of spore-crystal mixture solution,
left to absorb the toxin for 20 minutes and allowed
to dry at room temperature. The mixture was then
transferred into Petri dishes (90x15 mm) together
with 10 larvae (25 days old) per each and left in an
acclimatized chamber at 27±1 °C and 60±5 RH with
a photoperiod of 14:10 (L:D) h for 10 days. Sterile
(1H20 was used as a control instead of spor-crystal
mixture and three replicates were set up for each
treatment.
Statistical analysis
Data from the bioassay experiments were
subjected to the analysis of variance (ANOVA)
using SPSS for Windows (SPSS, 2001) and means
were separated at the 5% significance level by
145
the least significant difference (LSD) test. The
data were subjected to probit analyses using the
same statistical program to estimate LC50 and
LC95 values for E. kuehniella and P. interpunctella
larvae.
RESULTS AND DISCUSSION
Bt isolation from soil samples
Soil samples from different agricultural fields
were screened for the presence of Bt isolates. Totally
sixty different bacterial colonies were analyzed with
PCR method using cryl C, cry1B, crylAblAd and
cry2 primer pairs. Of all these isolates U14.2 were
positive for cry1C, U14.4 and U6.6 were positive for
cry1B, U14.1 and U14.5 were positive for both
cry1C and cry1B (Fig.1). Neither of the isolates
produced expected PCR product with crylAblAd and
cry2 primer pairs.
Scanning electron micrograph of spore-crystal
mixture
Spore-crystal samples were examined under
scanning electron microscope to show detailed
view of Bt kurstaki and, products of U6.6, U14.1,
U14.2, U14.4 and U14.5 isolates. It was evident
that local isolates produced bipyramidal, spherical
and cubic crystal proteins with different sizes
(Fig. 2).
Plasmid profile of isolates and reference strain
Plasmids were resolved by agarose (0.5%) gel
electrophoresis and visualized under UV
luminescence (Fig. 3). U14.1, U14.4 and U14.5
isolates had similar banding patterns with Btk. All
strains produced plasmid bands larger than 19.3 kb
except U14.2. The isolate U.6.6 did not contain
smaller plasmid bands under 19.3kb.
3000 by
2000
1500
1200
1000
500400300200100
Fig. (1): Agarose gel (0.8%) electrophoresis of the PCR products amplified by using cryl primers (cry.1C
130 and cry1B 367).
U14.1 U14.2 U14.4
MAC; = 10.00 K X 24m Detector = SE1
ENT = 20 00 IN IIDale 26 Mar 2010
U6.6
!MG 1000 K X
ENT 20 00 kV
Fig. (2): Electron micrograph of isolates spore-crystal mixture.
U14.5
Detector =.5E1
0-Me 26 Mar 2010
MAG 10001C X
EMT 20.0014V
0Mottor = SE
Dm. 26 Mat 2010
Bacillus thuringiensis subsp. kurstaki
Fig. (3): Plasmid profile of local Bt isolates.
147
Fig.(4): SDS-PAGE (12%) profile of local Bt
isolates.
100
80
60
040
20
0
Control X250 mgml 1-11000 g m 1 1.41500 g in 1
U 6.6 U 14.1 U14.2 U14.4
Br isolates U 14.5 B tk
Fig. (5): Percent mortality of E. kuehniella larvae after exposure to spore-crystal mixture of local
Bt isolates. Bars with the same letter are not significantly different for each dose. Error bars
indicate standard errors of means.
"Control "250 perril L--'1000 [renal "1500 Wird
U6.6 U14.1 U14.2 U14.4 U14.5
Et isolate s
Fig. (6): Percent mortality of P. interpunctella larvae after exposure to spore-crystal mixture of
local Bt isolates. Bars with the same letter are not significantly different for each dose. Error bars
indicate standard errors of means
148
SDS-PAGE analysis
The crystal protein profile of the isolates was
determined by SDS-PAGE analysis. Each isolate
produced a characteristic banding pattern with some
differences. U14.1, U14.4 and U14.5 isolates
produced around 45, 70 and 130 kDa proteins.
However,. U14.2 and U6.6 produced only 60 kDa
major protein band (Fig. 4). Some of the isolates
also produced bands less than 45 kDa showing the
presence of some other low molecular weight
proteins.
Bioassay
Toxicity of isolates against E. kuehniella and
P. interpunctella larvae are shown in figures 5 and 6.
When the larvae of E. kuehniella were exposed to
250 gg ml"' spore-crystal mixtures of Btk and U14.1
isolate, 43.34% and 33.34 mortality percentages
were observed, respectively (Fig. 5). The mortality
resulted by other isolates were significantly lower
than that with U14.1 and Btk. At the highest
concentration (1500 gg m11), mortality rates were
40.00, 46.67 and 66.67% for U14.1, U6.6 and Btk,
respectively. Btk showed highest insecticidal activity
(33.34%) against P. interpunctella larvae at 250 lag
mr1 concentrations (Fig. 6). The insecticidal activity
of U6.6, U14.1, U14.4 and U14.5 isolates was nearly
20% at the same concentration. However, U14.2
isolate did not show any toxicity against the larvae
compared to other isolates (F= 4.113; df = 5; P
<0.021). Higher larval mortality due to higher toxin
concentration was obvious in most of the isolates.
Mortality rates caused by U6.6, U14.4 and Btk at the
1500 pg ml-' concentration were 53.34, 50.00 and
56.67%, respectively, but_the mortality caused by
U14.2 isolate was only 20% at the same
concentration (P<0.016).
Lethal concentrations (LC50 and LC95) of Btk,
U6.6, U14.1, U14.2, U14.4 and U14.5 for E.
kuehniella and P. interpunctella are shown in Table
2. They were 1032.63 and 2834.46 for Btk, and
1524.33 and 3288.34 gg ml"' for U6.6, respectively.
The LC50 and LC95 values required for P.
interpunctella were similar to E. kuehniella for Btk
(1084.63, 1032.63 and 2837.94, 2834.46 gg tnrlfor
P. interpunctella and E. kuehniella respectively).
However, corresponding values for U6.6 isolate
against the P. interpunctella larvae were 1197.84
and 2833.78 gg m11.
Bt products display high toxicity against a wide
range of lepidopteran, coleopteran, and dipteran
pests (Zi-Quan et aL, 2008). Our local isolates were
characterized by PCR and SDS-PAGE analysis by
determining the presence of cryl genes and Cry
proteins. PCR analysis revealed that U14.1 and
U14.5 were positive for cry1B and cry1C, and
Table (2): LC50 and LC95 (gg/m1) values of isolates
against E. kuehniella and P. interpunctella larvae
LC50 LC95 x2 Df P
Isolates E. kuehniella
U6.6 1524.33 3288.34 05.52 20.063
U14.1 1726.47 4532.37 25.30 20.000
U14.2 2474.34 5951.75 16.47 20.000
U14.4 2256.60 4729.10 03.98 20.136
U14.5 2780.43 5602.88 02.23 20.312
Btk 1032.63 2834.46 36.01 20.000
P. interpunctella
U6.6 1197.84 2833.78 17.58 20.000
U14.1 1723.02 3827.54 10.28 20.006
U14.2 2822.20 5156.24 02.06 20.357
U14.4 1280.04 3032.87 16.94 20.000
U14.5 1565.36 3542.64 09.77 20.000
Btk 1084.63 2837.94 26.29 20.000
U14.2, U14.4, and U6.6 were positive for cry1B
gene having insecticidal activity primarily against
lepidopteran pests. Wang et al., (2003) reported that
especially Cryl, Cry2, and Cry9 group of proteins
display strongest activity against lepidopteran pests.
The results of the present study show that isolates
carrying cryl type genes show consid;rable
mortality effect at the highest concentrations of
spore crystal mixture against the larvae of
E. kuehniella and P. interpunctella. We used spore-
crystal mixture in bioassay studies because spores
and crystals included in the suspensions produce a
higher level of mortality than either crystals or
spores alone (Crickmore, 2006). Mortality rates of
local isolates were found to be lower than those of
Btk. The mortality rates of U14.1 and U6.6 were
40 and 47% against E. kuehniella larvae at the
highest concentration, respectively. Moreover.,
U14.4 and U6.6 showed 50 and 54% mortality
against P. interpunctella larvae. Santos() et al.,
(2004) reported that eight out of twelve tested toxins
killed nearly 50% of Conopomorpha cramerella
(Snellen 1904, Lepidoptera: Gracillaridae) in the 3rd_
4th instar larvae. Although U14.5 harbors both cry 1 C
and cry1B genes and corresponding Cry protein
bands, it exhibited fairly low toxicity against E.
kuehniella larvae when compared to U14.1, U14.2,
U14.4, and U6.6. Nevertheless, while U14.4 and
U6.6 having the same type of cry gene, their
insecticidal activity was found to be different even
against the same type of larvae. U14.1, U14.4 and -
U14.5 isolates exhibited highly similar plasmid
profile resembling Btk, but U6.6 produced only one
type of plasmid band bigger than 19.3 kb.
Our local isolates produced cuboidal, bi-
pyramidal and spherical shaped crystal proteins it
varying sizes around 45, 60, 70 and 130 kDa.
Obeidat et al., (2004) stated that strains producing
bi-pyramidal and cuboidal crystal proteins showed
similar protein profiles. Although both U6.6 and
U14.2 had spherical crystal protein and similar SDS-
PAGE protein profile, their insecticidal activity was
not identical. Isolates of U14.1 and U14.5 produced
bipyramidal and spherical Cry proteins and showed
similar SDS-PAGE profile as well. Furthermore.,
U14.1 and U14.5 bear both cry1C and cry1B genes,
but insecticidal activity caused on the larvae was not
equal. Our results are in agreement with the study of
some researchers who stated that strains sharing the
same cry genes showed significantly different
insecticidal potency (Du image, 1981; Ceron et al.,
1995; Martinez et al., 2005 and Bozlagan et al.,
2010). In another study Hongyu et al., (2001)
reported that 71% of their 122 isolates belonged
to different mortality groups with 60% against
Spodoptera exugia (Hubner, Lepidoptera:
Noctuidae).
Yilmaz (2010) also reported that their local
isolates collected from soil samples in Adana
carrying the same type of cry genes exhibited
different insecticidal activity (20 to 80%) against the
larvae of the same species. These kinds of results are
attributed to environmental factors, the target insect
species, concentration and distribution methods of
the product of different strains of microorganisms
(Bauce et al.,. 2002; Carisey et al., 2004; Kouassi,
2001). Insect metabolism may also have a profound
effect on the efficacy of the toxins.
Results of this study suggest that local Bt isolates
exhibit toxic effects on the survival and development
of the E. kuehniella and P. interpunctella larvae.
Although the spore-crystal mixture of the local Bt
isolates did not display mortality higher than 54% at
the highest experimental concentration, they caused
a considerable decrease in the rate of development in
the treated pest larvae. It can be suggested that
spore-crystal mixture of these isolates can be used to
reduce the damage on stored products caused by
these important pest insects.
ACKNOWLEDGEMENT
This study was supported by the Erciyes
University Research Fund (grant No. FBY-08-568).
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