216

Bacillus thuringiensis - An eco-friendly biopesticide JBiopest, 5 (Supplementary) : 216-222 (2012) 216

Characterization of locally isolated Bacillus thuringiensis for the

Development of Eco-friendly Biopesticides in Bangladesh
Asaduzzaman Shishir, Asma Akter, Md. Hasibul Hassan, Golam Kibria, Mohammad Ilias, Shakila Nargis
Khan and Md. Mozammel Hoq*
ABSTRACT
Bacillus thuringiensis (Bt) was investigated in three different habitats (vegetable and crops-cultivated soils,
phylloplanes and insect guts) of Bangladesh. A total of 61 Bacillus cereus-like isolates were obtained by
selective methods and 57 of those were identified as Bacillus thuringiensis isolates based on their hemolytic
activity, presence of parasporal crystal proteins, plasmid profile and crystal protein profile. The prevalence of Bt
was highest (60%) in soil samples followed by leaf and insects. Five different types of parasporal crystal
proteins (spherical, bipyramidal, irregular pointed, cuboidal and irregular shaped) were observed among the
isolates which indicates the diversity of the local Bt isolates examined. In addition, confirmation of the strain
identification was done using 16S rDNA gene sequencing. Plasmid analysis recovered from the isolates yielded
at least one 15kb DNA, which is well comparable to the reference strain, B. thuriginesis kurstaki var. HD-73.
Further similarity between the test and standard strains was observed while analyzing crystal proteins on a
SDS-polyacrylamide gel that produced major bands of Cry1, Cry2 and Cry9 type proteins. Bioassay performed
with isolates Bt-01i, Bt-25f and reference strain, Bt kurstaki var. HD-73 against pulse beetles Callosobrochus
chinensis resulted LC50 values of 0.30, 0.72 and 0.22 mg/ml crude proteins respectively which demonstrated their
competency in Biopesticide production and application.
Key words: Bt, biopesticides, crystal proteins, 16S rDNA
fact, each habitat may contain a novel Bt strains awaiting
gene sequencing, SDS-PAGE.
discovery which has a toxic effect on a target insect group.
INTRODUCTION Therefore, Bt strains have been collected and characterized in
order to evaluate their toxic potential against various insect
Bacillus thuringiensis (Bt) is a gram-positive, rod-shaped,
orders (Chak et al., 1994; Theunis et al., 1998; Bravo et al.,
motile, facultative anaerobic, spore-forming bacterium widely
1998; Forsyth and Logan 2000; Uribe et al., 2003; Özgür et al.
used as biocontrol agent against pests (Fernando et. al.,
2005; Glen et al., 2006). Powdered Bt biopesticide formulation
2010). Bt produce parasporal crystalline inclusion bodies
and Bt transgenic plants are common measures in different
constituted by highly specific insecticidal toxins which are
parts of the world at present. Transgenic Bt cotton containing
protein by nature. These toxins are mainly active against
cry1Ac gene which offers resistance to major bollworms was
lepidopteran species and some also shows toxicity against
first commercially released in the world in 1996 and during
dipteran and coleopteran species and other organisms
2002 in India (Prasad et al., 2009). Steven and Naranjo
(Vidyarthi et al., 2002; Martin et al., 2010).
highlighted the impact of Bt on various organisms in an
So far more than 50,000 Bt strains have been isolated from elaborate manner (Nethravathi et al., 2010). Identification of
different environments (Sadder et al., 2006). It has been cry gene content by PCR is the most effective techniques in
reported that Bt can be present in several different habitats screening large native collection when predicting insecticidal
such as soil, stored product dust, insect cadavers, grains, activities of individual strains (Ben-Dov et al., 1997; Porcar et
agricultural soils, olive tree related habitats, different plant al., 2003). Biological activity tests, plasmid contents, 16S rDNA
and aquatic environments (Martin and Travers, 1989; analysis, PFGE analysis of chromosomal DNA, crystal
Meadows et al., 1992; Ben-Dov et al., 1997; Theunis et al. morphology and protein profiling of Bt are also currently in
1998; Bravo et al., 1998; Bel et al., 1997; Mizuki et al., 1999; use as complementary methods in the search for novel strains.
Iriarte et al., 2000, Maher et al., 2004; Xavier et al., 2007). In

© JBiopest. 313
 Asma Akter et al. 217
The use of Bt product is gradually being increased as the incubated overnight at 30°C. Then the cultures were plated
alternative to chemical pesticides, as organic pesticides cause on MYP agar from broth and incubated at 30°C for 24 hrs.
too many ill effects to human beings when they consume Those which formed irregular white colonies with a pink
insecticides treated products (Kandibane et al., 2010). background, similar to reference isolates were primarily
Therefore, the aim of this study is to initiate the establishment identified as Bacillus thuringiensis. Similar method was
of the collection of indigenous Bt strains in Bangladesh, followed for leaf and insect samples. The acetate selection
determining diversity among them and sorting out of the method described by Travers et al. (1987) was used to further
potential strains that are active against vegetable pests for screening whether or not germination of their spores was
their large scale production. inhibited at 0.25M acetate. Isolates were then streaked on 5%
Sheep blood agar plate and incubated overnight at 30ºC.
MATERIALS AND METHODS Isolates possessing â-hemolytic activity were sub-cultured
on Acras agar medium (Wei-Ming et al., 1995) plates and
Sample collection
incubated at 30oC for 18-48 hrs at 30ºC allowing sporulation.
A total of 99 samples were collected from 11 different places They were then observed under Phase contrast microscope
of Bangladesh targeting soil, leaf and insect as their habitat (Zeiss) at 1000 magnification for the presence of parasporal
as shown in Table 1. Soil samples were collected from the crystal protein. Observation for all isolates was recorded
places not exposed to sunlight or at 2 to 5 cm depth by scarping considering the presence of crystal proteins and crystal
off surface material and were placed in plastic bags aseptically, shapes.
transported to the laboratory and stored at 4°C until
processed. 16S rDNA gene sequencing
Bacterial strains: Colony PCR was performed with universal primers
complementary to phylogenetically conserved portions of
B. thuringiensis subsp. k u r s t a k i HD-73 (cry1Ac ), B. the 5' and 3' ends of the 16s rDNAs of Bacillus thuringiensis.
thuringiensis subsp. japonensis Buibui (cry8Ca ), B. Primers, 20F 5'-GAGTTTGATCCTGGCTCAG-3' (position 9-
thuringiensis subsp. sotto (BGSC T84, cry1Aa) were received 27), and 1500R 5'-GTTACCTTGTTACGACTT-3' (position
in slant culture, provided by Okayama University, Japan and 1509-1492) were used for 16S rDNA gene sequencing. The
used as reference strains. amplification and sequencing was carried out according to
the method described by Ben-Dov et al. (1997). The 16S
Isolation and analysis of Bacillus thuringiensis
rDNA gene sequence of the purified PCR product was
Isolation was carried out using two different methods with determined using 3 primers 20 F, 520 F and 920 F with an
some minor modifications. To isolate B. thuringiensis, a 10% Applied Biosystems model 3130 DNA sequencer and the ABI
soil suspension in 0.9% NaCl was preheated at 80°C for 10 PRISM cycle sequencing kit. The sequence obtained was
min and 100 µL of each samples was then transferred to 900µl compared with those acquired from GenBank using the BLAST
Mannitol-egg yolk-polymyxin (MYP) broth for enrichment of program (Altschul et al., 1990).
environmental samples, a selective medium for the Bacillus
cereus group (Oxoid, Ltd., Basingstoke, England), and Plasmid profiling

Table 1. Sampling sites, origin of samples collected and number of samples yielding Bacillius thuringiensis (Bt).

Type of Number of Type of Number of

Name of localities Name of localities
sample sample sample sample
Soil 11 Lakshmirchor, Soil 11
Savar
Leaves 03 Jamalpur Leaves 11
Keraniganj, Dhaka Soil 04 Soil 04
Ati, Dhaka
Jessore Soil 01 Leaves 04
Soil 02 Soil 08
Agargaon, Dhaka
Leaves 01 Sonargaon Leaves 08
Comilla Soil 02 Insects 07
Soil 04 Norsinghdi Soil 07
Nandipara, Dhaka
Leaves 04 Chuadanga Soil 07
 Bacillus thuringiensis - An eco-friendly biopesticide 218
Plasmid of the isolates was purified by alkaline lysis method insects. The food was then dried and placed in a petri dish
(Sabine et al., 2003) with some modifications. 2 ml of overnight where 10 adult beetles were placed. The toxicity of each strain
culture grown in LB broth at 30 o C was pelleted and was assayed in triplicate for either the original toxin-spore
resuspended in 100µL of TE buffer (40 mM Tris-HCl, 2 mM suspension or the dilutions. The transformation was confirmed
EDTA, pH 7.9). 200 µL of lysis solution [3% (w/v) SDS, 15% carefully to the test cases with soft brush to avoid physical
(w/v) sucrose, 50 mM Tris-hydroxide, pH 12.5] and was trauma. Mortality was scored comparison with parallel control
incubated for 30 min at 60°C. Then 5U of proteinase-K was in which pulse grains were soaked in sterile distilled water
added and mixed gently and incubated for another 90 min at instead of bacterial suspension and used to correct the test
37°C. 1 mL of phenol: chloroform: isoamyl alcohol (25:24:1) mortality by using Abbot’s formula. The LC50 values were
was added to the mixture and the tubes were inverted carefully determined by probit analysis using SPSS for Windows. When
several times. The samples were then centrifuged for 15 min the control mortality were more than 10%, the percentage
at 13000 rpm and the supernatant fluid harboring plasmid mortalities was corrected following Abbott’s (1925) formula.
DNA was separated carefully. Plasmids of the isolates were The dose response data were analyzed (LC50, LC90) and
then electrophoresed through 0.5% agarose gel prepared in presented using SPSS-16, Biostat-2009, Microsoft Excel
1× TBE and visualized against UV in Gel Documentation Program-2007. Computations performed by this program were
machine (Bio-Rad). based on Finney (1964).

Crystal protein weight determination RESULTS

100 µL of sporulated culture grown in T3- medium (per liter: 3 Bacillus thuringiensis distribution
g tryptone, 2 g tryptose, 1.5 g yeast extract, 0.05 M phosphate A total of 99 samples were examined in this study (Table 1)
buffer pH 6.8, and 0.005 g of MnCl2) was pelleted and washed and growth on MYP medium plate permitted 99 to cosidered
with 1.0M NaCl containing 5mM EDTA and then with 5mM as Bacilli. Acetate slection and hemolysis as Bt test, a means
EDTA alone (Ozturk F. et al. 2008). Washed crystals and spores to distinguish between B. anthracis, B.cereus (Bc) and B.
was resuspended in 100 µL of SDS sample loading buffer (50 thuringiensis, identified 57 isolates as Bt and restof them as
mM Tris-HCl, pH 7.5; 2% (w/v) SDS, 0.05% (w/v) bromophenol Bc. Randomly selected 15 isolates were allowed to sporulate
blue, 1mM EDTA, 10% (v/v) glycerol, 15 mM DTT) and boiled and observed under Phase contrast microscope and presence
for 5 min at 100°C. Insoluble material was removed by of parasporal crystal proteins was confirmed. For these
centrifugation and supernatant containing ä–endotoxin was randomly selected 26 isolates, Bt index was calculated 0.6
analyzed by SDS-PAGE loading 20ìl aliquots onto 7.5% ranging from 0.4 for leaf to 0.67 for soil.
acrylamide gels. Following electrophoresis, the gels were
stained for 1 hr in 0.1% Coomassie Brilliant Blue G-250 and Crystal composition of the isolates
destained overnight in a solution containing 6.75% (v/v) Fifteen out of randomly selected 26 isolates from a total of 99,
glacial acetic acid and 9.45% (v/v) methanol. The molecular were observed under Phase contrast microscopy to be
weights of proteins were determined by using protein parasporal crystal protein producing (Fig.3). Five different
molecular standards (Invitrogen, USA). types of crystal shapes were observed among the isolates
Bioassay such as spherical, cubical, bipyramidal, irregular pointed and
irregular shaped. More than one type of crystal proteins were
The adult pulse beetles (Callosobrochus chinensis,
also found in some isolates (Table 2).
Tribolium castaneum and Sitophilus oryzea) were collected
and kept at 28-32ºC and 70-80% RH for both multiplication 16S rDNA gene sequencing for identification
and bioassay. Isolates with parasporal bodies were cultured
16S rDNA gene sequencing of isolates, numbered 01i, 25f
in 100 ml of T3- liquid medium and incubated for 7 days at
and 48s yielded an 897 nucleotide-long amplicon with
30°C with continuous shaking at 250 rpm. Liquid cultures
universal primer (5’-GAGTTTGATCCTGGCTCAG-3’) which
were centrifuged at 5000 rpm for 15 min. Pellets (spores and
was compared with the sequences reported in Gene Bank of
parasporal protein crystals) were washed in 20 mL sterile
NCBI (National Centre for Biotechnology Information, http:/
distilled water and centrifuged at 5000 rpm for 5 minute.
/www.ncbi.nih.gov/). From the BLAST search (Basic Local
Washing procedure was repeated twice. The pellets were
Alignment Search Tool) that provides a method for rapid
resuspended in 20 mL of sterile distilled water and kept at
searching of related sequences, the 16S rDNA sequences
4°C. Different concentrations of spore-crystal mixture of Bt
showed 99% sequence match with Bt strain BAC3042. The
was prepared by diluting with sterilized distilled water and
partial sequence of this strain was aligned with related
the fresh pulse grain were soaked in it as food for the test
 Asma Akter et al. 219
Figure 1. Presence of crystals under Phase-contrast microscope (A): 24 hrs (B): 48 hrs

Spore
Spore
Crystal

Crystal

A
B

Table 2. Classification of Bt isolates based on crystal morphology and comparison with reference strains.

Similarity with
Morphology of crystals Bt Isolates % of samples
reference strains
Bipyrmidal(BP) 01i, 26s, 45s Sotto , HD-73 20%
BP, Spherical(SP) 06i, 27s, 48s, Sotto , Buibui , HD-73 20%
SP 03i, 25f, 41s, 49s, 51s Sotto , Buibui, HD-73 33%
SP, Irregular pointed(IP) 47s Buibui, HD-73 7%
IP, Irregular shape(IS) 40f Buibui 7%
BP , Cubic 44f Sotto, HD-73 7%
BP , IP 47f Sotto , Buibui, HD-73 7%

sequences (obtained from BLASTn) by CLUSTAL W 2.0.12 4 plasmid bands of 8kb, 10kb- 15kb, and 22kb resulting in a
program CLUSTAL 2.0.12 multiple sequence alignment, unique array (lane 10) (Benintende et al., 1999).
resulted in the generation of highest score of 99% among the
Characterization of crystal protein of the isolates
sequences.
Crystal protein profile of thirteen native isolates and reference
Plasmid profiles of the isolates strains were analyzed by discontinuous SDS-PAGE (Laemmli,
The plasmid profiles of the Bt strains were compared to the 1970) which revealed protein bands with molecular weight
reference strains which revealed a major plasmid band of 15 ranging from 19kDa to ~195kDa (Fig. 5). Analysis showed the
kb size was present in all isolates (Figure 4). In addition, presence of several major polypeptide bands within the
plasmid bands varying in length between 10 and 22 kb were isolates 25f, 1i, 27s, 48s, 47f, 45s, 49s, 51s and reference strains
also observed in some local strains. For 49s strain, there were Btk HD-73 and Btsotto, where three common prominent bands

Figure 2. Plasmid profiling of the Bt isolates; lane 1: Bt-40f; lane 2: Bt-6i; lane 3: Bt-47s; lane 4: Bt-25f: lane 5: Bt-48s; lane 6: Bt-
1i; lane 7: Bt-27s; lane 8: Marker (PDK-9); lane 9: Bt japonensis buibui; lane 10: Bt-49s; lane 11: Bt-51s; lane 12: Bt- sotto; lane 13:
Btk HD-73.
 Bacillus thuringiensis - An eco-friendly biopesticide 220

1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9
200kDa
200kDa
116.3kDa 116.3kDa
97.4kDa
66.3kDa
55.4kDa

36.1kDa
30.0kDa 36.5kDa
31.0kDa

Figure 3. A B
Gel (A): 1: Marker; 2: HD-73; 3: Sotto; 4: 25f; 5: 1i; 6: 3i; 7: 47s; 8: 6i; 9:40f; 10: 44f
Gel (B): 1:47s; 2: 49s; 3: 27s; 4: Sotto; 5: 48s; 6: 51s; 7: 47f; 8: 45s; 9: Marker.

of ~65kDa, ~130kDa and ~135kDa suggest the presence of antibiotic selection, no desired growth was observed in 34
Cry1, Cry2 and Cry9 proteins (Crickmore et al., 1998). (16 phylloplane, 4 insect and 14 soil) samples. Though
Bioassay polymyxin B sulfate is used to inhibit most of the Gram
negative and Gram positive bacteria, S. aureus, B. subtilis
Static bioassay was conducted to determine the susceptibility
and some other soil bacteria can grow along with B. cereus
level of the adult pulse beetles (Callosobrochus chiniensis, and B. thuringiensis. Crystal protein observation and analysis
Sitophilus oryzea and Tribolium castaneum) against two Bt was performed under Phase contrast microscope to
isolates (1i, 25f) and one reference strain Btk HD-73 where C. distinguish Bt and others. Bt was found in all selected habitats
chiniensis was found to be more susceptible than Sitophilus which suggests the ubiquity of Bt in Bangladesh. The
oryzea and T. castaneum. Comparing LC50 and LC90 value at abundance of B. thuringiensis was the highest in all soil
72, 96 and 120 hrs it was found that strain Btk HD-73 was samples with a Bt index maximum of 0.67.
most active and 01i isolate’s activity was impressive as its
Crystal morphology of Bt can provide valuable information
very close to the activity of reference strain. 25f isolate was on target insect spectra (Maeda et al. 2000). Spherical shaped
also found to have good activity after 5 days exposure to the crystal morphology was observed mostly among the isolates
insects in comparison to the reference strain (Table 3). In C. tested which indicate their toxicity to the dipterans. On the
chiniensis significant difference of efficacy among the strains basis of the content of protein crystals the isolates were
was found only at 24 hrs (Test of Homogeneity, p=0.017). divided into 5 different groups (Table 2). 16S rDNA gene
Significant variation (5%) among different doses was also sequence analysis was performed to identify Bt isolates.
found except 24 and 48 hours (p=0.729 and 0.242). Universal forward primers for 16S rDNA gene sequencing
DISCUSSION were used and three Bt isolates were identified as Bt. The
genes coding for the insecticidal Cry proteins are normally
A total of 99 samples (61 from soil, 31 from phylloplanes and
associated with plasmid of large molecular mass (Gonzales
7 from insect) were collected from local agricultural lands in
and Carlton, 1980). Plasmid profiling was performed with nine
Bangladesh mainly of different common vegetables. After
Table 3. Susceptibility of four Bt isolates (LC50 and LC90 mg/mL) against Callosobrochus chiniensis.

1i 25f HD-73
Time (hours)
LC 50 LC90 LC50 LC90 LC50 LC90
72 h 0.30 0.72 0.73 2.02 0.22 0.74
96 h 0.22 0.83 0.09 0.85 0.08 0.58
120 h 0.14 0.78 0.04 0.31 0.02 0.48
144 h 0.12 0.59 0.06 0.31 0.01 0.42
LC= lethal concentration (mg/ml)
 Asma Akter et al. 221
Bt isolates and the result showed bands of different molecular Strains of Bacillus thuringiensis. A p p l i e d a n d
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because they produce higher level of mortality than either Nethravathi, C. J., Hugar P. S., Krishnaraj, P. U., Vastrad, A. S.
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Received: November 14, 2011 Revised: January 18, 2012 Accepted: February 6, 2012