Bt Research, 2015, Vol.6, No.

8, 1-16
http://bt.biopublisher.ca

Research Article

Open Access

Genetic Diversity of Indigenous Bacillus thuringiensis Strains by RAPD-PCR to

Combat Pest Resistance
Shishir M.A., Pervin S., Sultana M., Khan S.N., Md Hoq M.
Department of Microbiology, University of Dhaka, Dhaka- 1000, Bangladesh
Corresponding author email: mhoq@du.ac.bd
Bt Research, 2015, Vol.6, No.8 doi: 10.5376/bt.2015.06.0008
Received: 08 Sep., 2015
Accepted: 15 Oct., 2015
Published: 16 Nov., 2015
Copyright 2015 Shishir et al, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Shishir M.A., Pervin S., Sultana M., Khan S.N., and Md Hoq M., 2015, Genetic Diversity of Indigenous Bacillus thuringiensis Strains by RAPD-PCR to
Combat Pest Resistance, Bt Research, 6(8): 1-16 (doi: 10.5376/bt.2015.06.0008 )

Abstract Genetic diversity is highly relevant and significant in discovering novel insecticidal genes in Bacillus thuringiensis (Bt)
strains and to deal with the problems of emerging insect resistance towards Bt biopesticides. In view of this, Random Amplified
Polymorphic DNA (RAPD)-PCR analysis was performed with a decamer AGCTCAGCCA for molecular typing of 177 Bt strains of
Bangladesh to determine their genetic diversity. These Bt strains were allocated into 15 genomic types with their binary matrices as
determined from the dendrogram based on a standardized distance in scale bar. Genotype 9 and 11 were the largest among others,
each containing more than 25% of the Bt strains. The average diversity index, as deduced for each group by cluster: isolate ratio at a
specific distance, was higher for locations (0.27 0.098) than that for biotypes (0.23 0.046) which indicates an unmingled and
vertical transfer of biochemical properties among the strains. Prevalence of agriculturally important subgroups of cry1 gene in
indigenous Bt strains was also determined where cry1Aa and cry1Ca gene were found to be the most prevalent (21.74%). While
analyzing the distribution pattern of cry genes, they were observed to be present in all RAPD- genotypes but genotype 10 and were
most prevalent in genotypes 1, 6 and 9. The phylogeny reconstruction among the strains was performed by neighbor-joining method
with the 16S rRNA gene sequences and the correlation among the phylogeny, RAPD genotypes, Biotypes and presence of cry genes
were analyzed.
Keywords Bacillus thuringiensis; cry genes; Genotyping; resistance; RAPD-PCR

Background

inverted repeats flanking the endotoxin genes in high

frequency causing DNA rearrangements etc and some
extrinsic factors like mutation, nutritional influences
etc (Kaur et al., 2006). Genetic diversity among the Bt
strains is, therefore, beneficent which enhances the
scope of discovering novel toxins and urges for
extensive exploration for more strains.

The key to the toxicity of Bacillus thuringiensis

against the insect larvae is the specific molecular
interactions of the insecticidal proteins with the membrane
receptors followed by pore formation in the insect
mid-gut epithelium. The degree and spectrum of
toxicity of Bt insecticidal proteins against different
insect species are variable. There are currently around
75 primary subgroups of Cry toxins, 3 for Cyt toxins
and 4 for Vip toxins (Adang et al., 2014) and more
than 300 different members so far reported are present
in these subgroups (Crickmore et al., 2 014,
http://www.btnomenclature.info/). The remarkable
diversity is because of a high degree of gen etic
plasticity or variations that occurs among the Bt
strains due to many intrinsic factors like the presence
of many different plasmids in each strain and their
conjugal transfer, recombination between chromosomal
DNA and plasmids, involvement of transposon-like

Again, resistance development in insects against any

insecticide is a common occurrence with no exception
for Bt toxins. The facts behind the resistance and
cross-resistance of insect pests to Bt toxins as reported
are, i) reduction of binding of toxins to receptors in
the midgut of insects, ii) reduced solubilisation of
protoxin, iii) alteration of proteolytic processing of
protoxins and iv) toxin degradation and o r
precipitation by proteases etc (Bruce et al., 2007). Few
additional virulence factors such as, phospholipase C
(Palvannan and Boopathy 2005; Martin et al., 2010),
proteases (Hajaij-Ellouze et al., 2006; Brar et al., 2009;
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and McClelland, 1990; Williams et al., 1990). Besides
this taxon-specific amplicon generation for the
detection of diversity, RAPD-PCR fingerprinting
technique is faster, less labor-intensive and more
reliable in comparison to other molecular typing
methods (Bostock et al., 1993; Sikora et al., 1997)
and thus was found to be more feasible method for
this study.

Infante et al., 2010) and hemolysins (Gominet et al.,

2001; Nisnevitch et al., 2010) etc that influence the
toxicity, are also variable among the strains due to the
genetic variations beside the insecticidal proteins.
Efforts should, therefore, be continued in discovering
more Bt strains with genetic diversities to discover
novel toxins with improved activity, to widen the
spectrum and to overcome the resistance problems
besides protein engineering with the existing pool. So,
a good number of Bt strains were previously isolated
by us from different regions of Bangladesh and
studied in terms of their abundances, distribution
patterns as well as diversities connected t o
biochemical properties, plasmid and cry genes profile
(Shishir et al., 2014). Potentiality of these strains,
presumed through insecticidal gene and protein profile
analysis, was established for certain strains against
Melon fruit fly (Bactrocera cucurbitae) upon bioassay
and this novel toxicity was reported (Shishir et al.,
2015).

Considering the high relevance of the above facts, the

present study was designed to determine the genetic
diversity among 177 Bt strains of Bangladesh and to
reveal the correlation between the distribution pattern
of cry genes to the diversity.

1 Results and Discussion

1.1 RAPD profile based genotyping
The specific typing of B. thuringiensis enables
tracking of strains dispersed in the environment and
assist in the discovery of new strains. The existing
serotyping scheme, while having provided an
invaluable basis for Bt classification for a long time,
provides no information about the genetic relatedness
of strains within groups and between groups and does
not necessarily indicates the degree and spectrum of
toxicity (Gaviria- Rivera and Priest, 2003). Contrarily,
RAPD is a modified PCR method with a single
arbitrary primer that recognizes differences in the
prevalence and positions of annealing sites in the
genome producing a spectrum of amplicons that are
considered to reflect the genomic composition of the
strain and may vary along the strains (Welsh and
McClelland, 1990; Williams et al., 1990). The
advantage of this method is that no prior knowledge of
the genome under research is necessary (Bostock et al.,
1993; Sikora et al., 1997) and it was found from
several studies that the RAPD analysis could
effectively distinguish between the Bt strains (Kumar
et al., 2010). Hence, the analysis of genetic diversity
among the indigenous Bt strains was done by
RAPD-PCR method in this study.

Thus, more Bt strains and the genetic diversity

analysis among them is highly significant for
maximum utilization of the resources and to combat
the resistance problem. Several different techniques
were reported for genetic diversity analysis, like M13
fingerprinting (Miteva et al., 1991), arbitrary primer
PCR (Brousseau et al., 1993), PCR using conserved
primers for 16S to 23S ribosomal intergenic spacer
sequences (Bourque et al., 1995), DNA hybridization
using variable region of 16S rRNA gene (te Giffel et
al., 1997), AFLP fingerprinting, RAPD-PCR (Welsh
and McClelland, 1990) etc. Ribotyping, either by PCR
or DNA hybridization, failed to detect the diversity
among Bt strains which could be because of the use of
one single gene or operon and the evolutionarily
conserved nature of rRNA gene. However, when the
whole genome was used for identification of Bt strains
by M13 DNA fingerprinting and arbitrarily primed
PCR, considerable diversity among the Bt serovars
representing different serotypes had been detected.
Random amplification of polymorphic DNA (RAPD)
is a modified method of PCR with a single arbitrary
primer that recognizes differences in the prevalence
and positions of annealing sites in the genome and
produces a varied spectrum of amplicons reflecting
the genomic composition of the strains (Welsh

A total of 177 Bt strains were employed for

RAPD-PCR amplification with decamer OPA 03 (5'AGCTCAGCCA -3') which was reported to produce
100% polymorphism (Kumar et al., 2010) and also
observed to be efficient in few initial screenings in
this study (Data not shown). Molecular weights
of these bands were then estimated comparing with
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the DNA standard (100 bp DNA ladder, Bioneer,
Korea) and the presence of 16 different bands (100,
150, 200, 225, 275, 300, 350, 400, 450, 500, 600, 700,
800, 900, 1000 and 1100 bp) were observed at varied
numbers and combinations in the strains. By
scrutinizing these bands, binary matrices for the
strains were obtained where the presence was scored
with 1 and absence with 0. No strain was observed
with all 16 bands and maximum 11 bands were
present for certain strains. Polymorphism based on
these 16 individual bands was calculated and 100%
polymorphism was not observed in any strain.
Maximum 68.8% polymorphism was seen in 0.4% of
the test strains whereas 25% polymorphism was most
prevalent followed by 31.3% (15.3% of test strains)
and 18.8% (13.1% of test strains) (Table 1).

As binary matrix was prepared for each strain based

on the 16 polymorphic DNA bands, 256 (16 2)
numbers of different banding patterns are possible.
Therefore, 15 major RAPD pattern i.e. 15 genotyps
were presumed from all these binary matrices with
proximities to the major patterns which was derived
from the dendrogram based on the heights of the
clades (Fig 1- sup). Dendrogram (Fig 1- sup) was
constructed by UPGMA clustering method with the
distance matrix and similarity matrix among
the strains prepared by dice coefficient
comparison method from their binary matrices i.e.
the numerical RAPD profiles (Table 1- sup). The
heights of the clades in scale bar, an indication
of distance among

Table 1 Prevalence of polymorphism at different level among the indigenous Bt strains.

Polymorphism (%)

6.3

12.5

18.8

25

31.3 37.5 43.8

50

56.3 62.5 68.8

75

Prevalence (%)

2.5

10.6

13.1

16.1

15.3 12.3 12.7

5.5

3.8

1.3

0.4

81.3 87.5 93.8

0

100
0

Fig. 1 Representative gel images showing random amplified polymorphic DNA (RAPD) genotypes (numbered 1- 15 in the left alignment
of the nodes) produced from Bacillus thuringiensis strains with the primer OPA 03. (Marker: 100 bp DNA ladder, Bioneer, Korea).

the strains was standardized in this case. A middle

height at 0.2 was presumed as the threshold point

from the scale bar where the start point extended up to

0.45 to distinguish the major clusters as separate
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genotype throughout the whole study. Thus the height
of any clade (cluster) exceeding 0.2 (Fig 1-sup) was
considered as separate genotype. RAPD pattern of Bt
strains representing these 15 genotypes are as shown
in Figure 1. This enabled quantitative comparison of
genetic diversities among different sets of strains e.g.
biotypes or locations upon standardization.

of a certain location is not resulted from the influence

of abiotic factors only such as UV, salinity, trace
elements, pH, organic maters etc rather a phenotypical
pattern was found to be maintained as the DI among
the strains with similar biochemical properties was
found to be lower across different locations.

The Bt strains were found to be divided into two

major clusters, A and B in the dendrogram (Fig 1sup). Cluster B was smaller comprising of only
14 strains and A was larger with 163 strains, hence
subdivided further into subclusters A1 and A2.
Sub-cluster A2 was large enough and found to be
further branched into clusters with significant number
of strains denoted as A2a and A2b.
Fig 2 Prevalence of different genotypes among the indigenous
Bacillus thuringiensis strains of Bangladesh.

Genotype 2, 4 and 10 were simplicifolious (single

leafed), genotype 14 was bifolious (two leaved),
genotype 8 and 15 were trifolious (three leaved) and
rest others were polyfolious (more than three leaved).
The genotypes were observed to contain the strains in
a mingling manner with respect to their biotypes
except genotype 12 which contained 80% of strains
from biotype kurstaki. And for the locations, strains
from the same location appeared to be closely related
even though their biochemical characteristics differed
(Fig 1- sup).

The prevalence of Bt strains in different genotype was

also calculated and genotype 9 and 11 were found to
be the largest, each containing more than 25% of the
strains (Fig 2).
1.2 Comparison of diversity between biotypes and
locations
Based on the threshold height or distance of the clades
in the scale bar, the diversity indices (DI) were
calculated as the ratio between the number of major
clusters and the number of strains, within biotypes and
locations. In case of biotypes, genetic diversity was
maximum in Bt israelensis followed by sotto, eleven
and minimum was in biotype 13, ten and nine (Fig
3A). In case of locations, maximum diversity was
observed among the strains of Narshingdi and the
minimum was for Munshigonj (Fig 3B).

Fig. 3 Comparison of diversity indices (DI) as calculated based

on the ratio of number of genotypic clusters beyond the
threshold level and number of strains for A) the selected
biotypes B) the selected locations.

1.3 Prevalence of cry genes

Detection of major cry genes (viz. cry1, cry2, cry3,
cry4, cry8, cry9, cry10 and cry11) in the indigenous Bt
strains was reported earlier (Shishir et al., 2014).
Besides the prevalence of primary subgroups (Fig 4A),

The average diversity index for locations (0.270.098)

was higher than that for biotypes (0.230.046) which
indicates that the genetic diversity among the strains
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1.4 Pattern of cry genes distribution within
RAPD-genotyes
As the genotypes and cry gene profiles of the strains
were thus retrieved, it was analyzed whether the
distribution of cry genes was random or genotype
oriented. So, the distribution of different cry genes in
different genotypes was analyzed and from the
graphical presentation (Fig 5) it was revealed that cry
genes were present in all genotypes except genotype
10. The abundance of these cry genes were maximum
in genotypes 1, 6, 9, 11 and 12. Though genotypes 9
and 11 were found to be the largest containing more
than 25% of the strains, only genotype 9 of them was
significant with different cry genes besides genotypes
1 and 6 as compared to the number of strains.

tertiary subgroups of cry1 gene (cry1Aa, cry1Ac,

cry1Ba, cry1Ca belonging to cry1 as it was the most
prevalent) were also detected (Fig 2A- sup, 2B- sup,
2C- sup and 2D- sup) and their prevalence was
determined where cry1Aa and cry1Ca were found to
be the most prevalent (Fig 4B).

Comparing the ratio between the number of cry genes

and strains in the genotypes, genotype 6 (2.167) was
found to be most significant followed by genotype 1
(1.285), genotype 9 (0.29), genotype 11 (0.18) and
genotype 3 (0.14). On the other hand, maximum 6
types of cry genes were present in genotypes 1, 6, 9
and 11. Thus, it was clear from this analysis that
though the cry genes were observed in varied
frequencies in most of the genotypes, cry genes were
found to be most abundant in terms of number and
type in genotype 6, 1 and 9. Again, the presence of
same cry gene in different genotypes increased the
chances that the degree and spectrum of toxicity might
be variable i.e. genes except cry4, cry8 and cry10
were found to be present in multiple genotypes.

Fig.4 Prevalence of different cry genes in the indigenous Bt

strains (n: Number of organisms). A) Primary subgroups, B)
Tertiary subgroups.

1.5 Comparison between different similarity

parameters
Another comparison was performed with 20 Bt strains
(indigenous- 19, reference- 1) in terms of their 16S
rRNA gene sequence based phylogeny (Shishir et al.,
2014), Biotype, RAPD based genotype and number of
available cry genes (Fig 6) which revealed that
phylogenetically close strains were similar in biochemical
properties. Phylogenetically close 12 strains as in
sub-cluster A1 were observed to have similar biochemical
properties since from same biotype kurstaki except
strain DSf3 (non-hemolytic), strain CiSa5 (biotype ten)
and KSa2 (dendrolimus). Again in sub-cluster A2, 3
strains out of 5 were non- hemolytic and 2 out of 3
strains in cluster B were from biotype kurstaki.
Though the biochemical properties of most of them

Fig.5 Distribution of different cry genes in the different

genotypes those were established in this study by RAPD-PCR
method. Prevalence of different genes in each genotyp e is
indicated with same colored column i.e. color varied with
genotypes not genes.

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(Carlson and Kolst, 1993). Other comparisons of soil
isolates of B. cereus and B. thuringiensis strains,
selected with no regard for insect toxicity, have
demonstrated extensive chromosomal DNA exchange
with no apparent clonal population structure (Hu et al.,
2004). On the other hand, correlation persisted for the
highly conserved phenotypes like biochemical
properties and genotypes such as 16S rRNA etc. as
these are regulated by the in house conserved genes.

conformed to the phylogenetic relatedness, their

RAPD genotypes were variable e. g. 5 genotypes (1, 5,
6, 11 and 12) were visible among the strains of
sub-cluster A1 which were from the biotype kurstaki.
This genetic diversity might be due to the presence of
many different plasmids in each strain and high
frequency of DNA rearrangements in variable regions
by conjugation transfer mechanism and the transposon-like
inverted repeats flanking the endotoxin genes. Plasmid
DNA exchange in nature is well documented in B.
thuringiensis strains and has been implicated as the
source of the remarkable diversity of cry genes

The number of available cry genes among these

strains was also variable. It can, therefore, be said that

Fig. 6 Comparison of relatedness between the Bt strains determined by 16S rRNA gene sequence, biochemical properties, RAPD
genotyping and availability of cry genes.

the report of conformity between phylogenetic

and phenotypic i.e. biotype or serotype (biotype in
this case) relatedness (Shishir et al., 2014) was also
evidenced in this study though RAPD- genotyping and

of Dhaka and the reference Bt kurstaki HD-73, Bt

sotto T84A1 and Bt japonensis Buibui strains
collected from Bt stock collection of Okayama University,
Japan were used in this study. LB agar (per litre: tryptone
10 g, yeast extract 5 g, NaCl 10 g, agar 15 g) and LB
broth were used for culture maintenance, propagation and
subculture throughout the study.

cry gene profile did not follow the pattern.

2 Materials and methods

2.1 Bacterial strains and growth conditions
Indigenous Bt strains (n=177) preserved at Bt resource Centre
in the Department of Microbiology, University

2.2 Total DNA extraction

Total DNA was extracted from the indigenous Bt
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isolates streaked on LB agar medium (Bravo et al.,
1998). After 12 hours of incubation at 30C, a single
colony, transferred into 100 l of sterile de-ionized
water in a microfuge tube, was vortexed and kept at
-70C for 30 min. It was then incubated in boiling
water for 10 min to lyse the cells and briefly
centrifuged for 20 s at 12,000g (Eppendorf
centrifuge, 5415D). The upper aqueous phase
transferred into sterile microfuge tubes was used as
template and preserved at -30C for further use.
50-100 ng of DNA from this suspension was used as
template in RAPD- PCR analysis.

with the same batch of PCR master mix.

2.5 Genotyping and estimation of diversity index
Throughout the whole study, threshold level was
chosen at 0.2 in the scale bar. Each cluster was
considered a separate genotype if distances among the
strains in that cluster were less than 0.2 in scale bar.
Thus the genotypes were identified among the tested
strains as a whole and also in terms of biotype and
location. Again, the ratio between the number of
clusters and isolates for a set of strains was considered
as their diversity index. Based on this criterion, the
diversity index for biotypes and locations were
estimated and compared.

2.3 RAPD-PCR analysis

RAPD-PCR was performed using the primer (OPA 03:
5'- AGCTCAGCCA- 3') with minimum 60% G+C
content and devoid of any internal repeat as maximum
polymorphism was reported for this decamer (Kumar
et al., 2010). PCR was carried out within a reaction
volume of 25 l [1 PCR Master mix (Promega,
USA), 2.0 M of primer, 50-100 ng of template DNA]
in a thermal cycler (MJ mini, BioRad, USA) by 35
cycles (95C for 1 min, 40C for 1 min, 72C for 1
min) with an initial denaturation step at 95C for 4
min and a final extension step at 72C for 15 min
(Kumar et al., 2010). PCR products (15 l) were then
analyzed in 1.5% (w/v) Agarose (Promega, USA) gel
by horizontal electrophoresis at 60V for 1h in 1 TBE
[89 mM Tris (pH 7.6), 89 mM boric acid, 2 mM
EDTA] buffer and gel images were captured after
visualization against UV trans-illumination in a gel
documentation system (Alpha imager mini, USA)
following staining in Ethidium Bromide (EtBr)
(Sigma, USA) solution (0.5 g/ ml) and destaining in
distilled water. Molecular weight of the DNA bands in
those gels was then determined by using Alphaview
SA software (version 3.4.0.0).

2.6 Detection of subgroups of cry1 genes

DNA templates (50-100 ng) from the Bt strains (n=
177) were mixed with PCR reaction mixture
containing 0.5 M of each primer, 1 PCR Master
mix (Promega, USA) in 25 l reaction volume and
amplification was performed in a DNA thermal cycler
to detect cry genes belonging to the cry1 family
(major group) such as cry1Aa, cry1Ac, cry1Ba,
cry1Ca. For all primer sets, PCR was carried out with
an initial single denaturation step at 95C for 2 min
and 30 amplification cycles including denaturation at
95C for 45 s, annealing at 53C for 45 s and extension at
72C for 60 s. Finally an extra extension step was
applied at 72C for 10 min. PCR products (10 l)
were then electrophoresed in 1.5% agarose (Promega,
USA) gel prepared and submerged in 1TBE buffer at
60V for 60 min. Gel was visualized in a gel documentation
system following staining and de-staining.
2.7 Determination of distribution of cry genes in
the genotypes
Detection of cry genes from major groups such as
cry1, cry2, cry3, cry4, cry8, cry9 and cry10 was
reported previously (Shishir et al., 2014). Certain
other subgroups of cry genes belonging to the cry1
family (major group) such as cry1Aa, cry1Ac, cry1Ba,
cry1Ca were also investigated. Combining the
presence of the above mentioned genes, cry gene
profile was obtained for each strains. Again, each
strain for its RAPD profile belongs to a cert ain
genotype. Thus, the number of cry genes in each
genotype and their distribution was determined.

2.4 Data analysis and dendrogram construction

Binary matrix was prepared for each strain from the
gel images based on the presence or absence as scored
1 or 0 respectively for the amplicons bands. Based on
the binary matrices, similarity and distance matrices
were calculated following dice coefficient method.
These data were used in cluster analysis by
UPGMA method to construct the dendrograms
(http://insilico.ehu.es/dice_upgma/). For maximum
accuracy of comparison, all isolates were processed
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te Giffel, M. C., Beumer, R. R., Klijn, N., Wagendorp, A. and Rombouts, F.
M. 1997, Discrimination between Bacillus cereus and Bacillus

3 Conclusion
This study will facilitate the research on Bacillus
thuringiensis in Bangladesh by providing valuable
information to find out diverse Bt strains those which
can be used in biotechnological purposes. The results
also suggest that the degree and spectrum of the
toxicity of indigenous Bt strains could be diverse to be
used as efficient weapons to fight the resistance
problems with pests.
Acknowledgements
This work was supported by a Grant-in-Aid from the USDA as
a project entitled "Production of Bacillus thuringiensis
biopesticides by biotechnological approach for the control of
vegetable pests in Bangladesh". We thank Okayama University,
Japan for providing reference strain.
References
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Bacillus thuringiensis Crystal Toxins and Mechanism of Action, p.
39-87. In: T. S. Dhadialla and S. S. Gill (ed.). Advances in Insect
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Bravo, A., Sarabia, S., Lopez, L., Ontiveros, H., Abarca, C., Ortiz, A., Ortiz,
M., Lina, L., Villalobos, F. J., Pena, G., Nunez-Valdez, M. E., Soberon,
M. and Quintero, R. 1998, Characterization of cry genes in a Mexican
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Bruce, M. J., Gatsi, R., Crickmore, N. and Sayyed, A. H. 2007, Mechanisms
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Schnepf, E., Sun, M. and Zeigler, D. R. 2014, Bacillus thuringiensis
toxin nomenclature. http://www.btnomenclature.info/
Gominet, M., Slamti, L., Gilois, N., Rose, M. and Lereclus, D.
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Supplementary Figures and Tables:

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Fig. (supplementary) 1 Dendrogram exhibiting the genetic distance among the selected strains of Bacillus thuringiensis based on
their RAPD-PCR patterns in the range from 100 bp to 1.1 kb which were compared using the Dice coefficient and the UPGMA
clustering algorithm.

Figure 2 (supplementary): Presence of cry1Aa, cry1Ac, cry1Ba, cry1Ca genes in indigenous Bt strains was detected by agarose gel
electrophoresis of the PCR products. A) cry1Aa-type genes, B) cry1Ac-type genes, C) cry1Ba-type genes, D) cry1Ca-type genes.
(Marker: 100 bp DNA ladder, Bioneer, Korea).
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Table 1 (supplementary): Data of presence or absence of certain amplicons mentioned with their sizes in the Bacillus thuringiensis
strains (presence= 1, absence= 0)

Molecular weight (bp)

700

600

500

450

400

350

300

275

225

200

150

100

Biotype

800

Name

900

notype

1000

Strain

1100

RAPD-Ge

NaSd1

galleriae

MyIb1

indiana

RaSa1

indiana

RaSa2

indiana

JDc1

kurstaki

JSc1

kurstaki

JSd1

kurstaki

KSe2

kurstaki

NaL1

kurstaki

Soi1

kurstaki

ChSa2

sotto

KuSa2

sotto

CgSe1

thuringiensis

RhSd1

thuringiensis

JeSe1

thirteen

SySa2

fifteen

KbSc1

indiana

MeSb2

indiana

NaSc2

indiana

NsSb1

indiana

JaS7

kurstaki

KbSa1

morrisoni

MeSe2

morrisoni

NsSe1

sixteen

TaSc2

sixteen

DSh7

sotto

SySa1

thirteen

SSa1

thuringiensis

SSf4

thuringiensis

NoS4

kurstaki

SaSc1

indiana

SSc1

israelensis

JaL1

kurstaki

JDa1

kurstaki

JSc3

kurstaki

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JSd2

kurstaki

JaS10

ten

MeSa1

darmstadiensis

KSa2

dendrolimus

MuSa1

dendrolimus

MuSe4

eleven

MuSc2

israelensis

DSe6

kurstaki

JDb1

kurstaki

KSb1

kurstaki

MuSc4

kurstaki

SSb1

kurstaki

MeSb1

thuringiensis

MuSd2

thuringiensis

RaSd1

indiana

NaL2

kurstaki

JaL4

nine

Soi3

nine

SpSc1

sixteen

SgSm2

sotto

RhSa2

thuringiensis

TaSb3

eleven

TaSc1

eleven

JaS1

nine

FhSa3

darmstadiensis

FhSb1

dendrolimus

NaSd3

eleven

SgSn1

eleven

CgSd1

fifteen

KbSb2

fifteen

KkSa2

fifteen

KkSd1

fifteen

NsSe2

fifteen

SgSj2

fifteen

SpSd3

fifteen

RaSb2

galleriae

SaSa2

galleriae

CgSe3

indiana

DpSb1

indiana

MyLa1

indiana

MySb2

indiana

NaSc3

indiana

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RaSb1

indiana

SaSb1

indiana

SaSc2

indiana

SaSd1

indiana

KfSa1

israelensis

CiSa1

kurstaki

DSe1

kurstaki

JDc2

kurstaki

SaS7

kurstaki

SSb2

kurstaki

RaSc1

morrisoni

KfSa2

nine

KkSc2

nine

NaSa2

nine

SgSc1

nine

FhSa2

ostriniae

NaSa1

sixteen

NaSb2

sixteen

NsSd2

sixteen

KkSb1

sotto

CgSc2

thirteen

KbSb1

thirteen

KkSc1

thirteen

RpSb1

thirteen

RpSc1

thirteen

SgSm1

thirteen

CiSa2

thuringiensis

CiSa3

thuringiensis

USc3

thuringiensis

10

TaSe2

ostriniae

11

JSa1

dendrolimus

11

JSb2

dendrolimus

11

JSc2

dendrolimus

11

MaSc1

dendrolimus

11

MuSc3

dendrolimus

11

RpSc2

eleven

11

RhSc2

fifteen

11

NsSa1

indiana

11

RhSb1

indiana

11

TaSa4

indiana

11

KeS4

israelensis

11

SaS5

israelensis

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11

SaS1

israelensis

11

AtS1

kurstaki

11

DSa3

kurstaki

11

DSe4

kurstaki

11

JaS5

kurstaki

11

JDa2

kurstaki

11

JDb2

kurstaki

11

KSa1

kurstaki

11

NoS2

kurstaki

11

SaS6

kurstaki

11

SoS7

kurstaki

11

RhSd2

morrisoni

11

DSa7

nine

11

JeS1

nine

11

NaS2

nine

11

JaL3

sotto

11

MaSb1

sotto

11

MuSd3

sotto

11

AtS2

ten

11

JaL2

ten

11

JaS4

ten

11

SaS2

ten

11

MaSb2

thirteen

11

MaSb3

thirteen

11

SgSp1

thirteen

11

AtL1

thuringiensis

11

JaL6

thuringiensis

11

JSa3

thuringiensis

11

KeS1

thuringiensis

11

KSc1

thuringiensis

11

MuSd1

thuringiensis

11

NaS1

thuringiensis

11

SaS3

thuringiensis

11

SaS8

thuringiensis

11

SSd2

thuringiensis

11

SSf1

thuringiensis

11

USc1

thuringiensis

11

Japo

japonensis

11

HD-73

kurstaki

11

Sotto

sotto

12

JSb1

kurstaki

12

SSc2

kurstaki

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12

SSe2

kurstaki

12

SSf2

kurstaki

12

JaL3

sotto

13

ChSa1

indiana

13

FhSa1

indiana

13

MeSd2

indiana

13

SgSp2

indiana

13

SpSb3

indiana

13

CiSa5

kurstaki

13

NaSc1

sixteen

13

RaSd2

sixteen

13

DSc2

thuringiensis

14

FhSb3

eleven

14

FhSb2

indiana

15

NaSd2

indiana

15

Rhsb2

indiana

15

DSh4

sotto

16