Diversity Analysis of Bacillus thuringiensis Isolates Recovered from Diverse Habitats in India using Random Amplified Polymorphic DNA (RAPD) Markers
Nagendra K. Singh
Bacillus thuringiensis is a bacterium of huge agronomic and scientific interest. The subspecies of this bacterium colonize and kill a large variety of host insects and nematodes with a high degree of specificity. In the present investigation, 32 native isolates of Bacillus thuringiensis recovered from different regions in India and 8 known Bacillus thuringiensis strains were analyzed at the molecular level using random amplified polymorphic DNA (RAPD) markers. Seven RAPD markers were used for diversity analysis. The RAPD banding pattern data was subjected to dendrogram construction using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) analysis using NTSYSpc2.2 software. Eight main clusters were formed at 25% similarity level and four isolates were standalone of these clusters. Most of the isolates were found to be diverse, even though they were isolated from the same source or location. The RAPD markers were found to be effective to distinguish B. thuringiensis native isolates recovered from different sources and locations. The results of present investigation help to understand diversity of B. thuringiensis in India, which would be exploited to find new types of B. thuringiensis endotoxins.
to cite this article:
Jawahar Katara, Rupesh Deshmukh, Nagendra K. Singh and Sarvjeet Kaur, 2013. Diversity Analysis of Bacillus thuringiensis Isolates Recovered from Diverse Habitats in India using Random Amplified Polymorphic DNA (RAPD) Markers. Journal of Biological Sciences, 13: 514-520.
Received: March 15, 2013;
Accepted: April 09, 2013;
Published: July 12, 2013
B. thuringiensis is a gram-positive bacterium distinguishable from other
closely related 3 Bacillus spp. viz., B. cerus, B. anthracis
and B. mycoides, because of its ability to synthesize crystal (Cry) proteins
during sporulation (Hofte and Whiteley, 1989). B.
thuringiensis has been used to control insect pests due to its environmental
safety and target specificity. When Cry proteins in B. thuringiensis
are ingested by insect larvae, the protoxin is dissolved and activated under
alkaline conditions present in the midgut of target insects and the active toxin
is released. This active toxin then binds to specific receptor in the insects
midgut epithelial cells resulting in pore formation in the membrane which eventually
leads to disruption of osmotic balance, cell lysis and death (Kaur,
2000). The different Cry proteins are toxic to a variety of insects that
cause serious damage to crop and lead to decrease in crop yield (Martin
and Travers, 1989; Iriarte et al., 1998).
Genotyping of bacteria employing Polymerase Chain Reaction (PCR)-based randomly
amplified polymorphic DNA (RAPD) method (Williams et al.,
1990) is a commonly used approach for strain typing because of its simplicity
and cost effectiveness (Gurtler and Mayall, 2001, Ronimus
et al., 1997). It is useful, fast and informative in differentiating
B. thuringiensis strains (Hansen et al.,
1998; Gaviria and Priest, 2003).
Taxon-specific markers with different specificities can be generated by using
RAPD fingerprinting technique (Cherif et al., 2003;
La Duc et al., 2004). This technique has successfully
been used for grouping of highly related genotypes of B. thuringiensis
(Sadder et al., 2006). RAPD has been used to
differentiate Bacillus anthracis from B. cereus, B. mycoides
and B. thuringiensis by identifying unique bands from these species (Kim
et al., 2007). The unique PCR product was cloned to develop species-specific
marker for identification of B. anthracis. Therefore, RAPD is a suitable
technique to discriminate the B. thuringiensis isolates for their diversity
In this study, an attempt was made to characterize the genetic diversity of
32 native isolates of B. thuringiensis isolated from different sources
collected from various states in India and 8 type strains used as reference
by using RAPD markers. We have previously carried out molecular typing of native
B. thuringiensis isolates from diverse habitats in India by using repetitive
extragenic palindromic sequence (REP)-PCR and enterobacterial repetitive intergenic
consensus (ERIC) sequence (ERIC)-PCR analysis (Katara et
MATERIALS AND METHODS
Isolation and culture of B. thuringiensis strains: A total of
32 native isolates of B. thuringiensis collected from diverse habitats
of India (Kaur and Singh 2000a, b)
and a set of eight type strains obtained from the Bacillus Genetic Stock
Centre (BGSC), Department of Biochemistry, Ohio State University and Columbus,
Ohio 43210, USA, were used as reference in this study (Table 1).
All the strains were grown at 28°C for 14 h on Luria Bertani Agar (LA) containing
penicillin (10 μg μL-1) as the selective antibiotic. B.
thuringiensis isolates had characteristic resistance against penicillin
and this was used for initial screening. Single colony from LA plate was selected
to grow in the Luria Bertani broth (LB) to harvest cells for chromosomal DNA
isolation in each case.
DNA extraction: Bacteria were shaken in 10 mL of LB at 28°C for
14 h until cultures reached late exponential phase. DNA was isolated from the
cell suspensions (4.0 mL) by using OmniPrep kit for Gram Positive Bacteria,
in accordance with the manufacturers instructions (G-Biosciences, St Louis,
MO, USA). The concentration of DNA was determined spectrophotometrically at
260 nm and finally DNA samples were diluted to 10 ng μL-1 concentration.
RAPD-PCR amplification: In this study, RAPD markers were used for DNA
fingerprinting analysis of B. thuringiensis isolates. In preliminary
experiments, 12 RAPD primers were used for amplification of two native B.
thuringiensis isolates. Out of these, 7 primers were selected on the basis
of reproducibility and used for genotyping of all the B. thuringiensis
isolates. Thermo-cycling conditions for the RAPD markers system were: DNA denaturation
at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for
1 min, primer annealing at 36°C for 1 min and extension at 72°C for
2 min. It was followed by a final extension step at 72°C for 10 min and
then samples were stored at 4°C till used for electrophoresis.
Electrophoretic analysis: RAPD-PCR patterns were visualized by agarose
gel electrophoresis. Aliquots of 12.5 μL each of the amplification products
were loaded on to 0.8% agarose gel and run in 0.5X TBE buffer at 2 V cm-1
for 5 h. Thereafter, gel slabs were stained with 0.4 μg mL-1
of ethidium bromide and documented with Alpha Innotech Gel Doc System. Molecular
weight analysis of bands was performed using FluorChem 5500 software with 1Kb
DNA ladder (MBI Fermentas) as the molecular weight marker. All reactions were
repeated at least twice and only bands that were bright and reproducible, were
scored for analysis.
Statistical analysis of marker characteristics: All seven primers were
used twice for PCR amplification to detect reproducible bands. Only reproducible
bands were considered for analysis. Each band was considered as a marker and
scored 1 for presence of band and 0 for absence. A binary
data matrix of RAPD markers for all the isolates were recorded on Microsoft
office Excel spread sheet for further analysis.
Diversity analysis: Cluster analysis was used to examine genotypic relationships
among the tested strains. Amplification products were scored as either present
(1) or absent (0). A data matrix was prepared to determine similarities between
each pair of genotypes. This genotypic matrix was used for diversity analysis
by NTsys-2.1 software tool. In NTsys-pc, the matrix was subjected to unweighted
pairwise group method for arithmetic average analysis (UPGMA) to generate a
dendrogram using the SAHN subroutine and Tree plot of NTSYS-pc. Cophenetic correlation
was calculated as a measure of the faithfulness of cluster analysis and data
subjected to Principle Coordinates Analysis (PCA).
Discriminatory power of RAPD markers: The discriminatory power of RAPD
markers was evaluated by PIC (Polymorphic information content), RP (resolving
power) and MI (marker index). PIC for each marker was calculated as proposed
by Powell et al. (1996) as PIC = 1-ΣPi2
where Pi is the frequency of ith allele. As each band line has been considered
as a marker, PIC was separately calculated for each band line and averaged to
estimate PIC for the particular RAPD primer. Resolution power of the primer
was calculated by Rp = ΣIb where Ib (band informativeness) was calculated
by =1-[2x |0.5-p|], p is the proportion of genotype having a band. MI was calculated
according to Powell et al. (1996). MI is the product
between diversity index (equivalent to PIC) and Effective Multiplex Ratio (EMR)
where EMR is the product of fraction of polymorphic loci and number of polymorphic
RESULTS AND DISCUSSION
In present investigation, very wide diversity was observed among the B.
thuringiensis isolates which were isolated from different regions in India
(Table 1). All the primers used for RAPD analysis showed amplification
and generated RAPD fingerprint for B. thuringiensis isolates (Fig.
1a, b). A total of 108 bands were amplified from all these
||Agarose gel showing RAPD profiles generated using primers,
(a) RAPD11 and (b) RAPD14 for 32 native B. thuringiensis isolates
from diverse Indian habitats and 8 known strains. All the native isolates
numbers have SK prefix, M: 1kb molecular weight ladder
||Details of native B. thuringiensis isolates from different
sources from diverse locations of India and type strains used in this study
The discriminatory power of all 7 RAPD markers was evaluated by PIC (Polymorphic
information content), RP (resolving power) and MI (marker index). All 7 primers
were found polymorphic for all the amplified bands (Table 2).
Out of 7 markers, RAPD12 was giving maximum number (22) of polymorphic bands
having 0.18, 5.21 and 4.01 of PIC, RP and MI, respectively. An average of ±0.13
Polymorphic Information Content (PIC), 4.08 resolution power and 3.16 marker
index was obtained for individual band in 40 isolates.
Eight main clusters were observed in dendrogram produced by cluster analysis
(UPGMA) based on Jaccard coefficient by using molecular fingerprint of 32 native
isolates recovered from diverse locations of India and 8 type strains used as
reference at 25% similarity level (Fig. 2). One type strain
B. thuringiensis serovar finitimus (4B1) and three native isolates
SK-304, SK-935 and SK-722 were standalone of these clusters Cluster-I has three
native isolates and one reference strain. In this cluster, all 3 native isolates
are from the sources related to chickpea, namely 2 from the chickpea field soil
in Rohtak, Haryana and 1 from chickpea leaves from IARI field in New Delhi (Table
|| List of RAPD primers used for fingerprinting of native Bt
|*Average PIC of individual band in 40 isolates
All these isolates were collected from different regions. It is interesting
to get such similarity among isolates from very wide locations. In cluster-II,
two reference strains B. thuringiensis serovar aizawai/pacificus (4J3),
serovar finitimus (4B1) and a native isolate SK-1060 (obtained from barren
land in Dungarpur, Rajasthan) were grouped together. Cluster III has 2 native
B. thuringiensis isolates SK-4 and SK-758 which were collected from Chickpea
field in Rohtak, Haryana and Jowar grain dust in Guntur, Andhra Pradesh respectively.
||Grouping of B. thuringiensis isolates and type strains
as per dendrogram of RAPD profiles at 25% similarity
||Dendrogram produced by cluster analysis (UPGMA) based on Jaccard
coefficient by using molecular fingerprint of 40 isolates including 32 native
isolated from diverse locations of India and 8 type strains used as reference
obtained from BGSC
Cluster IV has highest number of isolates containing 4 reference strains and
15 native isolates, representing different regions namely, New Delhi, Uttar
Pradesh, West Bengal and Rajasthan. This cluster was further divided into 3
subgroups IVa, IVb and IVc. While IVa and IVb had 5 isolates each from different
regions, the subgroup IVc had 4 isolates from different regions and 5 type strains.
Cluster V had two isolates SK-229 and SK-729, collected from phyllosphere in
a pigeonpea field in IARI, New Delhi and Cotton seeds, Guntur, Andhra Pradesh
respectively. Cluster VI has two native isolates SK-5 (recovered from Cotton
field in Hissar, Haryana) and SK-1055 (recovered from Maize field in Udaipur,
Rajasthan). Cluster VII has three native isolates SK-51, SK-63 (collected from
Orchard soil in Shimla, Himachal Pradesh) and SK-700 (cotton field soil in Guntur,
Andhra Pradesh). Cluster VIII having two native isolates SK-94 and SK-680 obtained
from Grain dust in Shimla, Himachal Pradesh and a soil sample from Ganges Sangam
river bank, Uttar Pradesh, respectively.
It was observed that B. thuringiensis isolates are not clustered according
to their geographical locations and sources. Not a single cluster occurred according
to geographic location or source except cluster I where all 3 native isolate
were from chickpea soil and leaf samples. However, native isolate SK-4, also
collected from chickpea field, Rohtak, Haryana, fell in different cluster (cluster
III). Some isolates collected from same source, such as SK-51 and SK-63, collected
from orchard soil, Shimla, Himachal Pradesh showed only 26% similarity.
The eight clusters were subdivided into subgroups at 50% similarity level and
analyzed to find any basis behind grouping. However no correlation was observed
among the isolates in any of the subgroup. A 3D plot of principal coordinate
analysis showing the diversity of 32 native isolates and 8 type strains is depicted
in Fig. 3. In the present study, native strains of B. thuringiensis
were isolated from different places and sources. Thirty two B. thuringiensis
native isolates and 8 type strains were characterized by RAPD markers which
differentiate the isolates recovered from different places.
||3D plot of principal coordinate analysis showing the diversity
of 40 isolates including 32 native isolated from diverse locations of India
and 8 type strains obtained from BGSC
The RAPD analysis is considered to be a fast and simple method. Once the primers
revealing the polymorphism were identified and PCR conditions optimized, slight
differences in primer sequences caused significantly different RAPD patterns
and enabled the easy discrimination among the strains (Sikora
et al., 1997).
The technique of RAPD has been used for characterization of B. thuringiensis
isolates by some other workers (Sadder et al., 2006;
Zara et al., 2006; Konecka
et al., 2007; Manzano et al. 2009).
Sadder et al. (2006) carried out RAPD analysis
on 7 B. thuringiensis isolates recovered from Jordan and 5 reference
B. thuringiensis strains and found high degree of polymorphism. A clearly
defined habitats locational pattern was also not observed by Malkawi
et al. (1999) in their RAPD-PCR analysis of 16 B. thuringiensis
isolates recovered from different Jordanian habitats. Pattanayak
et al. (2001) also performed RAPD based fingerprinting of 21 serovars
of B. thuringiensis using 19 random decamer primers and found very low
similarity values range from 3-68% indicating high genetic divergence. Gaviria
and Priest (2003) carried out RAPD analysis of 126 B. thuringiensis
representing 57 serovars, which were allowed to 58 genomic type based RAPD patterns.
It was concluded that while the species is genomically diverse, the presence
of one or more homologous serovar represents clonal lineages of successful pathogens.
Vilas-Baas and Lemos (2004) also observed remarkable
genetic diversity among 218 B. thuirngiensis isolates from Brazil suggesting
that the diversity resulted from influence of different ecological factors and
spatial separation between strains generated by conquest of different habitats.
A high degree of genetic divergence with only 16 per cent similarity among B.
thuringiensis serovars was observed in RAPD analysis and was recommended
as a complement to flagellar serology for genetic characterization of B.
thuringiensis (Chaves et al., 2010). Kumar
et al. (2010) have also carried out RAPD-PCR of 70 B. thuringiensis
isolates collected from soil samples of water field and found that RAPD provided
a high degree of discrimination.
RAPD analysis is considered an important molecular biology technique for the
identification of indigenous B. thuringiensis isolates. Our study showed
that the regional B. thuringiensis isolates had a high degree of genetic
diversity. Most of the isolates were found to be diverse even though they were
isolated from the same source or location. The results indicate that the population
of native Bt isolates from different geographical locations and sources is very
Thus, RAPD analysis could distinguish the native isolates of B. thuringiensis
recovered from different regions and sources. It has contributed to our efforts
to understand the diversity of the B. thuringiensis isolates in various
climatic zones of India and will help in selection of isolates for screening
of new sources of cry gene alleles.
Wide diversity was observed among 32 native B. thuringiensis isolates
recovered from different regions in India and 8 known B. thuringiensis
strains used as reference by RAPD analysis. Out of 7 RAPD markers employed in
this study, marker RAPD12 was found to yield maximum number of polymorphic bands.
The isolates and reference strains fell into 8 main clusters at 25% similarity
level in UPGMA clustering. B. thuringiensis isolates were not clustered
according to their geographical locations and sources. B. thuringiensis
isolates had a high degree of genetic diversity as most of the isolates were
found to be diverse even though recovered from same habitat. Thus, RAPD analysis
has been useful to distinguish native isolates of B. thuringiensis isolated
from different regions and sources.
JK was recipient of fellowship from the Council of Scientific and Industrial
Research (CSIR), India. Authors are grateful to Rakesh K. Narula for excellent
1: Chaves, J.Q., F.A. Stringuini, E.S. Pires and C.F.G. Cavados, 2010. Genotypic analysis of Bacillus thuringiensis serovars by RAPD-PCR. Neotrop. Biol. Conserv., 5: 106-112.
Direct Link |
2: Cherif, A.L., S. Brusetti, A. Borin, A. Rizzi, A. Boudabous, H. Khyami-Horani and D. Daffonchio, 2003. Genetic relationship in the Bacillus cereus group by rep-PCR fingerprinting and sequencing of a Bacillus anthracis-specific rep-PCR fragment. J. Applied Microbiol., 94: 1108-1119.
3: Gaviria, R.A.M. and F.G. Priest, 2003. Molecular typing of Bacillus thuringiensis serovars by RAPD-PCR. Syst. Applied Microbiol., 26: 254-261.
Direct Link |
4: Gurtler, V. and B.C. Mayall, 2001. Genomic approaches to typing taxonomy and evolution of bacterial isolates. Int. J. Syst. Evol. Microbiol., 51: 3-16.
5: Hansen, B.M., P.H. Damgaard, J. Eilenberg and J.C. Pederson, 1998. Molecular and phenotypic characterisation of Bacillus thurigiensis isolated from leaves and insects. J. Invert. Pathol., 71: 106-114.
PubMed | Direct Link |
6: Hofte, H. and H.R. Whiteley, 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev., 53: 242-255.
Direct Link |
7: Iriarte, J., Y. Bel, M.D. Ferrandis, R. Andrew, J. Murillo and P. Caballero, 1998. Environmental distribution and diversity of Bacillus thuringiensis in Spain. Syst. Applied Microbiol., 21: 97-106.
CrossRef | PubMed | Direct Link |
8: Katara, J., R. Deshmukh, N.K. Singh and S. Kaur, 2012. Molecular typing of native Bacillus thuringiensis isolates from diverse habitats in India using REP-PCR and ERIC-PCR analysis. J. Gen. Applied Microbiol., 58: 83-94.
9: Kaur, S. and A. Singh, 2000. Distribution of Bacillus thuringiensis isolates in different soil types from North India. Indian J. Ecol., 27: 52-60.
10: Kaur, S. and A. Singh, 2000. Natural occurrence of Bacillus thuringiensis in leguminous phylloplanes in the New Delhi region of India. World J. Microbiol. Biotech., 16: 679-682.
CrossRef | Direct Link |
11: Kaur, S., 2000. Molecular approaches towards development of novel Bacillus thuringiensis biopesticides. World J. Microbiol. Biotechnol., 16: 781-793.
CrossRef | Direct Link |
12: Kim, T.H., G.M. Seo, K.H. Jung, S.J. Kim and J.C. Kim et al., 2007. Generation of a specific marker to discriminate Gacillus anthracis from other bacteria of the Bacillus cereus group. J. Microbiol. Biotechnol., 17: 806-811.
PubMed | Direct Link |
13: Konecka, E., A. Kaznowski, J. Ziemnicka and K. Ziemnicki, 2007. Molecular and phenotypic characterization of Bacillus thuringiensis isolated during epizootics in Cydia pomonella. J. Invertebrate Pathol., 94: 56-63.
14: Kumar, D., K. Chaudhary and K.S. Boora, 2010. Characterization of native Bacillus thuringiensis strains by PCR-RAPD based fingerprinting. Indian J. Microbiol., 50: 27-32.
15: La Duc, M.T., M. Satomi, N. Agata and K. Venkateswaran, 2004. gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group. J. Microbiol. Methods, 56: 383-394.
CrossRef | Direct Link |
16: Malkawi, H.I., F. al-Momani, M.M. Meqdam, I. Saadoun and M.J. Mohammad, 1999. Detection of genetic polymorphism by RAPD-PCR among isolates of Bacillus thuringiensis. New Microbiol., 22: 241-247.
PubMed | Direct Link |
17: Manzano, M., C. Giusto, L. Iacumin, C. Cantoni and G. Comi, 2009. Molecular methods to evaluate biodiversity in Bacillus cereus and Bacillus thuringiensis strains from different origins. Food Microbiol., 26: 259-264.
18: Martin, P.A.W. and R.S. Travers, 1989. Worldwide abundance and distribution of Bacillus thuringiensis isolates. Applied Environ. Microbiol., 55: 2437-2442.
Direct Link |
19: Pattanayak, D., S.K. Chakrabarti, P.A. Kumar and P.S. Naik, 2001. Characterization of genetic diversity of some serovars of Bacillus thuringiensis by RAPD. Indian J. Exp. Biol., 39: 897-901.
20: Powell, W., M. Morgante, C. Andre, M. Hanafey, J. Vogel, S. Tingey and A. Rafalski, 1996. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol. Breed., 2: 225-238.
CrossRef | Direct Link |
21: Ronimus, R.S., L.E. Parker and H.W. Morgen, 1997. The utilization of RAPD-PCR for identifying thermophilic and mesophilic Bacillus species. FEMS Microbiol. Lett., 147: 75-79.
22: Sadder, M.T., H. Khyami-Horani and L. Al-Banna, 2006. Application of RAPD technique to study polymorphism among Bacillus thuringiensis isolates from Jordan. World J. Microbiol. Biotechnol., 22: 1307-1312.
23: Sikora, S., S. Redzepovic, I. Pejic and V. Kozumplik, 1997. Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting. J. Applied Microbiol., 82: 527-531.
24: Vilas-Baas, G.T. and M.V. Franco Lemos, 2004. Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil. Can. J. Microbiol., 50: 605-613.
CrossRef | Direct Link |
25: Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey, 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 18: 6531-6535.
CrossRef | PubMed | Direct Link |
26: Zara, G., S. Zara, N.P. Mangia, G. Garau, C. Pinna, G. Ladu and M. Budroni, 2006. PCR-based methods to discriminate Bacillus thuringiensis strains. Ann. Microbiol., 56: 71-76.