Bt Research, 2015, Vol.6, No.8, 1-16
1
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
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
)
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
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., 2014,
/). The remarkable
diversity is because of a high degree of genetic
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
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.
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;
