MOLECULAR CHARACTERIZATION OF INDIGENOUS BACILLUS THURINGIENSIS KURSTAKI

ISOLATES WITH EFFECTIVE PESTICIDAL ACTIVITY AGAINST BACTROCERA CUCURBITAE

FROM BANGLADESH
Asaduzzaman Shishir1, Asma Akter1, Bodiuzzaman1, Nasima Aktar1, Mushfiqur Rahman2, Shakil Ahmed Khan2, Mohammad
Ilias1, Shakila Nargis Khan1 and Mozammel Hoq*1
1. Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh
2. Atomic Energy Establishment, Savar, Dhaka, Bangladesh
*Corresponding author’s e-mail: mmzhoq@gmail.com

Abstract: The indiscriminate use of agricultural pesticides has created serious health and environmental problems in many developing
countries including Bangladesh. On the other hand, Bacillus thuringiensis kurstaki (Btk) has long been used commercially as pest control
agents particularly against lepidopteran vegetable pests. In this study, new potential Btk-like isolates were collected from different sources
(viz. soil, leaves, insects, stored product dust etc.) and regions of Bangladesh. Acetate selection-heat treatment-lecithinase production and
haemolytic activity were initially performed to narrow down the spectrum for selection of Btk-likeisolates. Specific biochemical tests (Es+, Sa+,
Le+, Su-) were performed to differentiate Btk from other subspecies. Based on these tests, of 148 Bt isolates, 27.7% (41 Bt isolates) were
confirmed as Bt kurstaki isolates. The 41 isolates were investigated for the presence of cry1, cry1A and cry2A genes by PCR identification
with gene specific primers and demonstrated some level of diversity with respect to major genes content in the strains. Nine of the isolates
were found to carry all three genes while another nine contained both cry1 and cry2A genes. 29, 22 and 16 of the isolates contained singly
cry1, cry2A or cry1A gene respectively. Plasmid DNA was purified which ranged from 12-15 kb in most of the isolates which were very similar
to the plasmid pattern of Btk HD-73. Bioassay was performed with the isolates harbouring Lepidoptera specific cry1A genes against
vegetable pest Bactrocera cucurbitae. Five of the isolates having higher activity close to Btk HD-73 were considered for determination of LC 50
and LC90. The results will be a useful basis for developing effective bio-pesticides to decrease the use of hazardous chemicals in Bangladesh
agriculture.

Key words: Molecular characterization, Bacillus thuringiensis kurstaki (Btk), pesticidal activity, vegetable pest, Bangladesh.

Introduction: recorded from Bangladesh [14]. Melon fly, Bactrocera

cucurbitae (Diptera: Tephritidae), is a detrimental insect of
Long residual action and toxicity to a wide spectrum of cucurbitaceae crops which damages over 81 plant species and
organisms have made the chemical insecticides useful and in based on the extensive surveys carried out in Asia and Hawaii,
the same time these have brought serious hazards to the plants belonging to the family Cucurbitaceae are most
environment and health [8, 13]. The long persistence in the vulnerable
environment and incorporation in the food chain is the major
cause of bioaccumulation of lipophilic pesticides like Until recently in Bangladesh, insecticides were the major
organochlorines and organophosphates which makes them means to control the insects in all crops including vegetables.
difficult to remove and thus induce long-term health effects. The use of insecticides in Bangladesh reached 14,312 metric
Evidence suggests that other pesticides may do the same and tons in 2004 which started its journey with the grant receipt of 3
spray drift contaminates surrounding areas and non-target metric tons of endrin in 1957/58 and its still in the increasing
species, including human being. So, eco-friendly pesticides trend [14].
have become very urgent to reduce the contamination and the
likelihood of insect resistance [8, 12, 15]. In a growing season of 4 to 6 months in Jessore district, as
many as 150 applications of insecticides with at least once a
Bangladesh, a country with subtropical climate produces day during peak period were required to suppress the insect
different vegetables including eggplant, cucurbits, country pests in eggplant.
bean, cabbage, cauliflower, tomato, etc which covers an area
of ca. 4,98,073 acres. But the yield per unit area is quite low Deployment of integrated pest management (IPM) strategies
such as a conservative estimate puts about annual yield losses for crop protection is the most discussed issue which can make
in vegetables at 25% due to insect pests alone. More than 200 significant contribution to food security in the developing
major species among 700 insect and mite pest species of countries. The use of biopesticides, as a component of IPM,
different field crops, fruit trees, and stored products have been has been gaining acceptance over the world. Bacillus
thuringiensis (Bt) is the major biopesticide for its advantages

AFSSA 143
like specific toxicity against target insects, lack of polluting
residues and safety to non-target organisms such as
mammals, birds, amphibians and reptiles. It has been used in
agriculture, forestry and mosquito control and accounts for
95% of the 1% market share of biopesticides in the total
pesticide market [7].
Bacillus thuringiensis (Bt) is a gram-positive bacterium that is
characterized by the production of insecticidal crystal proteins
or δ-endotoxins which can kill insects belonging to the
Lepidoptera, Coleoptera, Diptera, Hymenoptera, Homoptera,
and Mallophaga, as well as some invertebrates [1, 6, 16].
These Cry toxins are encoded by cry genes and more than six
hundreds of cry genes have been enlisted in the full list of
delta-
endotoxins.(http://www.lifesci.sussex.ac.uk/Home/Neil_Crickm
ore/Bt/)
Information about the distribution of cry genes is still limited
and does not cover many distinct geographic areas. So, it is
very essential to search for novel and more potent strains with
new pathogenic spectrum and wider host ranges, in parts of
the world that have not yet been adequately sampled,
especially Bangladesh.
Though research with Bt is not new, searching for more and
more potential Bt strains keeps tremendous importance as
resistance to the already used Bt products is emerging as a
common problem. As there are some reports against
transgenic Bt-crops for immunologic adverse effect, Bt
biopesticide is more safe as spray-on formulation in the field.
But still no such Bt products are available in Bangladesh and it
is also lacking in any kind of national work with Bt. Large scale
production of Bt using cheap substrate by fermentation is also
challenging and taking them to the field is a complete course
for Bt biopesticide.
The present study therefore deals with isolation, identification
and molecular characterization of the indigenous Bt kurstaki
like isolates with a view to selecting the potential candidates for
biopesticide production and formulation. At the same time,
discovering melon fly (Bactrocera cucurbitae) controlling Bt
strains is also an important objective of this work.
Method and Materials:
Bacterial strains:
Bacillus thuringiensis kurstaki HD-73, B. thuringiensis sotto, B. studies.
thuringiensis japonensis Buibui were used as reference strains
and were obtained from Bt stock collection of Okayama Biochemical typing:
University, Japan. All of the isolates of Bt were preserved in Four most relevant biochemical tests i.e. esculin utilization,
10% glycerol stock at -70oC freeze.
Sampling and Isolation of Btk:
Soil, leaf, insect cadavers and stored product dust samples Sa+, Le+, Su-) [11].
from different regions (Table.1) of Bangladesh were collected
where no Bt preparations have been applied before. Samples
were taken aseptically from 2-5cm below the surface of
shadowed and slightly moistened places which are generally
not exposed to sunlight. Samples (about 10.0g for soil) were
put into the sterile plastic bags and carried to the laboratory
and kept in dry places at room temperature. The samples
were then processed to isolate Bacillus thuringiensis kurstaki
and the isolation of Btk was accomplished in combination of
acetate selection and heat treatment [17] with an extra step of
screening for lecithin hydrolysis capability to exclude other Bt
isolates lacking this property. Thus, 1.0g of each sample (for
leaf, 1 in each flask and for insect, gut was dissected and put
into the medium by mashing) was added into 20ml of L-broth,
buffered with 0.25M Na-acetate (pH 6.8) in a 125ml
Erlenmeyer flask and incubated in an orbital shaker for 4 hour
at 200rpm at 30oC. 0.5ml of sample was transferred into sterile
test tubes and heat- treated for 10 minutes at 80oC in a water
bath. Heat treated suspension was then plated on Egg yolk
agar medium (per litre: 10g of tryptone, 5g of yeast extract, 5g
of NaCl, and 15g of agar, 100ml of egg yolk suspension) and
incubated overnight at 30oC. Colonies that produced white
surrounding of precipitation were then picked and sub-cultured
to obtain pure culture and preserved both in slant and in
glycerol stock for future works.
Phase contrast microscopy:
Isolates with lecithin hydrolysis capability were inoculated in T3-
agar medium (1.0 L: Tryptone 3.0g, tryptose 2.0g, yeast extract
1.5g, MnCl2 0.005g, phosphate buffer 50mM, agar 15.0g; pH:
6.8) and incubated at 30oC for 40-72 hours to allow sporulation.
Isolates with juxtaposed glowing spore and dark crystal protein
observed under Phase Contrast Microscope were considered
as Bacillus thuringiensis.
Hemolytic test:
The production of extracellular hemolysins, an indication of
insecticidal activity of Bt isolates [10], was tested on blood agar
(nutrient agar containing 5% (v/v) sheep erythrocytes). Bt
isolates were inoculated as a point onto blood agar plates by
needle and the formation of clear zone of hemolysis
surrounding Bt colonies was examined after incubation at 27°C
for 24 h [8]. The positive isolates thus were selected for further
acid formation from salicin and sucrose, and lecithinase
production were carried out to identify Bacillus thuringiensis
kurstaki subspecies with their characteristic test result (Es+,
AFSSA 144
Plasmid profiling: (Promega, USA) gel prepared and submerged in 1×TBE buffer
Plasmid DNA was prepared by using the method described by at 60V for 50min. Gel was visualized in gel documentation
Crosa and Falkow [3]. Briefly, The pellet of a 5.0ml culture system (Alphaimager mini, USA) following staining in 1×TBE
grown in LB broth was lysed with 0.85 ml of TE buffer (50 mM buffer-EtBr (Sigma, USA) solution and de-staining in distilled
Tris, 20 mM EDTA; pH 8.5) containing 2 mg/ml of lysozyme water.
(Wako, Japan), 0.05 ml of 20% SDS solution, and 5U
proteinase-K (Nacalai tesque, inc, Japan). After mixing by Bioassay:
gentle inversions, the cell suspension was incubated at 37°C The toxicity of the Btk isolates positive for cry1 and cry1A
for 30 min. 0.03 ml of 3.0N NaOH was added subsequently to genes were determined against the 3rd instar larvae of
the suspension and mixed gently for 3 min. The suspension Bactrocera cucurbitae. Spore-crystal mixture for bioassay was
was neutralized by addition of 0.06 ml of 2 M Tris-HCl (pH 7.0) prepared from the twenty nine cry1 gene positive isolates and
and mixed gently. 0.1ml of 5 M NaCl was then added, and the reference strain Btk HD-73 by inoculating them in 100ml of T3-
suspension was mixed by inversions, placed on ice for 15 min, liquid medium and incubating for 7 days at 30°C with
and then centrifuged at 12,000 x g for 15 min at 4oC (Tomy, continuous shaking at 250 rpm. Cultures were centrifuged at
MX-305, high speed Refrigerated micro centrifuge, Japan). The 5000 rpm for 15 min to separate the culture from medium.
supernatant was transferred into a fresh centrifuge tube, and 2 Pellets (spores and crystal protein mixture) were washed twice
volume of ice-cold ethanol was added. The tube was kept at - with 20mL of cold sterile distilled water and centrifuged at 5000
20°C for 15 min and then centrifuged at 12,000 x g for 15 min. rpm for 5 min. The pellets were re-suspended in 20mL of
The supernatant was discarded, and the residue was dried by sterile distilled water and stored at 4°C. Larval diet was
inverting the tube over a paper towel for a few minutes. The prepared by mixing spore-crystal suspension of each isolate
residue was dissolved in 50μl of TE (10mM Tris, 1mM EDTA) with 10g of semisolid paste of boiled pumpkin. 10 larvae were
buffer and kept at -20°C. Plasmid DNA was analyzed by placed in each petri-dish provided with spore-crystal
electrophoresis on horizontal 0.8% agarose (Promega, USA) suspension mixed diet. The toxicity of each strain was assayed
gels submerged in 1×TBE buffer at 70V for 3 hours. Gel was in triplicate. The petri dishes were incubated at 25±2°C
visualized in gel documentation system (Alpha imager mini) temperature and 70±10% RH, with a photoperiod of 16:8 (L: D)
following staining in EtBr solution and de-staining in distilled for 7 days. Mortality was scored in comparison with a parallel
water. control in which diet was mixed with sterile distilled water
instead of bacterial suspension and it was used to correct test
Detection of cry1, cry1A and cry2A genes in Btk like mortality using Abbot’s formula [4].
isolates:
To identify the potential most Btk like isolates against Results:
vegetable pests, cry1 and cry1A genes were searched by
Polymerase Chain Reaction (PCR) with Universal cry1 primer Identification of Bacillus thuringiensis Isolates:
set, forward (5’-CATGATTCATGCGGCAGATAAAC-3’) and Samples were collected from peripheral and central locations
reverse (5’-TTGTGACACTTCTGCTTCCCATT-3’); lepidoptera of Dhaka and different agricultural areas of Bangladesh. Total
specific cry1A primer set, forward (5’- 139 samples were processed comprising of 97 soil, 31 leaf, 7
CCGGTGCTGGATTTGTGTTA-3’) and reverse (5’- insect and 4 stored product dust. After acetate selection and
AATCCCGTATTGTACCAGCG-3’); cry2A specific primer sets, heat treatment, 186 Bt like isolates were obtained in the EYA
forward (5’-TAAAGAAAGTGGGGAGTCTT-3’) and reverse (5’- medium from 114 samples (82%) out of 139 samples
AACTCCATCGTTATTTGTAG-3’). DNA template was mixed however 25 samples did not produce growth of any isolate
with PCR reaction mixture i.e. per μl containing 0.2mM dNTPs, (Table 1) . 155 lecithin hydrolysing isolates (83.3%) were
0.5µM of each primer, 1x PCR buffer and 0.5 u of Taq DNA picked (Fig 1A) from these 186 Bt-like isolates and after
polymerase (Promega, USA) in 25μl reaction volume and sporulation in T3- agar medium 148 isolates were observed to
amplification was performed in a DNA thermal cycler (MJ, Bio- possess parasporal body containing Cry proteins as revealed
Rad). For both cry1 and cry1A primer sets, PCR was carried under phase contrast microscope (Fig 1B) and light
out with an initial single denaturation step at 95oC for 2min and microscope also following CBB staining. The haemolysis test
30 amplification cycles including denaturation at 95oC for 45s, revealed that out of 148 isolates, 115 (77.7%) was found to be
annealing at 51oC for 45s and extension at 72oC for 60s. For haemolytic (Fig 1C) hence considered to have insecticidal
cry2A, annealing at 45oC for 45s was carried out. Finally an activity.
extra extension step was applied at 72oC for 10 min. 5μl of the
PCR products were then electrophoresed in 1.5% agarose

AFSSA 145
 A B C
Bt
others
kurstaki
22%
27.7%
Bt
Bt dendroli
Bt
thuringie mus
israelens
nsis Bt sotto 12%
is
18% 10.3%
10%
Fig 1: A) Lecithin hydrolysis- white precipitation surrounding the colonies. B) Glowing
spores and dark crystal protein of Bt isolates under Phase contrast microscope, C)
Differentiation between hemolytic and non-hemolytic Bt isolates. n=148

Table 1: Btk index in different sampling locations. Fig 2: A) Distribution of different biotypes of Bacillus thuringiensis
isolates in the samples collected.

Fig 3: Plasmid pattern of Btk like isolates indicated over the lanes. Btk HD-73
is the reference strain. (M: Supercoiled DNA ladder, Invitrogen)

Biotyping:
A simple biotyping method which included lecithin hydrolysis Characterization of cry gene content:
(done in the isolation stage), esculin utilization, sucrose and
Forty one Btk like isolates were checked for the presence of
salicin fermentation tests revealed the abundance of differentcry1, cry1A and cry2A genes. Initially 29 isolates were
biotypes of Bt in the working samples and Btk like isolates considered positive for cry1 gene as PCR product of about 270
were obtained in this process. Bacillus thuringiensis kurstakibp was evident for them which is the desired product size for
(27.7%) was the most abundant biotype among other types universal cry1 primer sets. These twenty nine cry1 gene
(Fig 2A). Bt thuringiensis, Bt type ten and Bt dendrolimus were
positive isolates were then examined for the presence of
the next abundant isolates. Btk was most abundant (index- Lepidoptera specific cry1A gene and of them sixteen isolates
0.69) in the soil of Jhenaidah (Table 1). were found positive with expected 490 bp PCR product (Fig
4A). Twenty two isolates out of forty one were observed with
Plasmid profile: bands of different product size for cry2A specific primers
Bacillus thuringiensis commonly harbours a varied number of among which desired product of more than 1500 bp was not
large plasmids with different molecular mass. As most of the observed. So, though the isolates responded in the PCR, they
cry genes are located on these large plasmids [3]. Plasmids were not considered to harbour cry2A genes.
from Bt kurstaki like isolates were extracted and plasmid
pattern of these isolates were compared with reference Btk Bioassay:
HD-73. Plasmids ranging from 10-15 kb were observed for the The toxicity of spore-crystal mixture of twenty nine cry1 gene
Bt isolates and a 12kb plasmid band was evident for Btk HD-73 positive Btk like isolates was assayed against 3rd instar larvae
(Fig 3). of Bactrocera cucurbitae. A single dose (6x107spores ml-1)
assay in triplicate was employed to screen for potential most
Btk like isolates. The assay was repeated with ten potential
most isolates and the highest mortality scoring four isolates i.e.
JSc1 (96%), SSc2 (80%) and SSe2 (63%) and JaS8 (56%)
were recorded along with reference Btk HD-73 (100%

AFSSA 146
mortality) (Fig 5). Their performance was also analysed Su-) of which lecithin hydrolysis test was accomplished in the
statistically calculating standard deviation. isolation stage. Bacillus thuringiensis kurstaki biotyped isolates
were most abundant (27.7%) among other sup-species of Bt in
35 the sampling sites.

Number of isolates
30
25
20
15
10
5
0
A cry1 cry1A cry2A

Fig 4: A) cry1 (about 270bp product in the left direction of marker) and cry1A
(about 490bp product in the right direction of marker) gene screening by PCR A Larvae died
analysis. Isolates
B name as per labelled over the lane (100bp DNA ladder;
TaKaRa, Japan). B) Number of cry1 and cry1A genes in the Btk biotyped 120
isolates.
100

Discussion: 80

60
Average
The particular interest of this research work was to isolate and 40 Mortality
identify Bacillus thuringiensis kurstaki like isolates as this Percentage
20
subspecies of Bt has a long successful history to control the
vegetable pests belonging to Lepidoptera order. So after heat 0
B
treatment of the samples following acetate selection, they were -20
Control HD-73 JSc2 JSc1 JSa1 SSc2 SSe2 JaS8

directly inoculated onto Egg Yolk Agar medium by spread plate

technique. This helped to pick only lecithin hydrolysing Bt
isolates which is also a distinguishing characteristic of Bacillus
thuringiensis from B. sphaericus. It diminished the chances of
picking some Bs wrongly considering Bt as some Bs are
evidenced to produce parasporal crystal proteins (Mtx and
Bin). This slight difference in isolation technique helped to
overcome the confusion in identification of Bt from Bs after
phase contrast microscopy. This modification does not prevent
the growth of some Bt isolates which are not lecithin
hydrolysing rather allowed a biochemical test needed to be
done later for biochemical typing of the isolates. In the above
mentioned process, 148 Bt like isolates were obtained from
139 samples and inoculated in T3- sporulating medium to allow
spore and crystal protein formation. All of them were confirmed
as Bt isolates after Phase contrast microscopy.
When a large number of isolates are to be worked with,
serology is too cumbersome to identify thousands of isolates.
Alternatively a rapid method based on four most relevant
biochemical tests was found to be suitable for bio-typing of the
Bt isolates [11]. This involved esculin utilization, acid formation
from salicin and sucrose and lecithinase production. The
results enabled them to divide the B. thuringiensis isolates into
16 biochemical types.
In this study, forty one Bt kurstaki like isolates were identified
following four most relevant biochemical tests (Es+, Sa+, Le+,
Fig 7: A) larva died after being fed with the diet containing spore-crystal
mixture. B) Average mortality percentage of Bactrocera cucurbitae assayed
against different isolates.
cry genes are often carried on plasmids and plasmid
exchange between strains as well as recombination
between cry genes from different backgrounds occur in Bt
strains [5]. In this study, Plasmid pattern of the Isolates
biotyped as Btk was almost similar to the reference strain
Btk HD-73 as the super-coiled plasmids extracted from
them ranged from 12 to 15kb in size. The similarity in the
plasmid profile among the same subspecies was
desirable and it also supported the biotyping based on
four most relevant biochemical tests.
The plasmids were also found to carry the cry genes as both
total DNA and plasmid DNA were checked for the presence of
cry1, cry1A and cry2A genes. Bt kurstaki subspecies has been
reported to carry cry1, cry2 and cry9 major gene classes and in
this study the presence of cry1, cry1A and cry2A were
investigated as the gene-insect specificity cluster were much
dense in the datasheet for cry1 and cry2 genes. 29 out of 39
isolates biotyped as Btk were positive for cry1 genes and 16
out of 29 were positive for cry1A genes and 22 were cry2A
positive. PCR products obtained from cry1 and cry1A specific
primer sets were found to be as expected but for cry2A,
multiple unexpected products of different sizes were observed.

AFSSA 147
These isolates assuming to harbour cry1 gene were tested
against the 3rd instar larvae of Bactrocera cucurbitae (Melon
fly), a detrimental pest of cucurbitaceae or vine crop family.
The result of the bioassay revealed that indigenous Btk JSc1,
Btk SSc2 and Btk SSe2 are highly toxic against the melon fly
(Bactrocera cucurbitae) larvae. The bioassay was repeated for
thrice and each time it was done in triplicate to justify the
results statistically.
There were some efforts in controlling the melon fruit fly
(Bactrocera cucurbitae) chemically but those were not so
efficient and the most conventional method to control them is
radiation technique by which sterile fly are introduced in the
nature. But easy and ready to use control measures are more
preferable like formulation of biopesticide and its direct
application in the field. So the results obtained from the
bioassay are very promising and will be very useful to develop
efficient Bt biopesticides to control melon fly affects in
vegetables. This will thus help to reduce hazards in food chain
and thereby enhance the food safety which will consequently
decrease the health risk.
Acknowledgement:
This work was supported by a Grant-in-Aid from the USDA as
a project entitled ‘‘Production of Bacillus thuringiensis 12. Margalit, J., Becker, N., Back, C., Zaritsky, A. 1995. Bacillus
biopesticides by biotechnological approach for the control of thuringiensis subsp. israelensis as a biological control agent of
vegetable pests in Bangladesh.’’ mosquitoes and black flies. In: Feng, T. Y., Chak, K. F., Smith, R. A.,
References:
1. Ben-Dov, E., Manasherob, R., Zaritsky, R. A., Barak, Z., Margalith,
Y. 2001. PCR analysis of cry7 genes in Bacillus thuringiensis by the five
conserved blocks of toxins. CurrMicrobiol, 42: 96-99.
2. Carlson, C. R., Johansen, T., Kolsto, A. B. 1996. The chromosome
map of Bacillus thuringiensis subsp. canadensis HD224 is highly similar
to that of the Bacillus cereus type strain ATCC 14579. FEMS Microbiol.
Lett, 141: 163-167.
3. Crosa, J. H., Falkow, S. 1981. In Gerhardt, P., Murray, R. G. E. ,
Costilow, R. N. , Nester, E. W. , Wood, W. A., Krieg, N. R., Phillips, G. B.,
(ed.), Manual of methods for general bacteriology. American Society for
Microbiology, Washington, D.C. Plasmids, 266-282.
4. Daffonchio, D., Borin, S., Frova, G., Manachini, P. L., Sorlini, C.
1998. PCR fingerprinting of whole genomes: the spacers between the
16S and 23S rRNA genes and of intergenic tRNA gene regions reveal a
different intraspecific genomic variability of Bacillus cereus and Bacillus
licheniformis [corrected]. Int J Syst Bacteriol, 48(1): 107-116.
5. De Maagd, R. A., Bravo, A., Crickmore, N. 2001. How Bacillus
thuringiensis has evolved specific toxins to colonize the insect world.
Trends in Genetics, 17: 193–199.
6. Feitelson, J. S., Payne, J., Kim, L. 1999. Bacillus thuringiensis:
insects and beyond. Biotechnology, 10: 271–275
7. Flexner, J. L., Belnavis, D. L. 1999. Microbial insecticides. In:
Biological and Biotechnological Control of Insect Pests, eds. Rechcigl
J.E. & Rechcigl N.A. pp. 35–62. Boca Raton: Lewis Publishers. ISBN 1-
56670-4790.
8. Frankenhuyzen, K. V. 1993. The challenge of Bacillus thuringiensis.
In: Entwistle, P. F., Cory, J. S., Bailey, M. J., Higgs, S. R., (eds) Bacillus
thuringiensis, an environmental biopesticide: theory and practice. Wiley,
Chichester, pp 1-35.
9. Ichikawa, M., Uemori, A., Yasutak, K., Kagoshima, K., Mizuki, E.,
Ohba, M. 2008. Failure to phenotypically discriminate between non-
insecticidal Bacillus thuringiensis strains with anticancer parasporins
(PS2, PS3, and PS4) and Bacillus thuringiensis strains that produce
insecticidal Cry proteins. Appl. Entomol. Zool. 43: 421-426.
10. Kitada, S. et al., 2005. Molecular Identification and Cytocidal Action
of Parasporin, a Protein Group of Novel Crystal Toxins targeting human
cancer cells. 6th Pacific Rim Conference on the Biotechnology of Bacillus
thuringiensis and its Environmental Impact, Victoria BC.
11. Martin, P. A. W., Travers, R. S. 1989. Worlwide abundance and
distribution of Bacillus thuringiensis isolates. Applied and Environmental
Microbiology, 55: 2437-2442.
Yamamoto, T., Margalit, J., Chilcott, C., Rose, R. I. (eds) Bacillus
thuringiensis biotechnology and environmental benefits, Vol 1. Hua
Shiang Yuan Publishing Co, Taipei, pp 521–556
13. Marrone, P. G., MacIntosh, S. C. 1993. Resistance to Bacillus
thuringiensis and resistance management. In: Entwistle, P. F., Cory, J.
S., Bailey, M. J., Higgs, S. R. (eds) Bacillus thuringiensis, an
environmental biopesticide: theory and practice. Wiley, Chichester, pp
221–235.
14. Rahman, M. M. 2000. Pesticides: their uses and problems in context
of Bangladesh. In Proceedings of the National Workshop on
Conventional and Nuclear Technique for Pesticide Residue Studies in
Food and Environment held on 15-19 October, 2000 at IFRB, AERE,
Savar.
15. Salehi, J. G. R., Komakhin, R. A., Piruzian E. S. 2005. Comparative
study of the expression of the native modified and hybrid cry3a genes of
Bacillus thuringiensis in prokaryotic and eukaryotic cells. Russ. J. Genet.
41(2): 116-121.
16. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J.,
Feitelson, J., Zeigler, D. R., Dean, D. H. 1998. Bacillus thuringiensis and
its pesticidal crystal proteins. Microbiol Mol Biol Rev, 62:775–806.
17. Travers, R. S., Martin, P. A. W., Reichelderfer, C. F. 1987. Selective
process for efficient isolation of soil Bacillus spp. Applied and
Environmental Microbiology, 53: 1263–1266.
AFSSA 148