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AFSSA 143

MOLECULAR CHARACTERIZATION OF INDIGENOUS BACILLUS THURINGIENSIS KURSTAKI
ISOLATES WITH EFFECTIVE PESTICIDAL ACTIVITY AGAINST BACTROCERA CUCURBITAE
FROM BANGLADESH
Asaduzzaman Shishir
1
, Asma Akter
1
, Bodiuzzaman
1
, Nasima Aktar
1
, Mushfiqur Rahman
2
, Shakil Ahmed Khan
2
, Mohammad
Ilias
1
, Shakila Nargis Khan
1
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 LC50
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:
Long residual action and toxicity to a wide spectrum of
organisms have made the chemical insecticides useful and in
the same time these have brought serious hazards to the
environment and health [8, 13]. The long persistence in the
environment and incorporation in the food chain is the major
cause of bioaccumulation of lipophilic pesticides like
organochlorines and organophosphates which makes them
difficult to remove and thus induce long-term health effects.
Evidence suggests that other pesticides may do the same and
spray drift contaminates surrounding areas and non-target
species, including human being. So, eco-friendly pesticides
have become very urgent to reduce the contamination and the
likelihood of insect resistance [8, 12, 15].
Bangladesh, a country with subtropical climate produces
different vegetables including eggplant, cucurbits, country
bean, cabbage, cauliflower, tomato, etc which covers an area
of ca. 4,98,073 acres. But the yield per unit area is quite low
such as a conservative estimate puts about annual yield losses
in vegetables at 25% due to insect pests alone. More than 200
major species among 700 insect and mite pest species of
different field crops, fruit trees, and stored products have been
recorded from Bangladesh [14]. Melon fly, Bactrocera
cucurbitae (Diptera: Tephritidae), is a detrimental insect of
cucurbitaceae crops which damages over 81 plant species and
based on the extensive surveys carried out in Asia and Hawaii,
plants belonging to the family Cucurbitaceae are most
vulnerable
Until recently in Bangladesh, insecticides were the major
means to control the insects in all crops including vegetables.
The use of insecticides in Bangladesh reached 14,312 metric
tons in 2004 which started its journey with the grant receipt of 3
metric tons of endrin in 1957/58 and its still in the increasing
trend [14].
In a growing season of 4 to 6 months in Jessore district, as
many as 150 applications of insecticides with at least once a
day during peak period were required to suppress the insect
pests in eggplant.
Deployment of integrated pest management (IPM) strategies
for crop protection is the most discussed issue which can make
significant contribution to food security in the developing
countries. The use of biopesticides, as a component of IPM,
has been gaining acceptance over the world. Bacillus
thuringiensis (Bt) is the major biopesticide for its advantages
AFSSA 144
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.
thuringiensis japonensis Buibui were used as reference strains
and were obtained from Bt stock collection of Okayama
University, Japan. All of the isolates of Bt were preserved in
10% glycerol stock at -70
o
C freeze.

Sampling and Isolation of Btk:
Soil, leaf, insect cadavers and stored product dust samples
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 30
o
C. 0.5ml of sample was transferred into sterile
test tubes and heat- treated for 10 minutes at 80
o
C 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 30
o
C. 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 30
o
C 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
studies.

Biochemical typing:
Four most relevant biochemical tests i.e. esculin utilization,
acid formation from salicin and sucrose, and lecithinase
production were carried out to identify Bacillus thuringiensis
kurstaki subspecies with their characteristic test result (Es
+
,
Sa
+
, Le
+
, Su
-
) [11].

AFSSA 145
Plasmid profiling:
Plasmid DNA was prepared by using the method described by
Crosa and Falkow [3]. Briefly, The pellet of a 5.0ml culture
grown in LB broth was lysed with 0.85 ml of TE buffer (50 mM
Tris, 20 mM EDTA; pH 8.5) containing 2 mg/ml of lysozyme
(Wako, Japan), 0.05 ml of 20% SDS solution, and 5U
proteinase-K (Nacalai tesque, inc, Japan). After mixing by
gentle inversions, the cell suspension was incubated at 37°C
for 30 min. 0.03 ml of 3.0N NaOH was added subsequently to
the suspension and mixed gently for 3 min. The suspension
was neutralized by addition of 0.06 ml of 2 M Tris-HCl (pH 7.0)
and mixed gently. 0.1ml of 5 M NaCl was then added, and the
suspension was mixed by inversions, placed on ice for 15 min,
and then centrifuged at 12,000 x g for 15 min at 4
o
C (Tomy,
MX-305, high speed Refrigerated micro centrifuge, Japan). The
supernatant was transferred into a fresh centrifuge tube, and 2
volume of ice-cold ethanol was added. The tube was kept at -
20°C for 15 min and then centrifuged at 12,000 x g for 15 min.
The supernatant was discarded, and the residue was dried by
inverting the tube over a paper towel for a few minutes. The
residue was dissolved in 50μl of TE (10mM Tris, 1mM EDTA)
buffer and kept at -20°C. Plasmid DNA was analyzed by
electrophoresis on horizontal 0.8% agarose (Promega, USA)
gels submerged in 1×TBE buffer at 70V for 3 hours. Gel was
visualized in gel documentation system (Alpha imager mini)
following staining in EtBr solution and de-staining in distilled
water.

Detection of cry1, cry1A and cry2A genes in Btk like
isolates:
To identify the potential most Btk like isolates against
vegetable pests, cry1 and cry1A genes were searched by
Polymerase Chain Reaction (PCR) with Universal cry1 primer
set, forward (5’-CATGATTCATGCGGCAGATAAAC-3’) and
reverse (5’-TTGTGACACTTCTGCTTCCCATT-3’); lepidoptera
specific cry1A primer set, forward (5’-
CCGGTGCTGGATTTGTGTTA-3’) and reverse (5’-
AATCCCGTATTGTACCAGCG-3’); cry2A specific primer sets,
forward (5’-TAAAGAAAGTGGGGAGTCTT-3’) and reverse (5’-
AACTCCATCGTTATTTGTAG-3’). DNA template was mixed
with PCR reaction mixture i.e. per μl containing 0.2mM dNTPs,
0.5µM of each primer, 1x PCR buffer and 0.5 u of Taq DNA
polymerase (Promega, USA) in 25μl reaction volume and
amplification was performed in a DNA thermal cycler (MJ, Bio-
Rad). For both cry1 and cry1A primer sets, PCR was carried
out with an initial single denaturation step at 95
o
C for 2min and
30 amplification cycles including denaturation at 95
o
C for 45s,
annealing at 51
o
C for 45s and extension at 72
o
C for 60s. For
cry2A, annealing at 45
o
C for 45s was carried out. Finally an
extra extension step was applied at 72
o
C for 10 min. 5μl of the
PCR products were then electrophoresed in 1.5% agarose
(Promega, USA) gel prepared and submerged in 1×TBE buffer
at 60V for 50min. Gel was visualized in gel documentation
system (Alphaimager mini, USA) following staining in 1×TBE
buffer-EtBr (Sigma, USA) solution and de-staining in distilled
water.

Bioassay:
The toxicity of the Btk isolates positive for cry1 and cry1A
genes were determined against the 3
rd
instar larvae of
Bactrocera cucurbitae. Spore-crystal mixture for bioassay was
prepared from the twenty nine cry1 gene positive isolates and
reference strain Btk HD-73 by inoculating them in 100ml of T3-
liquid medium and incubating for 7 days at 30°C with
continuous shaking at 250 rpm. Cultures were centrifuged at
5000 rpm for 15 min to separate the culture from medium.
Pellets (spores and crystal protein mixture) were washed twice
with 20mL of cold sterile distilled water and centrifuged at 5000
rpm for 5 min. The pellets were re-suspended in 20mL of
sterile distilled water and stored at 4°C. Larval diet was
prepared by mixing spore-crystal suspension of each isolate
with 10g of semisolid paste of boiled pumpkin. 10 larvae were
placed in each petri-dish provided with spore-crystal
suspension mixed diet. The toxicity of each strain was assayed
in triplicate. The petri dishes were incubated at 25±2°C
temperature and 70±10% RH, with a photoperiod of 16:8 (L: D)
for 7 days. Mortality was scored in comparison with a parallel
control in which diet was mixed with sterile distilled water
instead of bacterial suspension and it was used to correct test
mortality using Abbot’s formula [4].
Results:
Identification of Bacillus thuringiensis Isolates:
Samples were collected from peripheral and central locations
of Dhaka and different agricultural areas of Bangladesh. Total
139 samples were processed comprising of 97 soil, 31 leaf, 7
insect and 4 stored product dust. After acetate selection and
heat treatment, 186 Bt like isolates were obtained in the EYA
medium from 114 samples (82%) out of 139 samples
however 25 samples did not produce growth of any isolate
(Table 1) . 155 lecithin hydrolysing isolates (83.3%) were
picked (Fig 1A) from these 186 Bt-like isolates and after
sporulation in T3- agar medium 148 isolates were observed to
possess parasporal body containing Cry proteins as revealed
under phase contrast microscope (Fig 1B) and light
microscope also following CBB staining. The haemolysis test
revealed that out of 148 isolates, 115 (77.7%) was found to be
haemolytic (Fig 1C) hence considered to have insecticidal
activity.

AFSSA 146

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.
Table 1: Btk index in different sampling locations.

Biotyping:
A simple biotyping method which included lecithin hydrolysis
(done in the isolation stage), esculin utilization, sucrose and
salicin fermentation tests revealed the abundance of different
biotypes of Bt in the working samples and Btk like isolates
were obtained in this process. Bacillus thuringiensis kurstaki
(27.7%) was the most abundant biotype among other types
(Fig 2A). Bt thuringiensis, Bt type ten and Bt dendrolimus were
the next abundant isolates. Btk was most abundant (index-
0.69) in the soil of Jhenaidah (Table 1).
Plasmid profile:
Bacillus thuringiensis commonly harbours a varied number of
large plasmids with different molecular mass. As most of the
cry genes are located on these large plasmids [3]. Plasmids
from Bt kurstaki like isolates were extracted and plasmid
pattern of these isolates were compared with reference Btk
HD-73. Plasmids ranging from 10-15 kb were observed for the
Bt isolates and a 12kb plasmid band was evident for Btk HD-73
(Fig 3).


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)

Characterization of cry gene content:
Forty one Btk like isolates were checked for the presence of
cry1, cry1A and cry2A genes. Initially 29 isolates were
considered positive for cry1 gene as PCR product of about 270
bp was evident for them which is the desired product size for
universal cry1 primer sets. These twenty nine cry1 gene
positive isolates were then examined for the presence of
Lepidoptera specific cry1A gene and of them sixteen isolates
were found positive with expected 490 bp PCR product (Fig
4A). Twenty two isolates out of forty one were observed with
bands of different product size for cry2A specific primers
among which desired product of more than 1500 bp was not
observed. So, though the isolates responded in the PCR, they
were not considered to harbour cry2A genes.
Bioassay:
The toxicity of spore-crystal mixture of twenty nine cry1 gene
positive Btk like isolates was assayed against 3
rd
instar larvae
of Bactrocera cucurbitae. A single dose (6x10
7
spores 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%
Bt
kurstaki
27.7%
Bt
dendroli
mus
12%
Bt sotto
10.3%
Bt
israelens
is
10%
Bt
thuringie
nsis
18%
others
22%
n=148
B
A
C
AFSSA 147
mortality) (Fig 5). Their performance was also analysed
statistically calculating standard deviation.


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
analysis. Isolates name as per labelled over the lane (100bp DNA ladder;
TaKaRa, Japan). B) Number of cry1 and cry1A genes in the Btk biotyped
isolates.
Discussion:
The particular interest of this research work was to isolate and
identify Bacillus thuringiensis kurstaki like isolates as this
subspecies of Bt has a long successful history to control the
vegetable pests belonging to Lepidoptera order. So after heat
treatment of the samples following acetate selection, they were
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
+
,
Su
-
) of which lecithin hydrolysis test was accomplished in the
isolation stage. Bacillus thuringiensis kurstaki biotyped isolates
were most abundant (27.7%) among other sup-species of Bt in
the sampling sites.

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.
-20
0
20
40
60
80
100
120
Control HD-73 JSc2 JSc1 JSa1 SSc2 SSe2 JaS8
Average
Mortality
Percentage
Larvae died
0
5
10
15
20
25
30
35
cry1 cry1A cry2A
N
u
m
b
e
r

o
f

i
s
o
l
a
t
e
s
A
B
A
B
AFSSA 148
These isolates assuming to harbour cry1 gene were tested
against the 3
rd
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
biopesticides by biotechnological approach for the control of
vegetable pests in Bangladesh.’’

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