Karnataka J. Agric. Sci.

,25 (2) : (199-202) 2012

Characterization of Bacillus thuringiensis isolates of western ghats and their insecticidal

activity against diamond back moth (Plutella xylostella L.)*
GEETA GOUDAR, A. R. ALAGAWADI, P. U. KRISHNARAJ AND K. BASAVANA GOUD
Department of Agricultural Microbiology
University of Agricultural Sciences, Dharwad-580 005, India
E-mail: geetagoudar@gmail.com
(Received: July, 2011 ; Accepted: May, 2012)
Abstract : An attempt was made to isolate, characterize Bacillus thuringiensis from western ghats of Uttara Kannada and
evaluate their insecticidal activity against DBM. Total of 204 samples comprising of 157 soil samples, 38 leaf, 5 leaf litter and
4 compost samples were collected from western ghat regions of Uttara Kannada district of Karnataka and used for the
isolation of B. thuringiensis. Out of 439 B. thuringiensis like isolates obtained, 44 were confirmed to be B. thuringiensis
through crystal staining. Diverse crystal morphologies were observed among the isolates. Among the 44 isolates, 25 isolates
had spherical shaped crystals, 13 with irregular type, two each cuboidal type and bipyramidal types and one with rectangular
type of crystal. Isolate UK-762D had two types of crystals viz., cuboidal and bipyramidal. All the 44 isolates showed
positive reaction for nitrate reduction, catalase production, Voges-Proskauer reaction and oxidase test, but all were negative for
acid and gas production from glucose, arginine hydrolysis, chitinase activity and esterase activity. Thirty eight and 37 isolates
were positive for starch and casein hydrolysis respectively. The bioassay studies against third instar larvae of DBM revealed
that one hundred per cent mortality of larvae was recorded in the isolates UK-13C, UK-762D as well as by the reference strain
HD1 after 72 hrs of treatment.
Key words: Bacillus thuringiensis, Bioassay, Crystal morphology, Diamond back moth

Introduction
Bacillus thuringiensis (Berliner), a soil bacterium, has been
commercialized to manage insect pests due to toxicity of the endotoxin they posses. B. thuringiensis (Bt) formulations have
a narrow host spectrum and have been found to be harmless to
humans, mammals and non-targeted insects. Compared with other
insect pathogens, its life cycle is simple. When nutrients are in
sufficient amounts and the environment is conducive, the spore
germinates producing a vegetative cell that grows and
reproduces by binary fission. The bacterium produces a spore
and parasporal body, the latter is composed primarily of
insecticidal protein toxins (Federici, 1999).
Among the lepidopteran insect pests of cultivated plants,
diamond back moth (Plutella xylostella) is a widely distributed,
serious pest of cruciferous crops (You and Wei, 2007). The pest
is distributed all over India and a direct crop loss due to pest
damage is worth several crores of rupees. Satpathy et al. (2005)
reported 50-80 per cent loss in marketable yield of cabbage due
to attack of P. xylostella. To combat the menace of this pest,
integrated pest management strategies have been developed
with little success. This is mainly because; the pest has developed
resistance not only to chemical insecticides but also to Bt having
certain individual toxins (Tabashnik et al., 1990; Georghiou and
Wirth, 1997). Similarly, if single toxin gene is cloned into another
organism/plant and used extensively for insect control, insect
may become resistant in the field. This necessitates the search
for Bt strains with novel toxins.
India possesses a great diversity of natural ecosystem. One
of the areas rich in endemism is the western ghats which is
recognized as one of the hot spots of biodiversity in the world
(Khoshoo, 1994). This stretch of undisturbed area would allow a
high degree of recombination of genetic content aided by
transposon inherent to B. thuringiensis. Systematic isolation of

Bt and the analysis of richness in terms of genes important to

agriculture are absolutely lacking. Hence, in the present study,
an attempt was made to isolate Bt from soil, leaf, leaf litter and
compost samples collected from the western ghat region,
characterize the isolates and evaluate their biocontrol potential
against diamond back moth.
Material and methods
Study was conducted to isolate and characterize the Bt
strains from western ghat regions of Uttara Kannada district of
Karnataka during the year 2006-07. Total of 204 samples
comprising of 157 soil samples, 38 leaf, 5 leaf litter and 4 compost
samples were collected and used for the isolation of Bt. The
isolation from soil, leaf litter and compost samples was carried
out by following sodium acetate selection method (Travers
et al., 1987) and shaken flask technique of Smith and Couche
(1991) was followed for the isolation of Bt from leaf samples.
The isolates were purified and stored at 4o C using dimethyl
sulfoxide in a refrigerator for further studies.
All the isolates were subjected for morphological and
biochemical characterization. Morphological characterization of
the isolates was done by comparing the colony morphology
and cell morphology with that of the reference B. thuringiensis
strain HD1. The isolates were subjected to various biochemical
tests such as nitrate reduction, catalase production, VogesProskauer reaction, oxidase test, acid and gas production from
glucose, arginine hydrolysis, chitinase activity, esterase activity,
starch and casein hydrolysis as per the procedures outlined by
Cappuccino and Sherman (1992) in comparison with the
reference strain HD1.
All the 44 B. thuringiensis isolates were used for bioassays
to ascertain their insecticidal activity against Plutella xylostella.
The effects of individual Bt isolates were evaluated according

* Part of Ph. D. thesis, submitted by the first author to the University of Agricultural Sciences, Dharwad - 580 005, India
199

Karnataka J. Agric. Sci., 25 (2) : 2012

Table 1. Morphological characteristics of native Bacillus thuringiensis
isolates of western ghat region
Colony Morphology
Cell size (m)
Isolate
Colour
Nature
(LxB)
No.
UK-13C
White
Rough
3.0x1.0
UK-23B
White
Rough
3.5x1.5
UK-25A
Creamy white
Rough
3.0x1.0
UK-25C
White
Rough
2.5x1.0
UK-26A
White
Rough
5.5x1.5
UK-26C
White
Rough
4.0x1.0
UK-28A
White
Rough
5.0x1.5
UK-34C
White
Rough
5.5x1.5
UK-38A
White
Rough
4.5x1.5
UK-38D
White
Rough
5.0x1.5
UK-39C
White
Rough
3.5x1.5
UK-42A
Creamy white
Rough
4.5x1.5
UK-42C
White
Rough
5.5x1.5
UK-43B
White
Rough
5.0x1.0
UK-44B
White
Rough
3.5x1.5
UK-45B
White
Rough
3.0x1.0
UK-46B
Creamy white
Rough
3.0x1.0
UK-46C
White
Rough
5.5x1.5
UK-46D
White
Rough
3.5x1.5
UK-47B
White
Rough
3.0x1.5
UK-48B
White
Rough
2.5x1.0
UK-50A
White
Rough
6.0x1.5
UK-52A
White
Rough
4.5x1.0
UK-53B
Creamy white
Rough
4.5x1.5
UK-55B
White
Rough
5.5x1.0
UK-56C
Creamy white
Rough
3.5x1.5
UK-57C
Creamy white
Rough
3.0x1.0
UK-58B
White
Rough
2.5x1.0
UK-71C
White
Rough
4.0x1.5
UK-74C
White
Rough
3.5x1.5
UK-159A
Creamy white
Rough
3.5x1.5
UK-190D
Creamy white
Rough
4.0x1.5
UK-194D
White
Rough
6.0x1.0
UK-195C
Creamy white
Rough
4.5x1.0
UK-214A
Creamy white
Rough
5.5x1.5
UK-216B
White
Rough
4.0x1.0
UK-230A
White
Rough
4.5x1.5
UK-745A
White
Rough
5.0x1.5
UK-748D
Creamy white
Rough
5.5x1.5
UK-748G
Creamy white
Rough
3.5x1.0
UK-760E
White
Rough
4.5x1.5
UK-762B
White
Rough
4.5x1.5
UK-762D
Creamy white
Rough
6.0x1.5
UK-774B
White
Rough
5.5x1.0
HD1
White
Rough
5.5x1.5

to standard bioassay (Vastrad, 2000). As cabbage is the most

preferred host plant for Plutella xylostella, cabbage seedlings
were raised in pots one month prior to rearing of the insect
larvae. Diamond back moth was mass cultured in the laboratory
following the method described by Liu and Sun (1984) with little
modification. The native B. thuringiensis isolates grown in the
modified glucose medium (MGM) were used for conducting the
initial bioassay. The test isolates were grown individually in
MGM broth for 3 days at 28 2o C. The population count was
taken by following standard serial dilution and plate count
technique.
Cabbage leaf discs measuring 7.5 cm diameter were dipped
in cell suspension with the population of 1.2 x 106 c.f.u/ml. The
leaf discs were dried aseptically and fed to larva. Three replications
were maintained for each isolate with ten larvae per replication.
The number of dead larvae was recorded at 24, 48 and 72 hrs
after treatment. Leaf dipped in water was maintained as untreated
control. Mortality was observed at 24, 48 and 72 hrs of treatment
and data were subjected to analysis of variance after arcsine
transformation and the means were separated by Duncans
Multiple Range Test (DMRT) (Duncan, 1955).
Results and discussion
Four hundred and thirty nine Bt like isolates were obtained
from 204 samples. Only 44 of them were confirmed to be Bt
through crystal staining. Out of these 44 isolates, 27 were
obtained from soil samples, 12 from leaf, one from leaf litter and
four isolates came from compost samples. Bt has been isolated
from various sources like soil, live and dead insects (Damagaard
et al., 1998), stored grains (Meadows et al., 1992) foliar surfaces
(Smith and Couche, 1991), water bodies (Ichimastu et al., 2000),
marine sediments (Maeda et al., 2000) and activated sludge
(Mizuki et al., 2001).
All the isolates were examined for their colony and cell
morphology, gram reaction, endospore formation, crystal
morphologies and biochemical characters. There were wide
differences in the colony morphology of the isolates indicating
high variability among the isolates. Out of 44 isolates, 31 showed
white colonies and the remaining 13 formed creamy white
colonies. (Table 1).
The cells of all the isolates were rod shaped and the average
dimension ranged from 2.5 m x 1.0 m to 6.0 m x 1.5 m. All the

Table 2 . Crystal morphology of native isolates obtained from western ghat regions (Isolates from Uttara Kannada)
Crystal morphology
Bipyramidal
Cuboidal
Irregular

Rectangular
Spherical

Bipyramidal and Cuboidal

Isolates
UK-13C, UK-23B
UK-42A, UK-760E
UK-26C,UK-34C,UK-38A, UK-43B, UK-44B,UK-46D,
UK-47B,UK-52A, UK-56C, UK-74C, UK-194D,
UK-214A,UK-774B
UK-195C
UK-25A, UK-25C, UK-26A,UK-28A,UK-38D,UK-39C,
UK-42C, UK-45B,UK-46B,UK-46C, UK-48B, UK-50A,
UK-53B,UK-55B,UK-57C, UK-58B,UK-71C,
UK-159A, UK-190D, UK-216B, UK-230A,
UK-745A, UK-748D, UK-748G, UK-762B,
UK-762D
200

Number of isolates
02
02

13
01

25
01

Characterization of Bacillus thuringiensis isolates of Western................

Table 3. Bioefficacy of Bacillus thuringiensis isolates against 3rd instar
larvae of Diamond back moth
Isolate
No.
UK-13C
UK-23B
UK-25A
UK-25C
UK-26A
UK-26C
UK-28A
UK-34C
UK-38A
UK-38D
UK-39C
UK-42A
UK-42C
UK-43B
UK-44B
UK-46B
UK-46C
UK-46D
UK-45B
UK-47B
UK-48B
UK-50A
UK-52A
UK-53B
UK-55B
UK-56C
UK-57C
UK-58B
UK-71C
UK-74C
UK-159A
UK-190D
UK-194D
UK-195C
UK-214A
UK-216B
UK-230A
UK-745A
UK-748D
UK-748G
UK-760E
UK-762B
UK-762D
UK-774B
HD1
Control
S.Em
C.D.@ 1%

No.of
larvae
treated
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

24 hrs

Per cent mortality after

48 hrs
72 hrs

30 (33.20)c
0 (0.00)l
26 (30.64)cd
0 (0.00)l
15 (22.78)g
10 (18.43)h
5 (12.92)j
0 (0.00)l
15 (22.78)g
0 (0.00)l
2 (8.13)k
0 (0.00)l
17 (24.34)fg
17 (24.34)fg
18 (25.09)fg
20 (26.55)ef
12 (20.26)h
19 (25.83)f
0 (0.00)l
02 (8.13)k
10 (18.43)h
10 (18.43)h
23 (28.65)de
0 (0.00)l
05 (12.92)j
10 (18.43)h
0 (0.00)l
03 (9.97)k
0 (0.00)l
0 (0.00)l
03 (9.97)k
07 (15.34)i
0 (0.00)l
0 (0.00)l
0 (0.00)
02 (8.13)k
05 (12.92)j
0 (0.00)l
07 (15.34)i
03 (9.97)k
15 (22.78)g
07 (15.34)i
33 (35.05)b
02 (8.13)k
37 (37.45)a
0 (0.00)l
0.73
1.93

75 (59.98)c
15 (22.78)t
70 (56.77)d
15 (22.78)t
47 (43.26)ij
37 (37.45)lm
30 (33.20)no
15 (22.78)t
47 (43.26)ij
0 (0.00)u
23 (28.65)qr
15 (22.78)t
53 (46.70)gh
50 (44.98)hi
53 (46.70)gh
60 (50.75)ef
43 (40.96)jk
57 (49.00)fg
15 (22.78)t
23 (28.65)qr
37 (37.45)lm
39 (38.63)kl
62 (51.92)e
17 (24.34)st
28 (31.94)op
37 (37.45)lm
17 (24.34)st
25 (29.99)pq
20 (26.55)rs
15 (22.78)t
25 (29.99)pq
33 (35.05)mn
20 (26.55)rs
17 (24.34)st
18 (25.09)st
23 (28.65)qr
30 (33.20)no
20 (26.55)rs
33 (35.05)mn
25 (29.99)pq
47 (43.26)ij
35 (36.26)lm
80 (63.41)b
23 (28.65)qr
87 (68.84)a
0 (0.00)u
0.82
2.16

100 (89.96)a
33 (35.05)op
87 (68.84)b
30 (33.20)p
63 (52.51)ef
57 (49.00)gh
50 (44.98) ij
33 (35.05)op
63 (52.51)ef
17 (24.34)q
43 (40.96)klm
30 (33.20)p
69 (56.14)d
67 (54.92)de
67 (54.92)de
77 (61.32)c
60 (50.75)fg
70 (56.77)d
30 (33.20)p
43 (40.96)klm
57 (49.00)gh
58 (49.58)fgh
77 (61.32)c
36 (36.86)no
50 (44.98)ij
58 (49.58)fgh
37 (37.45)no
47 (43.26)jk
40 (39.22)lmn
33 (35.05)op
47 (43.26)jk
53 (46.70)hi
40 (39.22)lmn
37 (37.45)no
39 (38.63)mn
45 (42.11)jkl
50 (44.98)ij
40 (39.22)lmn
53 (46.70)hi
47 (43.26)jk
63 (52.51)ef
53 (46.70)hi
100 (89.96)a
41 (39.80)lmn
100 (89.96)a
10 (18.43)r
0.94
2.48

Note:
Values in the parenthesis are arcsine transferred values.
The values represented by same alphabet are statistically on par with
each other by DMRT mean of three replications.

isolates were gram positive, endospore forming and showed

the presence of crystals. Among the 44 isolates, 25 had spherical
shaped crystals, 13 with irregular type, two with cuboidal type,
two with bipyramidal type, one with rectangular type and another
one (UK-762D) with both cuboidal and bipyramidal shaped
crystals (Table 2) indicating predominance of spherical crystals
in the isolates. The dominance of spherical crystals has been
reported by Arrieta et al. (2004) in the isolates of coffee
plantations whereas only bipyramidal inclusions were reported
by Wangondu et al. (2003).
All the 44 isolates showed positive reaction for nitrate
reduction, catalase production, Voges-Proskauers reaction and
oxidase test, but all were negative for acid and gas production
from glucose, arginine hydrolysis, chitinase activity and esterase
activity. Thirty eight and 37 isolates were positive for starch
and casein hydrolysis respectively. Similar observations have
been made by Kaur et al., (2006) who reported that the strains of
B. thuringiensis, besides producing parasporal crystal bodies,
were positive for catalase production, oxidase activity, nitrate
reduction, starch and casein hydrolysis.
Bioassay study was conducted with all the 44 isolates against
the third instar larvae of P. lutella xylostella and the results are
presented in Table 3. In general the per cent mortality increased
with incubation period and maximum mortality was recorded at
72 hrs after feeding.
The per cent mortality after 24 hrs of exposure of third instar
larvae of P. xylostella ranged from 0 to 33.00 per cent. The
highest larval mortality was recorded by the reference strain
HD1 with 37.00 per cent. Among the native isolates, UK-762D
recorded the highest mortality of 33.00 per cent followed by the
isolate UK-13C (30.00 %) which was on par with the isolate UK25A (26 %). The isolates UK-23B, UK-25C, UK-34C, UK-38D,
UK-42A, UK-45B, UK-53B, UK-57C, UK-71C, UK-74C, UK-194D,
UK-95C, UK-214A and UK-745A did not show mortality at 24 h.
After 48 hrs of exposure, the cumulative mortality ranged between
0 to 87.00 per cent. Among the isolates, the highest mortality of
80.00 per cent was brought about by the isolate UK-762D
followed by the isolate UK-13C (75.00 %), where as the reference
strain HD1 recorded mortality of 87.00 per cent. The least
mortality of 15 per cent was recorded by the isolates UK-23B,
UK-25C, UK-34C, UK-42A, UK-45B and UK-74C. No mortality
was observed in the control.
The cumulative mortality ranged from 17 to 100 per cent
after 72 hrs of exposure. The highest mortality of 100 per cent
was reported by the isolates UK-13C, UK-762D as well as by the
reference strain HD1. Other isolates which recorded more than
70 per cent mortality are UK-25A (87.00 %), UK-46B (77.00 %),
UK-52A (77.00 %) and UK-46D (70.00 %). The remaining isolates
showed less than 70 per cent mortality. The lowest mortality of
17 per cent was recorded by the isolate UK-38D. Control recorded
10 per cent mortality.
In the present investigation it was observed that the per
cent mortality was very low or almost nil after 24 hrs of feeding.
This might be due to the fact that B. thuringiensis being stomach
poison, when Bt enters in to the midgut of insect, it gets
dissolved in the alkaline pH, releasing - endotoxin (Schnepf
et al., 1998), there by killing the insect.

201

Karnataka J. Agric. Sci., 25 (2) : 2012

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