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Distribution and toxicity of Bacillus thuringiensis (Berliner) strains from

different crop rhizosphere in Indo-Gangetic plains against polyphagous
lepidopteran pests

Article  in  International Journal of Tropical Insect Science · March 2021

DOI: 10.1007/s42690-021-00451-5

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International Journal of Tropical Insect Science
https://doi.org/10.1007/s42690-021-00451-5

ORIGINAL RESEARCH ARTICLE

Distribution and toxicity of Bacillus thuringiensis (Berliner)

strains from different crop rhizosphere in Indo‑Gangetic plains
against polyphagous lepidopteran pests
G. K. Sujayanand1,2   · Mohd Akram1 · Aravind Konda2 · Ashish Nigam1 · Shripad Bhat3 · Jyotirmay Dubey1 ·
Krishna Kumar1,4 · Senthilkumar K. Muthusamy5

Received: 16 September 2020 / Accepted: 22 January 2021

© African Association of Insect Scientists 2021

Abstract
Rhizobacterial diversity is an indicator of soil health and in turn it is influenced by the host crops, edaphic factors and weather.
The present investigation reports the diversity of endospore forming, gram positive rhizobacteria (Bacillus megaterium, B.
thuringiensis, B. cereus and Lysinibacillus spp.) inhabiting 11 different agricultural crops in the IGP of India. Twelve Bacillus
thuringensis (Bt) isolates were identified by screening 62 g positive bacterial isolates from 63 rhizosperic soil samples
collected from 6 districts of IGP. The highest Bt index was recorded from Bhopal (0.25) followed by Fatehpur (0.17), Kanpur
dehat (0.10), Jalaun (0.08) and Hamirpur (0.06). The bacterial isolates were characterized based on the 16SrRNA gene and
phylogenetically grouped into 3 clades. The spore crystal mixture of 12 Bt isolates were subjected to insect bioassay and
it revealed, F8.IIPR has highest toxicity against Spilosoma obliqua Walker (100%), Olepa ricini Fabricius (91.67%) and
Helicoverpa armigera Hubner (100%) larva. Survival analysis ­(ST50) showed that the S. obliqua is highly susceptible than
O. ricini and H. armigera to F8.IIPR. Crystal staining and protein profiling showed the presence of cry 1 (135 kDa) and cry
2 (65 kDa) genes in 5 Bt isolates. PCR amplification of vip3A gene confirmed its presence in five Bt isolates. To conclude
F8.IIPR and Ak2.IIPR has the potential as a promising biopesticide for controlling the three lepidopteran insects tested.

Keywords  Biopesticide · Insecticidal protein · Crystal protein · Bt protein profiling · Biochemical type and vip3

Introduction structure play important role in regulating the various

Indo-Gangetic Plains (IGP) is one of the largest fertile
plains in the world that occupies 13% of total geographical
area of India and feeds 40% of its’ population (Malviya
et  al. 2011). Rhizospheric bacterial diversity and its
* G. K. Sujayanand
sujayanand.GK@icar.gov.in; sujay2020@gmail.com
1
Division of Crop Protection, ICAR-Indian Institute of Pulses
Research, Kalyanpur, Kanpur 208024, India
2
Division of Plant Biotechnology, ICAR-Indian Institute
of Pulses Research, Kalyanpur, Kanpur 208024, India
3
Division of Social Science, ICAR-Indian Institute of Pulses
Research, Kalyanpur, Kanpur 208024, India
4
Pandit Deen Dayal, Upadhyay College of Horticulture
and Forestry, Dr. RPCAU​, Bihar 843121 Muzaffarpur, India
5
Division of Crop Improvement, ICAR-Central Tuber Crops
Research Institute, Thiruvananthapuram 695017, India
edaphic parameters such as disease suppression, organic
matter decomposition, plant growth promotion, etc.
(Malviya et al. 2011; Bahadur et al. 2017; Kumar et al.
2017; Johri et al. 2003). Bacterial communities are known
to dominate the soil surface horizon (0-30 cm) in all the
soil profiles of IGP than actinomycetes and fungus, which
plays a major role in enhancing soil health (Srivastava
et al. 2014). Among soil-borne bacterial communities,
Bacillus spp. is a prominent aggressive colonizer in crop
rhizosphere and show a broad spectrum of antagonistic
activity against soil borne pathogens (Govindasamy et al.
2010). The insect pest alone inflicts a yield loss of 16.8%
in major agricultural crops and it amounts to 35,877.3
USD (Dhaliwal et al. 2010). Bihar hairy caterpillar (BHC),
Spilosoma obliqua Walker (Lepidoptera: Erebidae) is a
major insect pest in IGP region (Singh et al. 2015). It
is a polyphagous pest in Indian subcontinent (including
Pakistan, southeastern Afghanistan, Bhutan, Bangladesh
and Srilanka) inflicting heavy yield loss in legumes

13
Vol.:(0123456789)

(mungbean and urdbean), oilseeds (sunflower, rapeseed
& mustard), vegetable crops (cole crops) and fibre crop
(Jute) (Goel et al. 2004; Kumar and Srivastava 2016).
Helicoverpa armigera Hubner (Lepidoptera: Noctuidae)
is also an important polyphagous insect that inflicts heavy
yield loss of about 60–90% in pigeonpea (Sujithra and
Chander 2014). To manage these pest farmers rely on
several broad-spectrum synthetic insecticides including
Triazophos, λ Cyhalothrin, chlorantraniliprole, spinosad
and emamectin benzoate (Mandal et al. 2013; Gadhiya
et al. 2014). The castor hairy caterpillar, Olepa ricini
Fabricius (Lepidopetra: Erebidae) commonly known as
wooly bear is a polyphagous insect pest that skeletonizes
the leaf by scrapping the chlorophyll completely in
banana, cotton, castor, sesame, cowpea, common bean,
elephant foot yam, moringa, sweet potato, pumpkin etc.,
(Vazhacharickal et al. 332019; David and Anathakrishnan
32004; Nair 1970). Castor is more preferred host wherein
it was found to defoliate castor leaves at a rate of 2.31 g
per four larvae within 2  days (Mohamed and Kareem
2010). The wooly bear was found to cause a yield loss
of about 70–80% in country bean (Revathi and Kingsley
2008). In general, application of synthetic insecticides
destabilizes the microbial diversity in rhizosphere and
also it kills the non target predators and parasitoids
(Hariprasad et  al. 2009). Further, continuous use of
synthetic insecticides had resulted in development of
insecticide resistance, resurgence and also destabilizes
the rhizobacterial diversity (Nicholson 2002). The
decline in the rhizobacterial species richness in the
soil leads to decreased resistance in the rhizospheric
region to pathogenic bacteria invasion (Jacobsen and
Hjelmsø 2014). To overcome the ill effect of insecticides,
deployment of biopesticides helps in enriching the
existing rhizospheric bacteria, parasitoids, predators
and beneficial insects in agro-ecosystem along with
suppression of noxious insect pest (Mishra et  al.
2015). Biopesticides, especially Bt was integrated with
parasitoids as biointensive Integrated Pest Management
module for diamond back moth (Plutella xylostella)
management in several South and South East Asian
countries (Srinivasan 2012).
Among the biopesticides, Bacillus thuringiensis (Bt)
alone constitutes 75% of total biopesticide market (Olson
2015; Rogério et al. 2014). Bt is a ubiquitous, facultative
anaerobic gram-positive, rod shaped bacterium that differ-
entiates into ellipsoidal spore and characteristic parasporal
crystal protein (δ-endotoxin) during later growth stages
(Crickmore 2006). This crystal protein is toxic to insects,
nematodes and protozoan (Bravo et al. 2007). Even though
numerous efforts have been made for novel Bt strain isola-
tion in several countries, only scanty literature is avail-
able on Bt isolates and its toxicity against Bihar hairy
13
International Journal of Tropical Insect Science
caterpillar, pod borer and castor hairy caterpillar (Gorashi
et al. 2014; Shishir et al. 2014). Hence, in the present study
efforts were made to identify and isolate Bt colonies from
different crop rhizosphere in IGP through biochemical
characterization, molecular characterization and based on
their toxicity profiling against three lepidopteran pest viz.,
Bihar hairy caterpillar, gram pod borer and castor hairy
caterpillar.
Materials and methods
Sample collection and isolation of spore forming
Bacillus isolates
The soil samples were collected from the sixty three agri-
cultural fields comprising six different districts of the IGP
region viz., Kanpur Dehat, Varanasi, Fatehpur, Hamirpur,
Jalaun and Bhopal. Soil samples were collected from a
depth of 5 cm below the soil surface to capture the rhizos-
phere region. The samples were collected from only those
fields having no record of bio-pesticide application. Five
samples were collected randomly in each agricultural
field using a sterile soil scooper (Rabha et  al. 2017).
The details of the samples were given in Supplementary
Table 1. The collected samples were transported in sterile
polythene bag (10 × 10 cm) from the field to laboratory.
Soil samples collected from the five random locations
in the agricultural fields were mixed and sampled using
quartering method and stored at 4 °C for further analy-
sis (Campos and Campos 2017). 10 g of the soil sample
was used for isolation of B. thuringiensis (Bt) using heat
selection protocol (Renganathan et al. 2011). 1 ml of soil
suspension was pipetted out into a sterile 10 ml test tube
after 4 h of incubation at 30 °C. The test tubes containing
soil suspension were placed in a hot water bath for 10 min
at 80 °C and then they were diluted up to ­10–3 followed
by plating in the nutrient agar (NA) using spread plate
technique. The plated petridishes were incubated for 18 h
at 30 °C for colony growth. Colonies having Bacillus like
morphology such as off white colour with smooth edges,
flat to slightly raised elevation were selected and marked
on the Petri dish (Rampersad and Ammons 2005). A ref-
erence Bt strain, Bacillus thuringiensis var. kurstaki Z-52
was isolated from commercial Bt formulation Biolep®
WP for comparing the indigenous Bt strains. These colo-
nies were sub-cultured on new NA plates to obtain pure
cultures. Gram staining was carried out for selective iso-
lation of gram-positive colonies. The bacterial cells were
studied under Leica microscope at 10 X 40 magnification.
The Bt index was calculated as reported by Anandhi et al.
(2013) and Thappan et al. (2008).
International Journal of Tropical Insect Science

Table 1  Abundance of endospore forming rhizobacteria and Bt index of the soil collected from 6 different districts of Indo-Gangetic plains of
India
S District (Tehsil) No. of soil Sampled crop No. of endospore forming bacterial No. of Bt Bt index Endospore forming,
no. sample ana- rhizosphere isolates appeared isolates Gram + ve rhizos-
lyzed obtained (% pheric bacterial
G +  G- Total from soil species present
samples)

1 Kanpur Dehat 19 Chickpea, 19 18 37 4 (15.79) 0.10 B. thuringiensis

(Akbarpur) pigeonpea, mus- B. megaterium
tard, potato and B. cereus
chilli Lysinibacillus spp.
2 Fatehpur 10 Chickpea and 16 12 28 5 (50) 0.17 B. thuringiensis
(Fatehpur sadar pigeonpea B. megaterium
and Bindki) B. cereus
B. pseudomycoides
Lysinibacillus spp.
3 Varanasi 12 Brinjal, chilli, 10 6 16 0 (0) 0.00 B. subtilis
(Varanasi) tomato, B. cereus
cowpea and Lysinibacillus sp.
pigeonpea
4 Hamirpur 10 Chickpea, 10 6 16 1(10) 0.06 B. thuringiensis
(Sumerpur, pigeonpea, field B. megaterium
Muskara and pea and lentil B. subtilis
Sarila) B. cereus
Lysinibacillus spp.
5 Jalaun 5 Fieldpea, 5 7 12 1(20) 0.08 B. thuringiensis
(Jalaun) chickpea and B. cereus
pigeonpea Lysinibacillus spp.
6 Bhopal 7 Chickpea, 2 2 4 1(14.29) 0.25 B. thuringiensis
(Phanda) pigeonpea len- B. cereus
til and lathyrus
Total 63 62 (54.87%) 51 (45.13%) 113 12 0.10

Molecular characterization by 16S rRNA & vip genes
Genomic DNA of 62 g positive bacterial isolates were
extracted using the protocol given by Nucleopore gDNA
Fungal Bacterial Minikit (NP-7006D). The 16S rRNA
and vip3A genes regions were amplified from the gram-
positive bacterial isolates and Bt isolates using the
standard primers (Bernasconi et al. 2004; Franco-Rivera
et al. 2004). The amplicons were purified and sequenced
using sanger sequencing. The BLAST search tool (http://
www.ncbi.nlm.nih.gov/BLAST​/ ) was used to identify
the species on the basis of sequence similarity of > 97%
with the closest sequence retrieved from the GenBank
(ATCC 10,792) as described by Shishir et  al. (2012).
For phylogenetic analysis the 16S rRNA gene sequence
of 62 bacterial isolates, 7 standard bacterial strains
(2:B. thuringiensis, 2:B. cereus, 1:B. megaterium and
2:Lysinibacillus sp.) from NCBI database were deployed.
Apart from the 62 g positive bacterial isolates, a gram
negative bacterial isolates (F9.IIPR), was selected as an
out group and it was sequenced and submitted to NCBI.
The phylogenetic tree was constructed on the aligned data
sets using the neighbor-joining method implemented in the
program MEGA 6.0.2 (Tamura et al. 2013).
Biochemical characterization
Out of 62 bacterial isolates, six representative isolates
belonging to 4 different phylogenetic groups (based on 16S
rRNA) were subjected to biochemical tests such as catalase,
malonate, Voges-Proskauer’s test, carbohydrate utilization
test [glucose, sucrose, mannitol, arabinose, and trehalose],
citrate utilization and nitrate reduction were performed for
by using Hi-Bacillus Test Kit (Himedia, India).
Subsequently, to study the biochemical phenotypes
among the 12Bt isolates, some important biochemical
tests for Bt viz., lecithinase, esculinase, urease, catalase,
starch hydrolysis, acid production in sucrose, motility and
casein hydrolysis tests were also performed as described
by Martin et al. (2010).

13

Preparation of endospore‑crystal mixture for crystal
staining, PAGE analysis and insect bioassay
A loop full of Bt culture was inoculated in 250 ml conical
flasks containing 100 ml sterile nutrient broth and kept in
shaker incubator for 96 h at 30 °C, 200 rpm. All the 12
Bt isolates (based on 16srRNA analysis in present study)
were incubated in 12 different conical flasks for sporulation.
Parasporal crystal production is a characteristic feature of
Bacillus thuringiensis unlike B. cereus (Sanahuja et al. 2011;
de Maagd et al. 2003). The Bt Spore Crystal Mixture (SCM)
was extracted as described by Zothansanga et al. (2016).
The SCM were subjected to endospore-crystal staining
with coomasie brilliant blue as described by Laemmli (1970)
with minor modifications and were grouped according to the
crystal shape (Lopez-Pazos et al. 2009). The protein com-
position of 12 Bt isolates SCM were studied through SDS-
PAGE as described by Laemmli (1970).
In order to compare the toxicity of these Bt isolates the
SCM extracted from 100 ml broth were used for diet con-
tamination bioassay against three pests [Spilosoma obliqua
Walker, Helicoverpa armigera Hubner and Olepa ricini Fab-
ricius] and its cfu/ml was enumerated by inoculating the SCM
on nutrient agar by spread plate technique and incubating the
Petri plates for overnight at 30 °C.
Insect rearing
The lepidopteran insects’ viz. Bihar hairy caterpillar,
Spilosoma obliqua Walker and pod borer, Helicoverpa
armigera Hubner were maintained in the Bio-ecology
laboratory, Division of Crop Protection, IIPR, Kanpur. S.
obliqua larvae were reared on mungbean leaves maintained
at 25 ± 1ºC, 75 ± 2% RH with a photoperiod of 8:16 h (L:D)
in Biogen® biological oxygen demand (BOD) chamber. H.
armigera larvae reared on chickpea artificial diet maintained
at 25 ± 1ºC and 75 ± 2% RH with a photoperiod of 8:16 h
(L:D) in Remi® BOD (Armes et  al.  1992). The castor
hairy caterpillar, Olepa ricini Fabricius was collected from
IIPR main farm on castor and was reared in Bio-ecology
laboratory on castor leaves and maintained at 25 ± 1ºC,
75 ± 2% RH with a photoperiod of 8:16 h (L:D).
Insect bioassay
The insecticidal activity of the 12 Bt isolates’ spore crys-
tal mixture (SCM) were examined against S. obliqua lar-
vae. The experiment comprised of 12 treatments along
with a control and each treatment was replicated thrice.
The Leaf dip bio-assay was performed on the mungbean
leaves (IPM 2–3) and they were washed with tap water
13
International Journal of Tropical Insect Science
and subsequently sterilized with 0.5% sodium hypochlo-
rite and rinsed with sterile water and then dipped in
100% concentrated Bt SCM. The lower and upper sur-
face of mungbean leaves were surface contaminated with
bacteria endospores and crystals. The treated leaves were
air dried and then 3–4 leaves were clumped into a small
bunch. For control, instead of SCM, sterile water was
used. Each leaf bunch was inserted in a 25 ml sample
container with a small perforation at the centre of its lid.
The 25 ml sample container was filled with sterile water
to keep the leaf turgid for 7 days and the container is
placed inside a Genetix® insect breeding dish (120 mm
X 80 mm). Fifteen ­3rd instar larvae were pre-starved for
3 h and released on the leaves in each insect breeding
dish and three such dishes were maintained at 25 ± 1ºC,
75 ± 2% RH with a photoperiod of 8:16 h (L: D).The lar-
val mortality was recorded up to 7 days after treatment.
The entire experiment was repeated thrice. Further the
experiment was repeated with ­5th instar larva along with
2 additional treatments i.e. 2 different concentrations of
commercial Bt formulation (Biolep).
The insecticidal activity of SCM of 12 Bt isolates were
screened against castor hairy caterpillar, Olepa ricini Fab-
ricius (Lepidoptera: Erebidae) by artificial diet contami-
nation method (Ammouneh et al. 2011). The castor leaf
diet was prepared as described by Rahman et al. (2002)
with slight modifications. Four small pieces of castor leaf
diet (10 × 7 mm) was contaminated with 100 μl of Bt SCM
per diet piece and it was placed in the Genetix® insect
breeding box (72 × 72 × 100 mm) before releasing twelve
pre-starved (for 3 h) ­3rd instar larvae. In case of untreated
control, 100 μl of sterile water was used instead of Bt SCM.
Each treatment was replicated 4 times and they were main-
tained at 25 ± 1 °C and RH 70 ± 5%. The larval mortality
was recorded up to 7 days and the percent mortality was
calculated.
Diet contamination method was deployed for screening
the insecticidal activity of 12 Bt isolates SCM against gram
pod borer, H. armigera larva. Ten 3 h pre-starved ­3rd instar
larvae were used per replication and each treatment was rep-
licated4 times. A small diet piece (1.5 cm X 1.0 cm) was
contaminated with 50 μl of Bt SCM and placed in a 100 ml
sample container containing a H. armigera larva. The larval
mortality was recorded up to 7 days after treatment and the
percent mortality was calculated. Based on percent mortal-
ity the toxicity of Bt isolates were grouped as highly toxic
(70–100%), moderately toxic (50–69%), less toxic (20–49%)
and negligible / non-toxic (< 20%) as described by Meadows
et al. (1992). The Kaplan–Meier survival analysis was per-
formed to estimate the Survival time (­ ST50) of each Bt strain
International Journal of Tropical Insect Science
against all three pests. The ­ST50 helps in finding out the Bt
strains efficacy against the insect by virtue of its speed of kill.
Statistical analyses
The percent mortality was calculated based on the larval mor-
tality in each replication. The larval mortality of twelve bacte-
rial isolates was subjected to Kaplan–Meier survival analysis in
SPSS 16.0 to find out the potential Bt isolate that can kill the S.
obliqua, O. ricini and H. armigera larva quickly (SPSS 2007).
The per cent mortality data were arc sine transformed and sub-
jected to PROC ANOVA procedure in SAS 9.1 to compare the
toxicity of different Bt isolates (SAS 2006).
Results
Sample collection and isolation of endospore
forming bacteria
Surveys were conducted in six different districts in the Indo-
Gangetic plains region to collect 63 rhizospheric soil sam-
ples representing 3 different agro-ecological regions viz.,
central plain zone, eastern plain zone and Bundhelkhand
(Table 1). From the 63 soil samples, 113 endospore forming
Fig. 1  Bt isolates morphology in nutrient agar (a-c) and its microscopic analysis of gram staining (d-f)
rhizospheric bacterial isolates (Fig. 1a-c) was selectively iso-
lated by heat selection protocol (Renganathan et al. 2011).
The rhizospheric soil samples of 11 different agricultural
crops viz., chickpea, pigeonpea, field pea, cowpea, lentil,
lathyrus, mustard, potato, tomato, brinjal and chilli were
collected.
Selective isolation of B. thuringiensis and Bt index
B. thuringiensis is gram positive rod shaped bacteria. In our
study, the identified 113 endospore forming bacterial isolates
were screened in comparison to the colony morphology of the
representative Bt isolate, B. thuringiensis kurstaki Z-52 (iso-
lated from Biolep®) and by Gram staining. Out of 113 bacterial
isolates, 54.87% isolates (i.e. 62 isolates) are Gram positive
(Fig. 1d-f) while remaining 45.13% (51 isolates) were Gram
negative (Table 1). The colony morphology of the sixty two
gram positive bacterial isolates was studied using the morpho-
logical parameters. Also, the presence of parasporal body (crys-
tal protein) was screened using the phase contrast microscopy.
Among the sixty two gram positive isolates studied 12 Bt iso-
lates were found to have the colony morphology and parasporal
body similar to the reference strain B. thuringiensis kurstaki
Z-52 (Table 1). The distribution pattern of Bt isolates were
influenced by the host crop and their geographical location. In
the present study, Fatehpur had recorded the highest number
13
 International Journal of Tropical Insect Science

Table 2  Occurrence of Bt S.no. District Chickpea Pigeonpea Mustard Total Name of isolate

isolates in different locations (Cicer (Cajanus cajan) * (Brassica
of Indo-Gangetic plains’ crop areitinum)* nigra) *
rhizosphere
1 Kanpur Dehat 0 2 (10) 2 (3) 4 Ak2.IIPR; RoT61.IIPR
(Akbarpur) Pa1.IIPR; Pa2.IIPR
2 Fatehpur 5 (7) 0 0 5 F8.IIPR; F1.IIPR;
F5.IIPR; F6.IIPR;
F4.IIPR
3 Varanasi 0 0 0 0 -
4 Hamirpur 1 (2) 0 0 1 SgH5.IIPR
5 Jalaun 1(2) 0 0 1 J3.IIPR
6 Bhopal 1 (3) 0 0 1 B1.IIPR
8 (17) 2 (10) 2 (3) 12
*-
 Number of soil samples screened is depicted inside parenthesis

of Bt isolates (5) followed by Kanpur dehat (4), Hamirpur (1),
Jalaun (1) and Bhopal (1).The Bt isolation index varied from
0.0 to 0.25 and it is highest for the soil samples of Bhopal (0.25)
followed by Fatehpur (0.17), Kanpur dehat (0.10), Jalaun (0.08)
and Hamirpur (0.06). Fifty per cent of Fatehpur soil samples
were found to harbour Bt followed by Jalaun (20%), Kanpur
Dehat (15.79%), Bhopal (14.29%) and Hamirpur (10%). Even
though 16 endospore forming bacterial colonies were isolated
from the Varanasi district none of them was Bt. When we com-
pare the samples based on crop rhizosphere, chickpea rhizos-
phere had recorded the highest number of Bt isolates i.e. 8 Bt
isolates (F8.IIPR; F1.IIPR; F5.IIPR; F6.IIPR; F4.IIPR; SgH5.
IIPR; J3. IIPR and B1.IIPR) followed by pigeonpea (2) and
mustard (2) (Table 2).
Molecular characterization & phylogenetic analysis
of bacterial isolates
The 16S rRNA genic region of the sixty two gram positive
bacterial isolates was sequenced using the standard prim-
ers. The amplicon size varied from 1222 bp (F9.IIPR) to
1537 bp (V5.IIPR). The 16S rRNA gene sequences were sub-
mitted to the GenBank with NCBI accession numbers from
KU601912 to KU601952 and KX661352 to KX661373.
Similarity search of the 16S rRNA sequence of the sixty two
isolates against NCBI database using BLASTN tool revealed
that twelve isolates shared highest similarity to Bacillus
thuringiensis (Bt) (Table 3). In addition, the phylogenetic
analysis revealed that the majority of isolates belonged to
either B. cereus group (34) (comprising of B. cereus or
Bacillus thuringiensis or B. subtilis) or B. megaterium (5) or

Table 3  Sequence similarity search (BLASTn) results of 16srRNA genes of Bt isolates

Sl.No Isolate name Similar to (Accession no.) Maximum score Total score Query coverage E value Identity

1 Ak2.IIPR (KU601952) Bacillus thuringiensis strain ATCC 2401 2401 100% 0.0 99%
10,792
2 F4.IIPR (KU601938) Bacillus thuringiensis strain ATCC 2418 2418 100% 0.0 99%
10,792
3 F5.IIPR (KU601936) Bacillus thuringiensis strain c25 2405 33587 100% 0.0 99%
4 SgH5.IIPR (KU601920) Bacillus thuringiensis strain SJC34 2422 2422 98% 0.0 99%
5 F1.IIPR (KU601941) Bacillus thuringiensis strain YGd22-03 2418 28954 100% 0.0 100%
6 F6.IIPR (KU601935) Bacillus thuringiensis strain 2427 33859 100% 0.0 99%
BM-BT15426
7 F8.IIPR (KU601933) Bacillus thuringiensis strain GTG-29 2390 2390 95% 0.0 99%
8 J3.IIPR (KU601926) Bacillus thuringiensis strain Al Hakam 2366 32943 100% 0.0 99%
9 B1.IIPR (KU601945) Bacillus thuringiensis strain SY 2340 2340 99% 0.0 98%
10 RoT61.IIPR (KU601922) Bacillus thuringiensis strain ATCC 2350 2350 99% 0.0 99%
10,792
11 Pa1.IIPR (KU601924) Bacillus thuringiensis isolate SIMH002 2412 2412 99% 0.0 99%
12 Pa2.IIPR (KU601923) Bacillus thuringiensis strain SJC34 2420 2420 100% 0.0 99%
13 BtkZ52 ref Bacillus thuringiensis serovar kurstaki 2431 2431 100% 0.0 100%
strain Z52

13
International Journal of Tropical Insect Science

Fig. 2  Phylogenetic analysis of
rhizosphere bacterial isolates
from Indo-Gangetic soils
The evolutionary history was
inferred using the Neighbor-
Joining method [Saitou and
Nei 1987]. The optimal
tree with the sum of branch
length = 0.66036883 is shown.
The evolutionary distances were
computed using the Maximum
Composite Likelihood method
[Tamura et al. 2004] and are
in the units of the number of
base substitutions per site. The
analysis involved 70 nucleo-
tide sequences. All ambigu-
ous positions were removed
for each sequence pair. There
were a total of 1596 positions
in the final dataset. Evolution-
ary analyses were conducted in
MEGA6 [Tamura et al. 2013]

Lysinibacillus fusiformis or Lysinibacillus sp. (21). Whereas
Pseudomonas synxantha (1); Rhodococcus (2) formed a sep-
arate clade as they are non-Bacillus isolates (Fig. 2).
In our study, vip3A gene was found to present in 5 Bt
isolates viz., F8.IIPR (MF143591), F6.IIPR (MF143590),
AK2.IIPR (MF143589), F1.IIPR (MK124722) and F5.IIPR
(MK214423) and its amplicon size is ~ 1000 bp (Supple-
mentary Fig. 1). The vip3A gene is having broad insecti-
cidal spectrum against lepidopteran larvae and these proteins
does not share homology with any other known insecticidal
protein including cry genes. Thus the identified Bt isolates
possess multiple insecticidal action through cry and vip3A
genes.
Biochemical characterization
Six isolates representing the six divergent groups’ viz., B.
thuringiensis- AK2.IIPR, B. cereus -J2.IIPR, B. megaterium-
F4T10.IIPR, Lysinibacillus group -V5.IIPR and Ek.IIPR and
Pseudomonas synxantha- F9.IIPR were chosen for studying
the bacterial group’s biochemical characteristics (Table 4;
Fig. 3a, b). The following biochemical tests viz., malonate,
Voges proskauer’s, citrate, ONPG, nitrate reduction, catalase
activity, arginine metabolizing and sugar namely mannitol,
glucose, arabinose and trehalose metabolizing ability of the
six isolates were studied using the hi-bacillus kit (Table 4;
Fig. 3a, b). The Bt isolate, AK2.IIPR produced positive
reaction for glucose, trehalose utilization, catalase and cit-
rate test, while negative for arabinose and mannitol. These
biochemical results further assisted in confirming this iso-
late as B. thuringiensis (Table 4; Fig. 3a, b). Conversely,
B. megaterium (F4T10.IIPR) produced delayed reaction for
nitrate reduction test. All the 5 carbohydrate utilization tests
(sucrose, mannitol, glucose, arabinose and trehalose) were
positive in B. cereus (J2.IIPR) (Table 4). The other repre-
sentative isolates for Pseudomonas synxantha (F9.IIPR)
and Lysinibacillus fusiformis (V5.IIPR) produced negative
reaction for the entire 5 carbohydrate utilization test. While
Lysinibacillus sp. (Ek.IIPR) gave negative only for arabinose
remaining 4 carbohydrate tests gave positive reaction indi-
cating the variation within Lysinibacillus isolates (Table 4).
In order to study the biochemical variations among the
12 Bt isolates they were subjected to lecthinase (L), esculi-
nase (E), urease (U), starch hydrolysis (T), sucrose (S), salicin
(A), catalase, casein hydrolysis and motility tests. Some bio-
chemical reactions like urease test resulted in distinguishing

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 International Journal of Tropical Insect Science

Table 4  Biochemical test results for representative bacterial groups

S.no Test name Bacterial Strains
AK2.IIPR F4T10.IIPR F9.IIPR V5.IIPR J2.IIPR Ek.IIPR
B. thuringiensis B. megaterium Pseudomonas Lysininbacillus B. cereus Lysinibacillus sp.
xynsantha fusiformis

1 Malonate -  +  -  +  - -

2 Voges proskauer’s - - - -  +   + 
3 Citrate - - -  +  - -
4 ONPG - - - - -  + 
5 Nitrate reduction  +  - -  +  - -
6 Catalase  +   +   +   +   +   + 
7 Arginine -  +  -  +   +  -
8 Sucrose - - - -  +   + 
9 Mannitol - - - -  +   + 
10 Glucose  +  - - -  +   + 
11 Arabinose - - - -  +  -
12 Trehalose  +  - - -  +   + 

the native Bt isolates. 6 isolates were found to be positive for
urease test while F1. IIPR was found to have weak positive
reaction (Fig. 3c, d) while SgH5.IIPR, J3.IIPR, B1.IIPR, Pa1.
IIPR and Pa2.IIPR were found to be negative. All 12 iso-
lates were displayed positive activity to salicin (Fig. 3e, f),
lecthinase and catalase tests (Table 5). For esculinase test,
all the isolates except RoT61.IIPR were found to be posi-
tive. For starch hydrolysis, the isolates Ak2.IIPR, F6.IIPR,
F8.IIPR, F1.IIPR and RoT61.IIPR were found to be positive.
The TLUAE phenotype was found in AK2.IIPR, F6.IIPR and
F1.IIPR. The only isolate that had a phenotype of TLUSAE
is F8.IIPR. The LSAE phenotype is observed in SgH5.IIPR,
B1.IIPR, Pa1.IIPR and Pa2.IIPR. The reference strain Bt
kurstaki Z-52, F4.IIPR and F5.IIPR had a LUAE phenotype.
The present result lucidly depicts the phenotypic variation
that existed among the Bt isolates. Only two isolates, F4.IIPR
and RoT61.IIPR were found to be casein hydrolysis positive
(Table 5). Except 2 isolates B1.IIPRand RoT61.IIPR remain-
ing isolates were found to be motile.
Crystal protein shape and protein profiling
The coomasie blue staining of twelve Bt isolates depicted
variations in crystal protein shape such as bipyramidal
(AK2.IIPR, F8.IIPR, F5.IIPR, F6.IIPRand F1.IIPR), spheri-
cal (F4.IIPR, B1.IIPR and SgH5.IIPR), rectangular (RoT61.
IIPR), cuboidal (Ak2.IIPR, F8.IIPR, F6.IIPR, F5.IIPR and
F1.IIPR) and amorphous (B1.IIPR, J3.IIPR, Pa1.IIPR and
Pa2.IIPR). Interestingly, five isolates had both bipyrami-
dal and cuboidal [F8.IIPR (Fig. 4a), AK2.IIPR (Fig. 4b),
F6.IIPR (Fig. 4c), F1.IIPR, F5.IIPR] shapes (Table 5).
SDS-PAGE analysis of the spore crystal mixture (SCM)
demonstrated that Bt isolates contained crystal proteins
of various molecular weights viz., 175, 135, 65, 51, 35
& 21  kDa (Fig.  5). Among the 12 Bt isolates, F8.IIPR
was found to produce 4 sharp protein bands (45, 50, 65,
135 kDa), Ak2.IIPR (50, 60, 75, 135 kDa) and F5 (135–140,
70, 40 kDa) three sharp protein bands and two faint bands
at 75 kDa and 74 kDa approximately. The isolates having
protein bands at 135 and 65 kDa were of indication that it
is possessing Cry 1 and Cry 2 proteins. Most of the indig-
enous Bt isolates produced proteins in the range of 29 to
62 kDa. The reference strain Bt kurstaki-z 52 (Fig. 5) has
showed crystal proteins at 70 kDa. This diversity in proteins
indicated that Bt isolate may have diverse cry genes and
insecticidal activities.
Insect bioassay
The SCM of 12 Bt isolates were challenged against ­3rd and
­5th instar Bihar hairy caterpillar, Spilosoma obliqua lar-
vae belonging to Erebidae family. Among the twelve iso-
lates tested, F8.IIPR showed highest (100%) mean larval
mortality within 7 days of treatment in both larval stages
­(3rd and ­5th instar) and it has recorded significantly high-
est toxicity. The isolate F1.IIPR was on par with F8.IIPR
and showed highest (100%) mean larval mortality against
­5th instar (Fig. 6a) within 3 days after treatment while the
size of larvae had increased in control (Fig. 6b). Further-
more, F8.IIPR (100%) has resulted in highest larval mortal-
ity against ­3rd instar larva and it was on par with F5.IIPR
(95.56%) (Table 6). Based on the average percent mortality
of 12 Bt isolates only 3 isolates viz., F8.IIPR, F5.IIPR &
F6.IIPR were considered as highly toxic while F1.IIPR falls
under moderately toxic category to ­3rd instar. Similarly, for
­5th instar larvae F8.IIPR & F1.IIPR were found to be highly

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International Journal of Tropical Insect Science
Fig. 3  Biochemical analysis of bacterial isolates through Hibacillus kit (a,b), Urease test (c,d) and Salicin test (e,f)
toxic while F5.IIPR showed moderate toxicity alike the com-
mercial Bt formulation Biolep® which also showed mod-
erate toxicity (53.33 & 66.67%) at the two different doses
tested against ­5th instar.
The ­ST50 analysis implies that F8.IIPR has resulted in
50% mortality within 24 h after treatment for both stages (­ 3rd
and ­5th instar). Further, 100% larval mortality was recorded
within 48 h after treatment (Fig.  7a, b). The Bt isolates
F1.IIPR and F5.IIPR has recorded 50% mortality within 48 h
after treatment incase of ­3rd instar. While it is 72 h and 120 h
for respective isolates incase of ­5th instar larva. Hence, it is
evident from the ­ST50 analysis that among the 12 Bt isolates,
F8.IIPR is the best isolate for managing S. obliqua larva as
it had rapid knock down action.
The potency of 12 Bt isolates SCM was also tested
against another polyphagous pest belonging to Erebidae
commonly known as castor hairy caterpillar, Olepa ricini
F. Interestingly, on these insects also the F8.IIPR isolate
showed highest mortality (91.67%) within 168 h after
treatment (Figs. 6c and 7c) against 3­ rd instar larva by S
­ T50
analysis. The next best isolate ­SgH5.IIPR has resulted in
13


13
Table 5  Biochemical tests and protein shape of Bt isolates
S.No Isolate name (NCBI Accession No.) Casein Catalase Urease Casein Motility Starch Esculinase Lecithi- Salicin Sucrose Cry
Hydrolysis (U) Hydrolysis hydrolysis (E) nase (L) (A) (S) Protein
(T) ­shape$

1 Ak2.IIPR ˉ  +   +  ˉ  +   +   +   +   +  - B,C

(KU601952)
2 F4.IIPR (KU601938)  +   +   +   +   +  ˉ  +   +   +  - S
3 F5.IIPR (KU601936) ˉ  +   +  ˉ  +  ˉ  +   +   +  - B,C
4 SgH5.IIPR (KU601920) ˉ  +  ˉ ˉ  +  ˉ  +   +   +   +  S
5 F1.IIPR (KU601941) ˉ  +  W +  ˉ ˉ  +   +   +   +  - B,C
6 F6.IIPR (KU601935) ˉ  +   +  ˉ  +   +   +   +   +  - B,C
7 F8.IIPR (KU601933) ˉ  +   +  ˉ  +   +   +   +   +   +  B,C
8 J3.IIPR (KU601926) ˉ  +  ˉ ˉ  +  ˉ  +   +   +  - A
9 B1.IIPR (KU601945) ˉ  +  ˉ ˉ ˉ ˉ  +   +   +   +  A
10 RoT61.IIPR (KU601922)  +   +  ˉ  +  ˉ  +  ˉ  +   +  - R
11 Pa 1.IIPR (KU601924) ˉ  +  ˉ ˉ  +  ˉ  +   +   +   +  A
12 Pa2.IIPR (KU601923) ˉ  +  ˉ ˉ  +  ˉ  +   +   +   +  A
13 Bacillus thuringiensis kurstaki Z52  +   +   +   +   +  ˉ  +   +   +  - B
serotype H3a 3b
$
  W-weak, A amorphous, B bipyramidal, C cuboidal, S spherical, R rectangular
 + positive reaction; – negative reaction
International Journal of Tropical Insect Science
International Journal of Tropical Insect Science
Fig. 4  Coomaise blue staining
of Bt crystal protein
50% mortality within 168 h after treatment. Again, the
F8.IIPR isolate falls under the high toxic category accord-
ing to Meadows classification and also the best isolate for
managing O. ricini. The F8.IIPR has taken relatively more
time for killing O. ricini (168 h) than S. obliqua (48 h)
based on ­ST50 analysis.
The toxicity of 12 Bt isolates against gram pod borer
(H. armigera), a major polyphagous insect pest belonging
to Noctuidae was studied by artificial diet contamina-
tion method. The highest larval mortality in 3­ rd instar
larvae was recorded from the isolate F8.IIPR (100%) fol-
lowed by AK2.IIPR (88.75%) and they had significantly
higher toxicity than remaining isolates tested (Table 6).
Both these isolates fall under high toxic category. The
­ST 50 analysis indicated that F8.IIPR and AK2.IIPR had
achieved highest mortality (100% & 88.75%) within 168 h
Fig. 5  Protein profiling of spore crystal mixture of Bt isolates
after treatment (Fig. 6e and 7d). Hence both isolates are
found suitable for managing H. armigera.
Discussion
Deployment of biopesticides ensures the sustainable crop
production by restoring soil health thereby reducing the
application of insecticides and reduced toxicity to the
microbial communities (Yasin et  al. 2016). The native
Bacillus thuringiensis (Bt) isolates poses novel insecticidal
genes with wider toxicity and thus makes it to be more
effective than exotic strains. For instance, Bonmee et al.
(2019) had documented huge variation in the frequency of
occurrence of cry1A, cry2A, cry9 and vip3A genes in native
Bt isolates in the rhizosphere soil. Hence, it is essential to
13

Fig. 6  Insect bioassay of Bt isolates with Spilosoma obliqua larva
(a-b) Olepa ricini larva (c-d) and Helicoverpa armigera larva (e–f)
know the diversity of native Bt strains occurring in IGP for
unraveling its potential toxicity against indigenous insect
pest. The insecticide consumption in IGP was 545 g/ha of
gross cropped area and the Compounded Annual Growth
Rate of insecticide consumption increased by 2.95 from
2000–01 to 2012–13 was (Devi et al. 2017). In order to
minimize the insecticide consumption in IGP screening of
63 rhizospheric soils was done for isolating native Bt isolates
in the present study. Even though Bt have been isolated
from different environmental habitats such as, humus rich
soils of mountain and forest, leguminous phylloplanes,
immature leaf, mature leaf, senescent leaf and grass, it
was reported to be more uniform in soil compared to other
sample types (Hongyu et al. 2000; Kaur and Singh 2000;
Collier et al. 2005; Aramideh et al. 2010). Hence, we have
sampled 63 locations from 6 different districts of IGP and
isolated 113 bacterial isolates from the rhizospheric soils.
Out of 113 bacterial isolates, 62 (54.87%) bacterial isolates
were gram positive and 51 (45.13%) were gram negative
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International Journal of Tropical Insect Science
isolates (Table  1). Twelve isolates out of 113 bacterial
isolates were identified as Bt by heat treatment technique
accompanied with biochemical analysis and 16S rRNAgene-
based diversity (Table 3). The present result confirms the
findings of Srivastava et al. (2014) who had reported that
surface soils of IGP are dominated by bacteria. The Bt index
ranged from 0.00 to 0.25, Bhopal recorded highest Bt index
(0.25) followed by Fatehpur (0.17) and Kanpur dehat (0.10)
(Table 1). The Bt index in uncultivated soil from Karnataka
reported to be 0.06 to 0.33 (Asokan and Puttaswamy 2007).
One of the reasons for higher Bt index in Bhopal is due to
its soil texture (i.e. high clay content) that helped to retain
high moisture than remaining soil samples (i.e. sandy loam
texture). A similar observation of negative correlation of
Bt abundance and distribution with soil sand content was
reported by Hossain et al. (1997). In our study, apart from
Bacillus cereus group, Lysinibacillus and B. megaterium
were found to be most abundant rhizosphere inhabiting
gram positive bacteria. The majority of Bt isolates were
isolated from legume rhizosphere, eight isolates (F8.IIPR;
F1.IIPR; F5.IIPR; F6.IIPR; F4.IIPR; SgH5.IIPR; J3.IIPR
and B1.IIPR) from the rhizosphere soil of chickpea, two
isolates from the rhizosphere soil of pigeonpea (Ak2.IIPR;
RoT61.IIPR) and two from the rhizosphere soil of mustard
(Pa1.IIPR; Pa2.IIPR). The present finding contradicts with
findings of Anandhi et  al. (2013) wherein rhizospheric
soils of tobacco (1.00) and maize (0.97) were found to have
abundant Bt colonies based on the Bt index. The possible
reason for contradiction is that they had taken only 3 crops
(rice, maize and tobacco) rhizosphere soil sample and they
hadn’t included any legume crop in their study.
The phylogenetic analysis showed 3 distinct groups
viz., Bacillus cereus group comprising of B. thuringiensis
and B. cereus (34 isolates), B. megaterium (5 isolates)
and Lysinibacillus (21 isolates) apart from the out group
Pseudomonas synxantha and Rhodococcus (2 isolates)
(Fig. 3). The present finding is in congruence with Sharma
et al. (2015) who also had reported 21 different Bacillus
and Bacillus derived genera like Lysinibacillus sp. from
Indo-Gangetic plains of India based on 16S rRNA gene
sequencing. In the present study 5 out of 12 Bt isolates
(41.66%) were having vip3A gene (MF143591, MF143590,
MF143589, MK124722 and MK214423). Shingote et al.
(2013) reported that among the 40 Bt isolates examined from
Vidarbha region of Maharashtra only 12.5% isolates were
found to have vip3A. Further, Bhalla et al. (2005) (33.3%)
and Selvapandiyan et al. (2001) also had reported such low
frequency (2%) of vip3A among the native Bt isolates in
India. vip3 gene has no similarity with delta endotoxin
and has a broad-spectrum insecticidal activity. The vip3A
protein is a novel insecticidal protein toxic against a large
number of lepidopteran larvae (Milne et al. 2008) and it is
 Table 6  Efficacy of Bt SCM against S. obliqua, O. ricini and H. armigera 
Isolate Dose (cfu/ml) Accession no % mortality due to
Bt isolates at 7 days
after treatment of S.
obliqua*
3rd instar 5th instar
Toxicity Grouping % mortality due to Bt Toxicity Grouping % mortality due to Toxicity Grouping
isolates at 7 days after Bt isolates at 7 days
treatment of O. ricini* after treatment of H.
armigera*
3rd instar 5th instar 3rd instar 3rd instar 3rd instar 3rd instar

AK2.IIPR 2 × ­108 KU601952 0.00 4.44 NT NT 33.33 LT 88.75 HT

(0.00c) (9.97 g) (35.07c,d) (70.43b)
F4.IIPR 1.3 × ­108 KU601938 2.22 17.78 NT NT 25.00 LT 20.00 LT
(4.99c) (24.85e,f) (29.33d,e) (26.57f)
International Journal of Tropical Insect Science

F5.IIPR 1.2 × ­107 KU601936 95.56 53.33 HT MT 41.67 LT 46.88 LT

(82.86a) (46.91c) (40.05b,c) (43.43d)
SgH5.IIPR 7.75 × ­106 KU601920 11.11 40.00 NT LT 50.00 MT 10.00 NT
(15.35c) (39.19d) (45.00b) (18.36 g)
F1.IIPR 1.3 × ­107 KU601941 68.89 100.00 MT HT 33.33 LT 20.00 LT
(56.74b) (90.00a) (35.18c,d) (26.12f)
F6.IIPR 4.1 × ­107 KU601935 68.89 35.56 HT LT 8.33 NT 80.00 HT
(57.57b) (36.52d) (14.42 g) (63.45c)
F8.IIPR 7.3 × ­107 KU601933 100 100 HT HT 91.67 HT 100.00 HT
(90.00a) (90.00a) (75.59a) (90.0a)
J3.IIPR 5.25 × ­106 KU601926 4.44 11.11 NT NT 16.67 NT 0.00 NT
(7.14c) (19.27f) (23.74e,f) (0.00 h)
B1.IIPR 6.75 × ­106 KU601945 0 17.78 NT NT 25.00 LT 10.00 NT
(0.00c) (24.85e,f) (29.84d,e) (18.36 g)
RoT61.IIPR 2.7 × ­108 KU601922 4.44 4.44 NT NT 0.00 NT 0.00 NT
(7.14c) (9.97 g) (0.00 h) (0.00 h)
Pa1.IIPR 1.5 × ­107 KU601924 13.33 22.22 NT LT 0.00 NT 0.00 NT
(17.71c) (27.87e) (0.00 h) (0.00 h)
Pa2.IIPR 5.41 × ­106 KU601923 13.33 8.89 NT NT 8.33 NT 20.00 LT
(17.71c) (17.11f,g) (16.78f,g) (26.55f)
B.t. kurstaki Z-52, 1.4 g/L KU601943 NE 53.33 - MT NE - NE -
Biolep® (46.92c)
B.t. kurstaki Z-52, 2.0 g/L KU601943 NE 66.67 - MT NE - 30.00 LT
Biolep® (54.74b) (33.20e)
Control - - 0.00 0.00 - - 0.00 - 0.00 -
(0.00c) (0.00 h) (0.00 h) (0.00 h)
Sem ±  0.92 0.77 0.64 0.70

NT Non Toxic, LT Less toxic, MT Moderately Toxic, HT Highly Toxic, NE Not estimated
*Values in parenthesis are arcsin transformed. In a column means followed by same letter are not significantly different from each other (P > 0.05)

13

Fig. 7  Survival analysis of Spilosoma obliqua (a-3rd instar larva; b-5th instar larva), Olepa ricini (c) and Helicoverpa armigera (d) larva against
Bt isolates
also responsible for pathogenicity of Bt towards lepidopteran
larvae (Donovan et al. 2001). The mode of action of vip3A
is very much different from cry1Ab (Lee et  al. 2003).
Moreover, the Vip3A protein doesn’t compete with Cry
protein for binding site. Hence they are complimentary in
nature to Cry protein for killing the lepidopteran larvae.
The representative bacterial isolates of B. thuringiensis
(Ak2.IIPR), B. megaterium (F4T10.IIPR), B. cereus (J2.
IIPR), Lysinibacillus fusiformis (V5.IIPR), Lysinibacillus
sp (Ek.IIPR) and Pseudomonas synxantha (F9.IIPR)were
subjected to biochemical test using Hi-bacillus kit and found
that only Lysinibacillus fusiformis was positive for citrate
utilization. Similarly, for Malonate test, only B. megaterium
and L. fusiformis were found positive (Table 4; Fig. 2). Simi-
lar results were reported by Rajasekhar et al. (2017) and
El-kersh et al. (2012). The isolate RoT61.IIPR was found
to hydrolyse starch and casein (Table 5). This is in accord-
ance with Horani et al. (2003) wherein a novel Bt serotype
H71 from Jordan was also found to hydrolyse gelatin, starch
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International Journal of Tropical Insect Science
and casein and also found to produce cuboidal or spheri-
cal crystals. The 6 Bt isolates (Ak2.IIPR, F8.IIPR, F6.IIPR,
F5.IIPR, F1.IIPR and F4.IIPR) along with the reference
strain Bt kurstaki z-52 were found positive for urease test
also it was effective against lepidopteran insects tested. The
present result supports the findings of Martin et al. (2009).
Wherein they had reported a strong correlation between
urease production and successful replication of those Bt in
gypsy moth, a lepidopteran pest. In the present study, dif-
ferent biochemical phenotypes (TLUAE, TLUSAE, LSAE,
LUAE) existed among the Bt isolates, as reported by Martin
et al. (2010). Motility test is one of the key tests to identify
Bt (Logan 2005). In general, Bt isolates are motile by peri-
trichous flagellum. Nevertheless, non-motile Bt isolates were
also reported (Maheswaran et al. 2010). Two isolates in the
present study was found to be non-motile. Similar report of
non-motile Bt isolates (5%) was reported from Saudi Arabia
(El-Kersh et al. 2012).
International Journal of Tropical Insect Science
Five distinct crystal morphologies were observed from
12 Bt isolates by phase contrast microscopy. These were
bipyramidal, cuboidal, spherical, amorphous and rectan-
gular (Table 5). The molecular weight of F8.IIPR crystal
protein is 45 kDa, 50 kDa, 65 kDa and 135 kDa. Jung et al.
(1997) reported that Btk HD1 has two proteins, bipyrami-
dal and cuboidal and it has molecular weight of 135 and
65 kDa respectively; while Bt 14 has only bipyramidal pro-
tein and its molecular weight is 135 kDa. Bt strains with
spherical crystals lack insecticidal activity (Logan et al.
2005). A prominent band is present at 75 kDa in Ak2.IIPR,
B1.IIPR, F1.IIPR, F6.IIPR, F5.IIPR, F4.IIPR and SgH5.
IIPR (Fig. 5). The molecular weight of various crystal pro-
tein viz., Cry1, Cry2, Cry 3, Cry 4 and Cry 10 were reported
as 130–140 kDa; 65–75 & 135 kDa; 66–73 & 70–75 kDa;
125–145,68,72,78,128,130–140 &135  kDa and 80  kDa
(Swamy et al. 2013). The 130–138 kDa lepidopteran-active
Cry proteins are encoded by Cry1 genes. The Cry2 and Cry3
proteins are 65 and 70 kDa, respectively (Chambers et al.
1991).
Bacillus thuringiensis was reported to be ubiquitously
present in soil from different geographic distribution
(Armengol et al. 2007; Park et al. 2008; Saadaoui et al.
2009; Yasutake et al. 2006; Forsyth et al. 2000).Very
scanty reports are available on Bt presence in arable soils
and its distribution in different crop rhizosphere. Hence,
in the present study we had analysed rhizosphere soil
samples of 6 leguminous plants (pigeonpea, chickpea,
field pea, cowpea, lentil and lathyrus), four vegetable
crops (chilli, potato, tomato and brinjal) and one oilseed
crop (mustard) for the presence of Bt and its toxicity
potential against 3 lepidopteran pests (Fig. 6). Bt gener-
ally does not kill an insect larva that inhabits the soil
(Martin and Travers 1989), but it is toxic to insect larvae
that are aerial and waterborne (Wu and Chang 1985).
Consequently, three polyphagous insect pest infesting
leguminous crops and other important agricultural crops
in India viz., Bihar hairy caterpillar (Spilosoma obliqua),
castor hairy caterpillar (Olepa ricini) and gram pod borer
(Helicoverpa armigera) were chosen for assessing the
insect toxicity tests. Among the 12 Bt isolate, F8.IIPR
is highly toxic against ­3 rd and ­5 th instar BHC and it is
isolated from Fatehpur district along with F1.IIPR and
F5.IIPR (Table 6). The latter two isolates recorded in
high and medium toxicity against ­5th instar BHC. Inter-
estingly, Agrawal et al. (2010) has reported that among
the 22 districts surveyed in U.P., BHC is found to occur
only in Fatehpur district on Chickpea crop. Hence the
reason for the higher toxicity of Bt isolate might be due
to co-existence of the insect pest with the pathogen.
Wyres et al. (2010) also has reported that in Bt popu-
lations capacity to increase production of toxin seems
linked to the eventual presence of a host. The following
Bt isolates, F8.IIPR, Ak2.IIPR, F1.IIPR, F6.IIPR and
F5.IIPR has both bipyramidal and cuboidal cry protein
and it had exhibited higher toxicity than remaining iso-
lates (Table 6). The results are in agreement with those of
Ohba and Aizawa (1986a, b) who had reported relation-
ship between the shape of the parasporal bodies and its
toxicity. Federici et al. (2006) showed that bipyramidal
crystal proteins exhibited toxicity only to Lepidoptera
and were associated with cry1 toxins, whereas cuboidal
crystal proteins exhibited toxicity to Lepidoptera and
Diptera and were most probably associated with cry2
toxin. In general, the type of cry genes present in a strain
correlates to some extent with its insecticidal activity.
The ­ST 50 analysis had shown that F8.IIPR had resulted
in 100% mortality within 48 h after treatment in BHC
(Figs. 7a, b). The present result is in accordance with
Patel (2014) who had also reported 100% mortality of
­3rd instar BHC with in 48 h after treatment of Bt kurstaki
5%WP at 0.1%.
Interestingly, F8.IIPR is highly toxic to CHC (91%) followed
by SgH5.IIPR (50%) that falls under medium toxic category
(Table 6). The Bt isolates obtained from soil was found to cause
37% to 88% toxicity against lepidopteran larvae (Chilcott and
Wigley 1993). Spores and crystals present in the SCM might
have resulted in higher level of mortality as it was already
reported that both in combination is more toxic than either crys-
tals or spores alone (Crickmore 2006). The present findings are
in agreement with Al-Otaibi (2013) in terms of spore crystal
mixtures toxicity against second instar S. littoralis larvae.
The percent mortality of 12 Bt isolates ranged from 0
to 100%. Among the 12 isolates tested against ­3rd instar H.
armigera, F8.IIPR (100%) is found to have highest mortal-
ity followed by Ak2.IIPR (88.75%) and F6.IIPR (80.00%)
than the mortality rate of the standard isolate Bt kurstaki
Z-52 (30%) (Table 6). The native Bt isolates of Telangana,
India reported to cause 16.67 to 94.44% mortality of 2nd
instar H. armigera (Lalitha et al. 2012). The variation in
toxicity of the 2 Bt isolates (Ak2.IIPR and F8.IIPR) in our
study may be attributed to the variation in protein crystals
concentration or composition or geographical location from
where it was isolated or host crop rhizosphere or variation
in biochemical phenotype. As the former Bt isolate was
isolated from Akbarpur in Mustard rhizosphere while the
latter was isolated from Fatehpur in chickpea rhizosphere
(Table 3). Interestingly, these isolates were found to have dif-
ferent biochemical phenotypes viz., TLUAE and TLUSAE.
Both the Bt isolates, F8.IIPR and F1.IIPR have the highest
toxicity against the BHC. Further F8.IIPR is having high
toxicity against CHC also. F8.IIPR and Ak2.IIPR displayed
high toxicity towards gram pod borer (Fig. 6e). Thus, the two
isolates F8.IIPR and Ak2.IIPR has the potential as biopes-
ticide for the management of above mentioned lepidopteran
insects respectively.
13

Conclusion
The present study vividly documents the diversity of gram
positive, endospore forming rhizospheric bacteria inhabiting
11 different agricultural crops with special emphasis to
occurrence of Bt isolates in 6 districts of Indo-Gangetic
plains. Sixty-three rhizosphere soil samples from 6 districts
of Indo-Gangetic plains was analysed and 62  g positive
endospore forming bacteria was isolated. These bacterial
isolates were characterized based on biochemical and 16S
rRNA into 3 distinct groups viz., Bacillus cereus group
comprising of Bt and Bc (34 isolates), B. megaterium (5
isolates) and Lysinibacillus (21 isolates). Out of 34 isolates
in B. cereus group, 12 isolates shared their 16S rRNA gene
homology with Bt strains in BLAST analysis and supported
by their insecticidal crystal protein presence. The highest
Bt index was recorded from Bhopal (0.25) followed by
Fatehpur (0.17). Further the majority of Bt isolates were
found in chickpea rhizosphere (8) followed by pigeonpea (2)
and mustard (2) out of 11 crops screened. The 12 Bt isolates
were further subjected to SCM isolation and coomaise blue
staining which indicated presence of 5 different crystal
shapes in different isolates. Interestingly, 5 Bt isolates had
both bipyramidal and cuboidal shapes [F8.IIPR, AK2.IIPR,
F6.IIPR, F1.IIPR, F5.IIPR]. The Bt isolate, F8.IIPRwas found
to have higher toxicity ­against3rd and ­5th instar larva of S.
obliqua followed by 3­ rd instar of O. ricini and H. armigera
based on Meadows classification and survival analysis
­(ST50). Further, Ak2.IIPR is found to be effective against
H. armigera next to F8.IIPR. Similarly, the isolates F5.IIPR
and F1.IIPR were found to be effective against S. obliqua in
second & third order. The vip3Aa gene is also found to be
present in 5 out of 12 isolates having higher toxicity against
3 lepidopteran insects tested indicating synergism between
vip3Aa and cry genes. Thus the present isolates F8.IIPR
(an isolate from chickpea rhizosphere) and Ak2.IIPR can be
effectively developed as a biopesticide for managing Bihar
hairy caterpillar, S. obliqua Castor hairy caterpillar, O. ricini
and pod borer, H. armigera. Further it will help in reducing
pesticide consumption and residues in agro-ecosystem by
promoting microbial biopesticides. It also helps in preserving
the microbial diversity in crop rhizosphere.
Supplementary information  The online version contains supplemen-
tary material available at https:​ //doi.org/10.1007/s42690​ -021-00451-​ 5.
Declarations  in Bacillus thuringiensis strains from Thailand. 3 Biotech
Conflict of interest  Hereby, I Dr. Sujayanand, G.K., consciously assure
that for the manuscript “Distribution and toxicity of Bacillus thuringiensis
(Berliner) strains from different crop rhizosphere in Indo-Gangetic plains
against polyphagous lepidopteran pest” following is fulfilled,
i. This material is our own original work, which has
not been previously published elsewhere.
13
International Journal of Tropical Insect Science
ii. This is to certify that there is no potential conflict
of interest in the research work articles.
iii. This is to certify that there is no human or animal
were harmed in this research work.
iv. This is to certify that pre-informed consent were
obtained from all co-authors.
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