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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

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

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 International Journal of Tropical Insect Science

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

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

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 International Journal of Tropical Insect Science

Preparation of endospore‑crystal mixture for crystal and subsequently sterilized with 0.5% sodium hypochlo-
staining, PAGE analysis and insect bioassay rite and rinsed with sterile water and then dipped in
100% concentrated Bt SCM. The lower and upper sur-
A loop full of Bt culture was inoculated in 250 ml conical face of mungbean leaves were surface contaminated with
flasks containing 100 ml sterile nutrient broth and kept in bacteria endospores and crystals. The treated leaves were
shaker incubator for 96 h at 30 °C, 200 rpm. All the 12 air dried and then 3–4 leaves were clumped into a small
Bt isolates (based on 16srRNA analysis in present study) bunch. For control, instead of SCM, sterile water was
were incubated in 12 different conical flasks for sporulation. used. Each leaf bunch was inserted in a 25 ml sample
Parasporal crystal production is a characteristic feature of container with a small perforation at the centre of its lid.
Bacillus thuringiensis unlike B. cereus (Sanahuja et al. 2011; The 25 ml sample container was filled with sterile water
de Maagd et al. 2003). The Bt Spore Crystal Mixture (SCM) to keep the leaf turgid for 7 days and the container is
was extracted as described by Zothansanga et al. (2016). placed inside a Genetix® insect breeding dish (120 mm
The SCM were subjected to endospore-crystal staining X 80 mm). Fifteen ­3rd instar larvae were pre-starved for
with coomasie brilliant blue as described by Laemmli (1970) 3 h and released on the leaves in each insect breeding
with minor modifications and were grouped according to the dish and three such dishes were maintained at 25 ± 1ºC,
crystal shape (Lopez-Pazos et al. 2009). The protein com- 75 ± 2% RH with a photoperiod of 8:16 h (L: D).The lar-
position of 12 Bt isolates SCM were studied through SDS- val mortality was recorded up to 7 days after treatment.
PAGE as described by Laemmli (1970). The entire experiment was repeated thrice. Further the
In order to compare the toxicity of these Bt isolates the experiment was repeated with ­5th instar larva along with
SCM extracted from 100 ml broth were used for diet con- 2 additional treatments i.e. 2 different concentrations of
tamination bioassay against three pests [Spilosoma obliqua commercial Bt formulation (Biolep).
Walker, Helicoverpa armigera Hubner and Olepa ricini Fab- The insecticidal activity of SCM of 12 Bt isolates were
ricius] and its cfu/ml was enumerated by inoculating the SCM screened against castor hairy caterpillar, Olepa ricini Fab-
on nutrient agar by spread plate technique and incubating the ricius (Lepidoptera: Erebidae) by artificial diet contami-
Petri plates for overnight at 30 °C. 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
Insect rearing diet (10 × 7 mm) was contaminated with 100 μl of Bt SCM
per diet piece and it was placed in the Genetix® insect
The lepidopteran insects’ viz. Bihar hairy caterpillar, breeding box (72 × 72 × 100 mm) before releasing twelve
Spilosoma obliqua Walker and pod borer, Helicoverpa pre-starved (for 3 h) ­3rd instar larvae. In case of untreated
armigera Hubner were maintained in the Bio-ecology control, 100 μl of sterile water was used instead of Bt SCM.
laboratory, Division of Crop Protection, IIPR, Kanpur. S. Each treatment was replicated 4 times and they were main-
obliqua larvae were reared on mungbean leaves maintained tained at 25 ± 1 °C and RH 70 ± 5%. The larval mortality
at 25 ± 1ºC, 75 ± 2% RH with a photoperiod of 8:16 h (L:D) was recorded up to 7 days and the percent mortality was
in Biogen® biological oxygen demand (BOD) chamber. H. calculated.
armigera larvae reared on chickpea artificial diet maintained Diet contamination method was deployed for screening
at 25 ± 1ºC and 75 ± 2% RH with a photoperiod of 8:16 h the insecticidal activity of 12 Bt isolates SCM against gram
(L:D) in Remi® BOD (Armes et  al.  1992). The castor
pod borer, H. armigera larva. Ten 3 h pre-starved ­3rd instar
hairy caterpillar, Olepa ricini Fabricius was collected from
larvae were used per replication and each treatment was rep-
IIPR main farm on castor and was reared in Bio-ecology
laboratory on castor leaves and maintained at 25 ± 1ºC, licated4 times. A small diet piece (1.5 cm X 1.0 cm) was
75 ± 2% RH with a photoperiod of 8:16 h (L:D). 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
Insect bioassay percent mortality was calculated. Based on percent mortal-
ity the toxicity of Bt isolates were grouped as highly toxic
The insecticidal activity of the 12 Bt isolates’ spore crys-
tal mixture (SCM) were examined against S. obliqua lar- (70–100%), moderately toxic (50–69%), less toxic (20–49%)
vae. The experiment comprised of 12 treatments along and negligible / non-toxic (< 20%) as described by Meadows
with a control and each treatment was replicated thrice. et al. (1992). The Kaplan–Meier survival analysis was per-
The Leaf dip bio-assay was performed on the mungbean formed to estimate the Survival time (­ ST50) of each Bt strain
leaves (IPM 2–3) and they were washed with tap water

13
International Journal of Tropical Insect Science

against all three pests. The ­ST50 helps in finding out the Bt rhizospheric bacterial isolates (Fig. 1a-c) was selectively iso-
strains efficacy against the insect by virtue of its speed of kill. lated by heat selection protocol (Renganathan et al. 2011).
The rhizospheric soil samples of 11 different agricultural
Statistical analyses crops viz., chickpea, pigeonpea, field pea, cowpea, lentil,
lathyrus, mustard, potato, tomato, brinjal and chilli were
The percent mortality was calculated based on the larval mor- collected.
tality in each replication. The larval mortality of twelve bacte-
rial isolates was subjected to Kaplan–Meier survival analysis in Selective isolation of B. thuringiensis and Bt index
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). B. thuringiensis is gram positive rod shaped bacteria. In our
The per cent mortality data were arc sine transformed and sub- study, the identified 113 endospore forming bacterial isolates
jected to PROC ANOVA procedure in SAS 9.1 to compare the were screened in comparison to the colony morphology of the
toxicity of different Bt isolates (SAS 2006). 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
Results (Fig. 1d-f) while remaining 45.13% (51 isolates) were Gram
negative (Table 1). The colony morphology of the sixty two
Sample collection and isolation of endospore gram positive bacterial isolates was studied using the morpho-
forming bacteria logical parameters. Also, the presence of parasporal body (crys-
tal protein) was screened using the phase contrast microscopy.
Surveys were conducted in six different districts in the Indo- Among the sixty two gram positive isolates studied 12 Bt iso-
Gangetic plains region to collect 63 rhizospheric soil sam- lates were found to have the colony morphology and parasporal
ples representing 3 different agro-ecological regions viz., body similar to the reference strain B. thuringiensis kurstaki
central plain zone, eastern plain zone and Bundhelkhand Z-52 (Table 1). The distribution pattern of Bt isolates were
(Table 1). From the 63 soil samples, 113 endospore forming influenced by the host crop and their geographical location. In
the present study, Fatehpur had recorded the highest number

Fig. 1  Bt isolates morphology in nutrient agar (a-c) and its microscopic analysis of gram staining (d-f)

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

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

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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 of various molecular weights viz., 175, 135, 65, 51, 35
urease test while F1. IIPR was found to have weak positive & 21  kDa (Fig.  5). Among the 12 Bt isolates, F8.IIPR
reaction (Fig. 3c, d) while SgH5.IIPR, J3.IIPR, B1.IIPR, Pa1. was found to produce 4 sharp protein bands (45, 50, 65,
IIPR and Pa2.IIPR were found to be negative. All 12 iso- 135 kDa), Ak2.IIPR (50, 60, 75, 135 kDa) and F5 (135–140,
lates were displayed positive activity to salicin (Fig. 3e, f), 70, 40 kDa) three sharp protein bands and two faint bands
lecthinase and catalase tests (Table 5). For esculinase test, at 75 kDa and 74 kDa approximately. The isolates having
all the isolates except RoT61.IIPR were found to be posi- protein bands at 135 and 65 kDa were of indication that it
tive. For starch hydrolysis, the isolates Ak2.IIPR, F6.IIPR, is possessing Cry 1 and Cry 2 proteins. Most of the indig-
F8.IIPR, F1.IIPR and RoT61.IIPR were found to be positive. enous Bt isolates produced proteins in the range of 29 to
The TLUAE phenotype was found in AK2.IIPR, F6.IIPR and 62 kDa. The reference strain Bt kurstaki-z 52 (Fig. 5) has
F1.IIPR. The only isolate that had a phenotype of TLUSAE showed crystal proteins at 70 kDa. This diversity in proteins
is F8.IIPR. The LSAE phenotype is observed in SgH5.IIPR, indicated that Bt isolate may have diverse cry genes and
B1.IIPR, Pa1.IIPR and Pa2.IIPR. The reference strain Bt insecticidal activities.
kurstaki Z-52, F4.IIPR and F5.IIPR had a LUAE phenotype.
The present result lucidly depicts the phenotypic variation Insect bioassay
that existed among the Bt isolates. Only two isolates, F4.IIPR
and RoT61.IIPR were found to be casein hydrolysis positive The SCM of 12 Bt isolates were challenged against ­3rd and
(Table 5). Except 2 isolates B1.IIPRand RoT61.IIPR remain- ­5th instar Bihar hairy caterpillar, Spilosoma obliqua lar-
ing isolates were found to be motile. vae belonging to Erebidae family. Among the twelve iso-
lates tested, F8.IIPR showed highest (100%) mean larval
Crystal protein shape and protein profiling mortality within 7 days of treatment in both larval stages
­(3rd and ­5th instar) and it has recorded significantly high-
The coomasie blue staining of twelve Bt isolates depicted est toxicity. The isolate F1.IIPR was on par with F8.IIPR
variations in crystal protein shape such as bipyramidal and showed highest (100%) mean larval mortality against
(AK2.IIPR, F8.IIPR, F5.IIPR, F6.IIPRand F1.IIPR), spheri- ­5th instar (Fig. 6a) within 3 days after treatment while the
cal (F4.IIPR, B1.IIPR and SgH5.IIPR), rectangular (RoT61. size of larvae had increased in control (Fig. 6b). Further-
IIPR), cuboidal (Ak2.IIPR, F8.IIPR, F6.IIPR, F5.IIPR and more, F8.IIPR (100%) has resulted in highest larval mortal-
F1.IIPR) and amorphous (B1.IIPR, J3.IIPR, Pa1.IIPR and ity against ­3rd instar larva and it was on par with F5.IIPR
Pa2.IIPR). Interestingly, five isolates had both bipyrami- (95.56%) (Table 6). Based on the average percent mortality
dal and cuboidal [F8.IIPR (Fig. 4a), AK2.IIPR (Fig. 4b), of 12 Bt isolates only 3 isolates viz., F8.IIPR, F5.IIPR &
F6.IIPR (Fig. 4c), F1.IIPR, F5.IIPR] shapes (Table 5). F6.IIPR were considered as highly toxic while F1.IIPR falls
SDS-PAGE analysis of the spore crystal mixture (SCM) under moderately toxic category to ­3rd instar. Similarly, for
demonstrated that Bt isolates contained crystal proteins ­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- evident from the ­ST50 analysis that among the 12 Bt isolates,
mercial Bt formulation Biolep® which also showed mod- F8.IIPR is the best isolate for managing S. obliqua larva as
erate toxicity (53.33 & 66.67%) at the two different doses it had rapid knock down action.
tested against ­5th instar. The potency of 12 Bt isolates SCM was also tested
The ­ST50 analysis implies that F8.IIPR has resulted in against another polyphagous pest belonging to Erebidae
50% mortality within 24 h after treatment for both stages (­ 3rd commonly known as castor hairy caterpillar, Olepa ricini
and ­5th instar). Further, 100% larval mortality was recorded F. Interestingly, on these insects also the F8.IIPR isolate
within 48 h after treatment (Fig.  7a, b). The Bt isolates showed highest mortality (91.67%) within 168 h after
F1.IIPR and F5.IIPR has recorded 50% mortality within 48 h treatment (Figs. 6c and 7c) against 3­ rd instar larva by S
­ T50
after treatment incase of ­3rd instar. While it is 72 h and 120 h analysis. The next best isolate ­SgH5.IIPR has resulted in
for respective isolates incase of ­5th instar larva. Hence, it is

13


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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 after treatment (Fig. 6e and 7d). Hence both isolates are
F8.IIPR isolate falls under the high toxic category accord- found suitable for managing H. armigera.
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) Discussion
based on ­ST50 analysis.
The toxicity of 12 Bt isolates against gram pod borer Deployment of biopesticides ensures the sustainable crop
(H. armigera), a major polyphagous insect pest belonging production by restoring soil health thereby reducing the
to Noctuidae was studied by artificial diet contamina- application of insecticides and reduced toxicity to the
tion method. The highest larval mortality in 3­ rd instar microbial communities (Yasin et  al. 2016). The native
larvae was recorded from the isolate F8.IIPR (100%) fol- Bacillus thuringiensis (Bt) isolates poses novel insecticidal
lowed by AK2.IIPR (88.75%) and they had significantly genes with wider toxicity and thus makes it to be more
higher toxicity than remaining isolates tested (Table 6). effective than exotic strains. For instance, Bonmee et al.
Both these isolates fall under high toxic category. The (2019) had documented huge variation in the frequency of
­ST 50 analysis indicated that F8.IIPR and AK2.IIPR had occurrence of cry1A, cry2A, cry9 and vip3A genes in native
achieved highest mortality (100% & 88.75%) within 168 h Bt isolates in the rhizosphere soil. Hence, it is essential to

Fig. 5  Protein profiling of spore crystal mixture of Bt isolates

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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
Fig. 6  Insect bioassay of Bt isolates with Spilosoma obliqua larva (rice, maize and tobacco) rhizosphere soil sample and they
(a-b) Olepa ricini larva (c-d) and Helicoverpa armigera larva (e–f) hadn’t included any legume crop in their study.
The phylogenetic analysis showed 3 distinct groups
viz., Bacillus cereus group comprising of B. thuringiensis
know the diversity of native Bt strains occurring in IGP for and B. cereus (34 isolates), B. megaterium (5 isolates)
unraveling its potential toxicity against indigenous insect and Lysinibacillus (21 isolates) apart from the out group
pest. The insecticide consumption in IGP was 545 g/ha of Pseudomonas synxantha and Rhodococcus (2 isolates)
gross cropped area and the Compounded Annual Growth (Fig. 3). The present finding is in congruence with Sharma
Rate of insecticide consumption increased by 2.95 from et al. (2015) who also had reported 21 different Bacillus
2000–01 to 2012–13 was (Devi et al. 2017). In order to and Bacillus derived genera like Lysinibacillus sp. from
minimize the insecticide consumption in IGP screening of Indo-Gangetic plains of India based on 16S rRNA gene
63 rhizospheric soils was done for isolating native Bt isolates sequencing. In the present study 5 out of 12 Bt isolates
in the present study. Even though Bt have been isolated (41.66%) were having vip3A gene (MF143591, MF143590,
from different environmental habitats such as, humus rich MF143589, MK124722 and MK214423). Shingote et al.
soils of mountain and forest, leguminous phylloplanes, (2013) reported that among the 40 Bt isolates examined from
immature leaf, mature leaf, senescent leaf and grass, it Vidarbha region of Maharashtra only 12.5% isolates were
was reported to be more uniform in soil compared to other found to have vip3A. Further, Bhalla et al. (2005) (33.3%)
sample types (Hongyu et al. 2000; Kaur and Singh 2000; and Selvapandiyan et al. (2001) also had reported such low
Collier et al. 2005; Aramideh et al. 2010). Hence, we have frequency (2%) of vip3A among the native Bt isolates in
sampled 63 locations from 6 different districts of IGP and India. vip3 gene has no similarity with delta endotoxin
isolated 113 bacterial isolates from the rhizospheric soils. and has a broad-spectrum insecticidal activity. The vip3A
Out of 113 bacterial isolates, 62 (54.87%) bacterial isolates protein is a novel insecticidal protein toxic against a large
were gram positive and 51 (45.13%) were gram negative number of lepidopteran larvae (Milne et al. 2008) and it is

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 Table 6  Efficacy of Bt SCM against S. obliqua, O. ricini and H. armigera 
Isolate Dose (cfu/ml) Accession no % mortality due to Toxicity Grouping % mortality due to Bt Toxicity Grouping % mortality due to Toxicity Grouping
Bt isolates at 7 days isolates at 7 days after Bt isolates at 7 days
after treatment of S. treatment of O. ricini* after treatment of H.
obliqua* armigera*
3rd instar 5th instar 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)

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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 and casein and also found to produce cuboidal or spheri-
larvae (Donovan et al. 2001). The mode of action of vip3A cal crystals. The 6 Bt isolates (Ak2.IIPR, F8.IIPR, F6.IIPR,
is very much different from cry1Ab (Lee et  al. 2003). F5.IIPR, F1.IIPR and F4.IIPR) along with the reference
Moreover, the Vip3A protein doesn’t compete with Cry strain Bt kurstaki z-52 were found positive for urease test
protein for binding site. Hence they are complimentary in also it was effective against lepidopteran insects tested. The
nature to Cry protein for killing the lepidopteran larvae. present result supports the findings of Martin et al. (2009).
The representative bacterial isolates of B. thuringiensis Wherein they had reported a strong correlation between
(Ak2.IIPR), B. megaterium (F4T10.IIPR), B. cereus (J2. urease production and successful replication of those Bt in
IIPR), Lysinibacillus fusiformis (V5.IIPR), Lysinibacillus gypsy moth, a lepidopteran pest. In the present study, dif-
sp (Ek.IIPR) and Pseudomonas synxantha (F9.IIPR)were ferent biochemical phenotypes (TLUAE, TLUSAE, LSAE,
subjected to biochemical test using Hi-bacillus kit and found LUAE) existed among the Bt isolates, as reported by Martin
that only Lysinibacillus fusiformis was positive for citrate et al. (2010). Motility test is one of the key tests to identify
utilization. Similarly, for Malonate test, only B. megaterium Bt (Logan 2005). In general, Bt isolates are motile by peri-
and L. fusiformis were found positive (Table 4; Fig. 2). Simi- trichous flagellum. Nevertheless, non-motile Bt isolates were
lar results were reported by Rajasekhar et al. (2017) and also reported (Maheswaran et al. 2010). Two isolates in the
El-kersh et al. (2012). The isolate RoT61.IIPR was found present study was found to be non-motile. Similar report of
to hydrolyse starch and casein (Table 5). This is in accord- non-motile Bt isolates (5%) was reported from Saudi Arabia
ance with Horani et al. (2003) wherein a novel Bt serotype (El-Kersh et al. 2012).
H71 from Jordan was also found to hydrolyse gelatin, starch

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International Journal of Tropical Insect Science

Five distinct crystal morphologies were observed from Bt isolates, F8.IIPR, Ak2.IIPR, F1.IIPR, F6.IIPR and
12 Bt isolates by phase contrast microscopy. These were F5.IIPR has both bipyramidal and cuboidal cry protein
bipyramidal, cuboidal, spherical, amorphous and rectan- and it had exhibited higher toxicity than remaining iso-
gular (Table 5). The molecular weight of F8.IIPR crystal lates (Table 6). The results are in agreement with those of
protein is 45 kDa, 50 kDa, 65 kDa and 135 kDa. Jung et al. Ohba and Aizawa (1986a, b) who had reported relation-
(1997) reported that Btk HD1 has two proteins, bipyrami- ship between the shape of the parasporal bodies and its
dal and cuboidal and it has molecular weight of 135 and toxicity. Federici et al. (2006) showed that bipyramidal
65 kDa respectively; while Bt 14 has only bipyramidal pro- crystal proteins exhibited toxicity only to Lepidoptera
tein and its molecular weight is 135 kDa. Bt strains with and were associated with cry1 toxins, whereas cuboidal
spherical crystals lack insecticidal activity (Logan et al. crystal proteins exhibited toxicity to Lepidoptera and
2005). A prominent band is present at 75 kDa in Ak2.IIPR, Diptera and were most probably associated with cry2
B1.IIPR, F1.IIPR, F6.IIPR, F5.IIPR, F4.IIPR and SgH5. toxin. In general, the type of cry genes present in a strain
IIPR (Fig. 5). The molecular weight of various crystal pro- correlates to some extent with its insecticidal activity.
tein viz., Cry1, Cry2, Cry 3, Cry 4 and Cry 10 were reported The ­ST 50 analysis had shown that F8.IIPR had resulted
as 130–140 kDa; 65–75 & 135 kDa; 66–73 & 70–75 kDa; in 100% mortality within 48 h after treatment in BHC
125–145,68,72,78,128,130–140 &135  kDa and 80  kDa (Figs. 7a, b). The present result is in accordance with
(Swamy et al. 2013). The 130–138 kDa lepidopteran-active Patel (2014) who had also reported 100% mortality of
Cry proteins are encoded by Cry1 genes. The Cry2 and Cry3 ­3rd instar BHC with in 48 h after treatment of Bt kurstaki
proteins are 65 and 70 kDa, respectively (Chambers et al. 5%WP at 0.1%.
1991). Interestingly, F8.IIPR is highly toxic to CHC (91%) followed
Bacillus thuringiensis was reported to be ubiquitously by SgH5.IIPR (50%) that falls under medium toxic category
present in soil from different geographic distribution (Table 6). The Bt isolates obtained from soil was found to cause
(Armengol et al. 2007; Park et al. 2008; Saadaoui et al. 37% to 88% toxicity against lepidopteran larvae (Chilcott and
2009; Yasutake et al. 2006; Forsyth et al. 2000).Very Wigley 1993). Spores and crystals present in the SCM might
scanty reports are available on Bt presence in arable soils have resulted in higher level of mortality as it was already
and its distribution in different crop rhizosphere. Hence, reported that both in combination is more toxic than either crys-
in the present study we had analysed rhizosphere soil tals or spores alone (Crickmore 2006). The present findings are
samples of 6 leguminous plants (pigeonpea, chickpea, in agreement with Al-Otaibi (2013) in terms of spore crystal
field pea, cowpea, lentil and lathyrus), four vegetable mixtures toxicity against second instar S. littoralis larvae.
crops (chilli, potato, tomato and brinjal) and one oilseed The percent mortality of 12 Bt isolates ranged from 0
crop (mustard) for the presence of Bt and its toxicity to 100%. Among the 12 isolates tested against ­3rd instar H.
potential against 3 lepidopteran pests (Fig. 6). Bt gener- armigera, F8.IIPR (100%) is found to have highest mortal-
ally does not kill an insect larva that inhabits the soil ity followed by Ak2.IIPR (88.75%) and F6.IIPR (80.00%)
(Martin and Travers 1989), but it is toxic to insect larvae than the mortality rate of the standard isolate Bt kurstaki
that are aerial and waterborne (Wu and Chang 1985). Z-52 (30%) (Table 6). The native Bt isolates of Telangana,
Consequently, three polyphagous insect pest infesting India reported to cause 16.67 to 94.44% mortality of 2nd
leguminous crops and other important agricultural crops instar H. armigera (Lalitha et al. 2012). The variation in
in India viz., Bihar hairy caterpillar (Spilosoma obliqua), toxicity of the 2 Bt isolates (Ak2.IIPR and F8.IIPR) in our
castor hairy caterpillar (Olepa ricini) and gram pod borer study may be attributed to the variation in protein crystals
(Helicoverpa armigera) were chosen for assessing the concentration or composition or geographical location from
insect toxicity tests. Among the 12 Bt isolate, F8.IIPR where it was isolated or host crop rhizosphere or variation
is highly toxic against ­3 rd and ­5 th instar BHC and it is in biochemical phenotype. As the former Bt isolate was
isolated from Fatehpur district along with F1.IIPR and isolated from Akbarpur in Mustard rhizosphere while the
F5.IIPR (Table 6). The latter two isolates recorded in latter was isolated from Fatehpur in chickpea rhizosphere
high and medium toxicity against ­5th instar BHC. Inter- (Table 3). Interestingly, these isolates were found to have dif-
estingly, Agrawal et al. (2010) has reported that among ferent biochemical phenotypes viz., TLUAE and TLUSAE.
the 22 districts surveyed in U.P., BHC is found to occur Both the Bt isolates, F8.IIPR and F1.IIPR have the highest
only in Fatehpur district on Chickpea crop. Hence the toxicity against the BHC. Further F8.IIPR is having high
reason for the higher toxicity of Bt isolate might be due toxicity against CHC also. F8.IIPR and Ak2.IIPR displayed
to co-existence of the insect pest with the pathogen. high toxicity towards gram pod borer (Fig. 6e). Thus, the two
Wyres et al. (2010) also has reported that in Bt popu- isolates F8.IIPR and Ak2.IIPR has the potential as biopes-
lations capacity to increase production of toxin seems ticide for the management of above mentioned lepidopteran
linked to the eventual presence of a host. The following insects respectively.

13
 International Journal of Tropical Insect Science

Conclusion 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
The present study vividly documents the diversity of gram were harmed in this research work.
positive, endospore forming rhizospheric bacteria inhabiting iv. This is to certify that pre-informed consent were
11 different agricultural crops with special emphasis to obtained from all co-authors.
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 References
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