World Journal of Microbiology and Biotechnology (2020) 36:78

https://doi.org/10.1007/s11274-020-02849-8

ORIGINAL PAPER

Novel formulations of Bacillus thuringiensis var. kurstaki:

an eco‑friendly approach for management of lepidopteran pests
P. S. Vimala Devi1 · P. Duraimurugan1 · K. S. V. Poorna Chandrika2 · V. Vineela1 · P. P. Hari1

Received: 31 October 2019 / Accepted: 4 May 2020

© Springer Nature B.V. 2020

Abstract
Bacillus thuringiensis (Bt) and entomopathogenic fungi (EPF) are in use for management of insect pests. Continuous use of
Bt can lead to problem of resistance development in insect pests. Hence use of combination formulations (CF) of microbials
with diverse modes of action has been attempted to slow down the process of resistance development. Suspension concentrate
(SC) formulations of a local strain of Bt var. kurstaki DOR Bt-127 were developed singly and in combination with conidia
of the EPF Nomuraea rileyi (Nr) and Beauveria bassiana (Bb). Electron microscopy of Bt + Bb CF treated larvae of Heli-
coverpa armigera revealed simultaneous infection by both microbials indicating their compatibility. Endotoxin contents in
Bt-SC, Bt + Bb and Bt + Nr CFs were 5.0, 4.7 and 4.7%, respectively. These formulations were effective against larvae of
Spodoptera litura, H. armigera and Achaea janata coupled with a lowering of the effective requirement of Bt and EPF. In
multi-location field trials, Bt-SC and Bt + Nr CF were highly effective against S. litura and A. janata on castor as well as H.
armigera and Thysanoplusia orichalcea on sunflower. However, Bt + Bb CF was highly effective only on sunflower against
H. armigera and T. orichalcea. All formulations had 24 months shelf-life at room temperature. DOR Bt-127 based SC for-
mulations developed singly and in combination with Nr and Bb were effective against major lepidopteran pests of castor
and sunflower and did not lose viability under storage at room temperature. The CFs of Bt with EPF could prove promising
for mitigating resistance development to Bt.
Graphic abstract

Keywords Bacillus thuringiensis var. kurstaki · Entomopathogenic fungi · Spodoptera litura · Helicoverpa armigera ·
Suspension concentrate · Combination formulations

Extended author information available on the last page of the article

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Introduction of the speed of kill. One potential threat for continuous use
of Bt for pest management is the problem of development
Agricultural crops are damaged globally by over 10,000 spe- of resistance in insect pests. Similarly, limitations of fungi
cies of insects that cause an estimated annual loss of 10.6% as microbial control agents is that, each species as well as
while the average annual losses due to insect pests in India strains within a species are usually effective against a par-
have been estimated to be 15.7% valued at US $ 36.0 billion ticular pest. Hence, if the two entomopathogens comple-
in major field crops (Dhaliwal et al. 2015). Among insect ment each other, or act synergistically, a beneficial effect
pests, the tobacco caterpillar, Spodoptera litura (Fabricius) can be obtained. This objective could be achieved through
and head borer, Helicoverpa armigera (Hübner) are key the development of an appropriate co-formulation of two
pests of several crops in India as well as globally causing or more entomopathogens with different host ranges and
significant damage to oilseeds, pulses, vegetables, cotton, ecological tolerances (Wang et al. 2002). Very few studies
etc. (CABI 2018). It is estimated that chemical insecticides have reported the combined application of entomopathogens
worth about Rs.1200 crores are used annually in Indian agri- with the aim of increasing efficacy (Glare 1994; Inglis et al.
culture for the control of insect pests (Agarwal and Pandey 1997, 1999, 2001). Using Bt var. tenebrionis in combina-
2017; Jayaraj et al. 2016). Excessive and indiscriminate use tion with B. bassiana produced a statistically significant
of insecticides against insect pests in field and horticultural 6–35% greater reduction in larval field populations of Colo-
crops resulted in development of insecticide resistance in rado potato beetle on potato than would have been predicted
several insect pests including S. litura and H. armigera to had the two biopesticides acted independently (Wraight
commonly used insecticides (Tong et al. 2013; Hussain et al. and Ramos 2005). Similarly additive interaction of Bt var.
2014). Increasing environmental concerns and requirement israelensis and B. bassiana was reported against housefly
of residue free produce have necessitated search for alternate (Mwamburi and Miller 2009). Research to date has focused
techniques that are eco-friendly, economically viable and on development of formulations singly for the various potent
socially acceptable for management of insect pests. microbials whose large-scale promotion is greatly hindered
Biocontrol agents, particularly bacteria and fungi, offer by the slow speed of kill, limited host range and dependence
considerable promise for insect pest management. Several on favourable environmental conditions. However, develop-
biological agents are commercially available for use in crop ment of storable combination formulations (CFs) of Bt and
protection, most notably products based on Bacillus thur- entomopathogenic fungi employing proven virulent isolates,
ingiensis (Bt), entomofungal pathogens Beauveria bassiana with a good shelf-life could lead to reliable results in the
(Bb) and Metarhizium anisopliae. Bt formulations have been field instead of using tank mix formulations of these patho-
employed globally as topical pesticides to protect crops gens in combination. Based on this hypothesis, we under-
from major insect pests like H. armigera, Plutella xylos- took studies to develop effective and viable CFs of Bt with
tella (Linnaeus), Ostrinia nubilalis (Hübner), Agrotis ipsilon entomopathogenic fungi.
(Hufnagel), S. exigua (Hübner) etc. (George and Crickmore In this paper, we present the results of studies carried
2012). Among the entomofungal pathogens, Bb is a poten- out for development and evaluation of SC formulations
tially versatile fungus because of its wide spread occurrence, using DOR Bt-127, a local strain of Bt var. kurstaki, singly
broad host range, ability to infect at different stages of its and in combination with Nr and Bb through laboratory
hosts and cause natural epizootics on major lepidopteran bioassays and field trials. This study is the first report of
pests (Vimala Devi and Duraimurugan 2013). Nomuraea development of storable CFs of Bt with the entomofungal
rileyi (Nr) is an entomopathogenic fungus with great poten- pathogens Bb (Indian patent no.315134) and Nr.
tial for the management of major lepidopteran noctuid pests
like S. litura, H. armigera, Plusia sp. etc. (Vimala Devi and
Prasad 2001). The fungus has the ability to cause epizootics
under ideal conditions of temperature and humidity (Edegar Materials and methods
et al. 2017; Vimala Devi and Prasad, 2001). Insecticide for-
mulations have been developed in the recent past as suspen- Mass production of Bt, Bb and Nr
sion concentrates (SC), which are defined as stable suspen-
sions of active ingredient(s) in a fluid (GIFAP 2012). SC Bt var. kurstaki strain DOR Bt-127 (MTCC 5976/
formulations are easy to measure, convenient to handle and NAIMCC-B-01463) from ICAR-IIOR collection was
less hazardous than pesticide dusts and wettable powders multiplied through solid state fermentation in polypropyl-
(Parmar and Tomar 2010). Hence, improved formulations of ene (PP) covers. Molasses (3.6 g), yeast extract (0.72 g),
the active ingredient are essential to make the bio-pesticide soybean meal (0.72 g), K 2HPO 4 (0.36 g) and KH 2PO 4
formulations comparable to chemical insecticides in terms (0.36 g) were mixed in 250 ml of distilled water and pH
was adjusted to 7.2. This solution was added to 150 g

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of wheat bran taken in a PP cover (35.6 × 50.8 cm, 150 (14.6 ml) and Span-20 (9.6 ml) were used as surfactants.
gauge). The open end of the PP cover was sealed and two Tween-80, Span-20 and mineral oil (196.0 ml) were vor-
ends were cut to give a hole of half inch size. Sponge plugs texed well. The conidial powders were thoroughly mixed
(7.6 × 7.6 cm) were inserted in these holes. This PP cover with the Bt powder and the wetting agent + mineral oil mix-
was autoclaved at 121 °C and 15 psi for 20 min. After ture was added. Formulations were developed in the manner
cooling, the cover was inoculated aseptically with 35 ml adopted for Bt-SC formulation. All the three SC formula-
of overnight seed culture of DOR Bt-127 strain (grown in tions (Bt, Bt + Bb and Bt + Nr) were transferred into high
nutrient broth) by opening one sponge plug and reinsert- density polyethylene bottles (HDPE) and used for further
ing it. The cover was shaken well to mix the contents and studies.
incubated at 31 ± 1 °C for 72 h. The cover was then cut
open, contents were mixed well in 800 ml distilled water Characterization of SC formulations
and filtered through a double layered muslin cloth. The
filtrate was centrifuged at 10,000 rpm for 10 min, super- SC formulations were characterized for various physico-
natant was discarded and the resultant pellet containing Bt chemical and biological parameters as detailed below.
spores and crystals was spread thinly on a sterile polythene
sheet, dried overnight at room temperature. Bt separated Physicochemical characterization
out as dry flakes that were powdered in a mixie jar and the
powder was passed through a sieve of 105 µm to get the Physicochemical characterization of SC formulations was
Bt technical powder (Bt-t) containing 105 µm particles. carried out in accordance with the CIPAC guidelines to
For comparison, Bt-127 strain was also multiplied in tubs determine pH, density and specific gravity, solution stabil-
according to Vimala Devi et al. (2005). ity as persistent foam, pourability, suspensibility and dis-
Bt-t was subjected to milling in a planetary ball mill persion ability (Dobrat and Martijn 1995). Colour of the
for one hour according to Vineela et al. (2017) and the formulations was determined by visible colour method using
resultant powder containing ~ 550 nm particles was used Munsell colour chart (Munsell 1905). Moisture content was
for development of the SC formulations. Bb (ITCC 4513) determined by moisture analyser (OHAUS MB25).
was multiplied on wheat bran based medium (Vimala Devi
and Hari 2009) while Nr (a local isolate) was multiplied Biological characterization
on broken sorghum grains (Vimala Devi 1994). Conidial
powder of these fungi was used for development of the SC Heat viable spore count of Bt in the SC formulations was
formulations. carried out by plating serially diluted suspensions on nutri-
ent agar (NA) plates in three replicates with 2 plates per
Development of SC formulations replicate. Total protein was quantified by Lowry et al. (1951)
while endotoxin was quantified by ELISA (Vineela et al.
Bt-127 SC formulation was developed as described by 2017). CFU of Bb and Nr were determined through plat-
Vineela et al. (2017) with a slight modification of the sur- ing of serially diluted suspensions on potato dextrose agar
factants. Two wetting agents Tween-80 (10.95 ml), Span-20 (PDA) and Saboraud’s maltose agar with yeast extract,
(7.2 ml) and the suspending agent oleic acid (5.9 ml) were respectively. Detection of bacterial and fungal contaminants
taken in a sterile beaker and mixed well using a high-speed was carried out on NA and PDA plates, respectively while
homogenizer at 10,000 rpm for 10 min to get a uniform human pathogens detection was carried out on organism-
mixture. To this mixture, light mineral oil (213.8 ml) was specific chromogenic media from Hi-Media viz. Simmons
added, and the contents of the beaker were mixed well to citrate agar, Salmonella differential agar, Shigella broth base,
obtain a uniform mixture. The above homogenized mixture Vibrio agar for Escherichia coli, Salmonella spp., Shigella
was added to 100 g of milled Bt powder, mixed well with spp. and Vibrio spp. respectively (Vimala Devi and Hari
a glass rod to form a uniform paste without any clumps. 2009).
This mixture was blended in a high-speed homogenizer for
15–20 min at 20,000 rpm to get a freely pourable SC for-
mulation. The final volume of the formulation was 300 ml Electron microscopic studies
weighing 318.0 g.
For development of CFs, milled Bt powder (100 g) was To elucidate the mode of action of the CF of Bt and an
used in formulation development along with pure conidial EPF, electron-microscopic (EM) studies were undertaken
powder of Bb (12.2 g) and Nr (16.0 g) while only Tween-80 at Ruska labs, Sri Venkateswara Veterinary University,

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Hyderabad using third instar larvae of H. armigera treated determined through bioassays at doses ranging 0.5–2.5 mg/
with Bt-t (1 g/l), Bb conidia (1010/l) and Bt + Bb CF (3 ml/l). ml.
CF and Bt treated larvae were dissected under stereo bin- Potencies of Bt-127 and SC formulations against the test
oculars at 12, 24 and 48 h after treatment (HAT), cuticle insects S. litura, H. armigera and A. janata were calculated
and mid gut were fixed in glutaraldehyde for further studies. using the formula:
Scanning electron microscopy (SEM) (Model: JOEL-JSM ( )
Potency (test material) = LC50 of standard∕LC50 of test material
5600) was carried out for cuticles of larvae treated with Bb
and CF at 24, 48 and 72 HAT while transmission electron × potency of standard
microscopy (TEM) (Model: Hitachi, H-7500) was carried
out with mid gut of larvae treated with Bt and Bt + Bb CF.
Field trials

Multi-location field trials were carried out during kharif

Efficacy of SC formulations
(rainy) season 2015–2016 to evaluate the efficacy of the SC
formulations against major lepidopteran pests of castor and
Laboratory bioassays
sunflower. Details of field trials conducted at three hot-spot
locations each for castor and sunflower crops are presented
Efficacy of SC formulations was studied through laboratory
in Table 1. The experiments were conducted in a randomized
bioassays against third instar larvae of H. armigera by diet
complete block design with eight treatments viz. SC for-
surface treatment (Vineela et al. 2017) and for S. litura and
mulations Bt-SC, Bt + Nr CF, Bt + Bb CF @ 3.0 ml/l, Bt-t
A. janata by leaf-spray technique (Vimala Devi et al. 2005)
@ 1.0 g/l, Nr and Bb @ 1 × 1010 spores/l, Profenofos @
in a completely randomized block design (CRBD). Three
1.0 ml/l and untreated control and replicated thrice. All the
replicates with 10 larvae per replicate were maintained for
plots were separated by a 1.8-m-wide buffer area and ade-
each treatment (Vimala Devi and Vineela 2016). Controls
quate care was taken to avoid drift of spray fluid to adjacent
were treated with sterile water. Observations of larval mor-
plots. Formulations were suspended in water and sprayed at
tality were recorded at 24 h intervals till 96 h. LC50 against
the volume rate of 500 l/ha using a hand operated knapsack
H. armigera and A. janata was determined for Btk refer-
sprayer. Water spray was given in untreated control. Standard
ence standard (potency 31,000 IU/mg against H. armigera)
agronomic practices as per local recommendations were fol-
supplied by M/s Wockhardt Life Sciences Ltd., Aurang-
lowed (Padmaiah et al. 2015; Padmavathi et al. 2015). Treat-
abad, India in bioassays with doses ranging 0.25–1.5 and
ments were imposed coinciding with the incidence of early
0.05–0.25 mg/ml, respectively. Since the Btk standard was
instars of lepidopteran pests during vegetative and star-bud
not effective against S. litura, a commercial Btk formulation
stages on castor and sunflower, respectively. Observations
Delfin of known potency (53,000 SU/mg against S. exigua)
on larvae of major lepidopteran pests (S. litura and A. janata
was used as reference standard against S. litura. LC50 was
on castor; H. armigera and T. orichalcea on sunflower) were

Table 1 Details of multi-location field trials on castor and sunflower

Particulars Field trials on castor
Hyderabad (Telangana state, India) Palem (Telangana state, India) Yethapur (Tamil Nadu state, India)

Cultivar DCH-519 DCH-519 DCH-519

Spacing 90 × 60 cm 90 × 60 cm 90 × 60 cm
Plot size 5.4 × 3.6 m 4.5 × 6.0 m 5.4 × 5.4 m
Date of sowing 3 August, 2015 16 July, 2015 24 August, 2015
Date of spraying 1 October, 2015 2 October, 2015 19 November, 2015
Particulars Field trials on sunflower
Hyderabad (Telangana state, India) Nandyal (Andhra Pradesh state, India) Latur (Maharashtra state, India)

Cultivar Morden NDSH-1 Morden

Spacing 60 × 30 cm 60 × 30 cm 60 × 30 cm
Plot size 4.2 × 4.5 m 3.0 × 3.6 m 4.2 × 4.5 m
Date of sowing 19 August, 2015 14 August, 2015 21 August, 2015
Date of spraying 2 October, 2015 30 September, 2015 6 October, 2015

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recorded from five randomly selected plants from each repli- Results
cation one day before and 5 days after spray (DAS) and the
per cent reduction over untreated control was worked out. Mass production of Bt

Statistical analysis Mass production of DOR Bt-127 in PP covers yielded

22 g of Bt powder per cover with spore count of 22.78 Log
Laboratory bioassay data was subjected to probit analysis CFU/g and endotoxin content of 15.7%. Multiplication of
for determination of LC50 using SPSS 16.0 software. Field the Bt strain in plastic tubs yielded 20 g of Bt powder with
data on the number of insect population was transformed a spore count of 15.48 Log CFU/g and endotoxin content of
into square root values and subjected to statistical analy- 11.3%. Thus multiplication of Bt in PP covers resulted in
sis. Following ANOVA, differences between datasets were higher yield, spore count and endotoxin content over mul-
determined using least significant difference at p ≤ 0.05. tiplication in tubs.

Shelf life studies Characterization of SC formulations

Shelf life of the formulations was determined from sam- The colour of formulations falls under brown colour for
ples stored in HDPE bottles at room temperature (30 ± 2 °C) Bt-SC, light greenish brown for Bt + Bb CF and greenish
drawn at 3 monthly intervals for 24 months in three repli- brown for Bt + Nr CF with variations in hue, value and
cates and studied for CFU of Bt, Nr and Bb, fungal and bac- chroma (Table 2). Moisture content in the formulations
terial contaminants and for presence of human pathogens. was very low ranging 0.20–0.42% which is not conducive
for the growth of any contaminants during storage. pH

Table 2 Physicochemical and biological properties of SC formulations

Parameter Bt-SC Bt + Bb CF Bt + Nr CF

Colour Hue: 7.5 YR; Value Hue: 5 YR; Value 4; Hue: 7.5 GY; Value 2; Chroma 2
3; Chroma 4 Chroma 2
Moisture content (%) 0.20 0.35 0.42
pH 6.63 6.25 6.51
Density (g/cm−3) 0.988 0.985 1.015
Suspensibility (%) 91.4 93.9 97.1
Dispersion (%) 93.9 91.3 90.1
Persistant foam (mm) Foam- nil; Oil- 4.3 Foam- nil; Oil- nil Foam- 1.075; Oil- nil
Pourability (%) 1.10 2.25 5.57
CFU (Log CFU/ml) Bt 17.56 (± 0.1) 17.47 (± 0.0) 17.30 (± 0.0)
Bb – 15.10(± 0.2) –
Nr – – 12.20 (± 0.2)
Protein content (mg/ml) 49.3 (± 1.7) 46.3 (± 1.7) 43.3 (± 1.7)
Toxin % by ELISA 5.0 4.7 4.7
Content of bio-control organism (%) 33.3 38.6 37.4
Human pathogens (37 °C) Escherichia coli Nil Nil Nil
Salmonella spp. Nil Nil Nil
Shigella spp. Nil Nil Nil
Vibrio spp. Nil Nil Nil
Other micro-organisms Nil Nil Nil

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Fig. 1 SEM of larval cuticle

(a) Cuticle of Bb treated larva
24 HAT, arrow indicates Bb
conidia 24 HAT (b) Cuticle of
Bb treated larva 72 HAT, arrow
indicates swollen Bb conidia
causing lesions on the cuticle
to enable penetration of germ
tube (c) Cuticle of Bt + Bb CF
treated larva 24 HAT, arrow
indicates swollen Bb conidia
causing lesions on the cuticle
to enable penetration of germ
tube (d) Cuticle of Bt + Bb CF
treated larva 48 HAT, arrow
indicates swollen Bb conidia
with germ tube penetrated into
cuticle

Fig. 2 TEM of mid gut of Bt

treated larvae and Bt + Bb CF
(1.5 kV) (a) midgut epithelium
of untreated larva (b) midgut
epithelium of Bt treated larva
24 HAT (c) midgut epithelium
of Bt + Bb CF treated larva 24
HAT. L-lumen, Mi-microvilli,
N-nucleus, Nm-nuclear mem-
brane, V- vacuole, Gc-goblet
cavity, Cr-chromatin, M-micto-
chondria, Db-dark bodies

of Bt-SC was slightly towards the alkaline neutral range ingredient to the target pests (Vineela et al. 2017). CFU
(6.63) while pH of the CFs, Bt + Bb SC (6.25) and Bt + Nr count of Bt in Bt-SC, Bt + Bb and Bt + Nr CFs were 17.56,
SC (6.51) were in acidic neutral range due to the fun- 17.47 and 17.30 Log CFU/ml, respectively. Spore counts
gal conidia. Density of the formulations (0.985–1.015 g/ of Bb and Nr in the respective CFs were 15.10 and 12.20
cm3) was in the range of water density implying that these log CFU/ml. Active ingredients (biocontrol organisms) in
formulations can be easily suspended in water enabling the Bt-SC, Bt + Bb and Bt + Nr CFs were 33.3, 38.6 and
spraying. SC formulations exhibited high suspensibility 37.4%, respectively with corresponding total protein con-
(91.4 to 97.1%) and dispersion ability (90.1 to 93.9%) in tents of 49.3, 46.3 and 43.3 mg/ml and δ-endotoxin con-
standard hard water, as the particle size of Bt was less tents of 5.0, 4.7 and 4.7% through ELISA. Human patho-
than 1 µm, thereby improving suspension of the formula- gens and other micro-organisms were completely absent
tions in any kind of water and bioavailability of active in the formulations (Table 2).

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Table 3 Laboratory efficacy of SC formulations against S. litura larvae

Test material HATa LC50 (mg) Bt (mg) Bb / Nr (conidia/ml) Confidence limits Potency (SU/mg)b
Lower Upper

Bt-t c 72 1.35 1.35 – 0.96 1.84 29,150

Bt-SC d 2.80 0.95 – 2.42 3.08 13,780
Bt + Bb CF d 3.56 1.21 1.7 × 108 3.29 3.96 11,130
Bt + Nr CF d 2.64 0.91 1010 2.44 3.32 14,840
Delfin e 0.74 – – 0.12 1.04 53,000
Nr f 96 4.08 × 107 – – 1.01 × 107 7.02 × 107 –
a
HAT Hours after treatment;
b
SU- S. exigua units;
c
Bt technical (mg/ml);
d
Bt-SC, Bt + Bb and Bt + Nr CFs (µl/ml);
e
Commercial Bt formulation;
f
N. rileyi

Electron microscopic studies TEM of the midgut of untreated H. armigera larva

showed two intact epithelial cells with nucleus and micro-
SEM of cuticle of H. armigera larva treated with Bb conidia villi. A clear nucleus with nuclear membrane, nucleolus
revealed that the conidia did not show signs of germination and chromatin was observed (Fig. 2a). In Bt treated larva
at 24 HAT (Fig. 1a). Initiation of germination was observed 24 HAT, TEM revealed degeneration of the midgut colum-
at 72 HAT with conidia becoming swollen and elongated nar epithelial cells with microvilli destroyed, vacuolation
with narrowing at one end indicating the beginning of germ in microvilli and cell cytoplasm. However goblet cavities
tube formation. In addition, lesions were observed on the lined with numerous cytoplasmic extensions and mitochon-
cuticle due to digestion of the cuticle for facilitating entry dria were observed which are typical to untreated larvae
of the germ tube (Fig. 1b). In contrast, cuticle of Bt + Bb showing incomplete degeneration (Fig. 2b). TEM of midgut
CF treated larvae 24 HAT revealed the presence of swollen of CF treated larvae revealed complete degeneration of the
and elongated conidia indicating beginning of germ tube columnar epithelial cells with intense vacuolation and round
formulation and lesions on the cuticle (Fig. 1c). By 48 HAT, dense bodies in cytoplasm by 24 HAT (Fig. 2c).
the germ tubes of the conidia were found to penetrate the
cuticle (Fig. 1d).

Table 4 Laboratory efficacy of SC formulations against H. armigera larvae

Test material HATa LC50 (mg) Bt (mg) Bb / Nr (conidia/ml) Confidence limits Potency (IU/mg)b
Lower Upper

Bt-tc 48 0.26 0.26 – − 0.17 0.43 44,020

Bt-SCd 0.84 0.28 – 0.34 1.14 13,640
Bt + Bb CFd 0.76 0.26 3.2 × 107 − 0.25 1.21 15,190
Bt + Nr CFd 0.86 0.29 2.8 × 107 0.13 1.25 13,330
Bt kurstaki standard 0.37 – – 0.13 0.54 31,000
Bbe 96 3.38 × 107 – – 2.09 × 107 6.57 × 107 –
Nrf 8.62 × 107 – – 1.01 × 107 7.2 × 107 –
a
HAT- Hours after treatment;
b
IU- International units;
c
Bt technical (mg/ml);
d
Bt-SC, Bt + Bb and Bt + Nr CFs (µl/ml);
e
B. bassiana and
f
N. rileyi

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Table 5 Laboratory efficacy Test material LC50 (mg) Bt (mg) Bb / Nr Confidence limits Potency (IU/mg)b
of SC formulations against A. (conidia/ ml)
janata larvae at 24 HATa Lower Upper

Bt-tc 0.16 0.16 – 0.14 0.19 38,750

Bt-SCd 0.17 0.06 – 0.14 0.19 36,580
Bt + Bb CFd 0.17 0.06 7.5 × 106 0.16 0.20 36,580
Bt + Nr CFd 0.16 0.05 5 × 108 0.10 0.24 38,750
Bt kurstaki standard 0.20 – – 0.08 0.28 31,000
a
HAT- Hours after treatment;
b
IU- International units;
c
Bt technical (mg/ml);
d
Bt-SC, Bt + Bb and Bt + Nr CFs (µl/ml)

Efficacy of Bt SC formulations Field trials

Laboratory bioassays In field trial on castor, larval population of S. litura and

A. janata were uniform in all the treatments before spray
Results of laboratory bioassays conducted with the SC and treatment differences were non-significant ranging
formulations for determination of LC50 are presented in 83.7–106.7 larvae per plant and 2.8–4.7 larvae per plant,
Tables 2. Bioassays with Bt-t, Bt-SC, Bt + Bb and Bt + Nr respectively. In all the three locations, there was a signifi-
CFs resulted in LC50 values of 1.35, 2.80, 3.56 and 2.64 mg/ cant reduction in S. litura and A. janata population over
ml, respectively at 72 HAT against third instar larvae of the untreated control following sprays of the formulations
S. litura with corresponding potencies of 29,150, 13,780, (Table 6). Per cent reduction over control of S. litura larvae
11,130 and 14,840 SU/mg using Delfin of 53,000 SU/mg at 5 days after spray (DAS) was highest in Bt-SC formulation
potency as the reference standard. Since S. litura was not (97.8 to 99.2%) and Bt + Nr CF (94.5 to 98.7%), which were
susceptible to the Bb isolate used, LC50 was generated only on par with profenofos (96.8 to 99.4%). Significant decrease
with N. rileyi conidia with the value being 4.08 × 107 conidia of 90.1 to 91.8% and 90.1 to 97.0% was also observed for
at 96 HAT (Table 3). Bt contents in the LC50 values of the Bt + Bb CF and Bt-t, respectively. In case of A. janata, the
three formulations viz. Bt-SC, Bt + Bb and Bt + Nr CFs were per cent reduction of larvae at 5 DAS over untreated control
lower than of Bt-t i.e., 0.95, 1.21 and 0.91 mg/ml, respec- was 99.3 to 100%, 86.1 to 98.5% and 90.6 to 94.4% in Bt SC,
tively revealing that the effective Bt requirement for S. litura Bt + Nr and Bt-t treatments respectively, while profenofos
was lowered by milling the Bt powder and formulating into recorded 100% larval reduction.
an SC (Table 3). In field trial on sunflower, incidence of H. armigera and
Bioassays with Bt-t, Bt-SC, Bt + Bb and Bt + Nr CFs T. orichalcea was observed when the crop was in the star
resulted in LC50 values of 0.26, 0.84, 0.76 and 0.37 mg/ml bud stage. Population of the pests before spray was uniform
respectively against third instar larvae of H. armigera at ranging 1.3 to 1.7 and 1.2 to 1.5 larvae per plant, respec-
48 HAT with corresponding potencies of 44,020, 13,640, tively. The per cent decrease of H. armigera larvae over con-
15,190 and 13,330 IU/mg using Wockhardt standard of trol by 5 DAS was highest in Bt-SC (95.0 to 100%) followed
31,000 IU/mg potency as the reference standard (Table 4). by 76.4 to 100% in Bt + Nr CF and 76.4 to 90.7% in Bt + Bb
LC50 of Bb and Nr were 3.38 × 107 and 8.62 × 107 conidia/ while the decrease was 85.7 to 100% in the chemical insec-
ml respectively at 96 HAT (Table 4). However, require- ticide, profenofos. Significant reduction of larval population
ment of the fungi was lowered even by 48 HAT in both of T. orichalcea was observed with Bt-SC (93.5 to 100%)
the CFs to 3.2 × 107 and 2.8 × 107 conidia/ml, respectively and CF of Bt + Nr (87.9 to 100%), on par with profenofos
for Bb and Nr revealing compatibility of the EPF with Bt. treatment (100% reduction over untreated control) (Table 7).
Bioassays with Bt-t, Bt-SC, Bt + Bb and Bt + Nr CFs
resulted in LC50 values of 0.16, 0.17, 0.17 and 0.16 mg/ Shelf‑life studies
ml, respectively against third instar larvae of A. janata at
24 HAT with corresponding potencies of 38,750, 36,580, Log CFU value of Bt spores in Bt-SC was 17.08 at zero day
36,580 and 38,750 IU/mg using Wockhardt standard of and did not decrease till 18 months of storage. Further, Log
31,000 IU/mg potency as the reference standard (Table 5). CFU values decreased slightly to 16.88 and 16.62 by 21
Since A. janata was not susceptible to both Bb and Nr, and 24 months, respectively (Fig. 3a). A similar trend was
LC50 values could not be generated. observed with respect to viability of Bt spores in the CFs.

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 World Journal of Microbiology and Biotechnology
Table 6 Field efficacy of Bt based SC formulations against major lepidopteran pests of castor (kharif, 2015–16)
Treatment Hyderabad Palem Yethapur
S. litura A. janata S. litura A. janata S. litura A. janata
b
Larvae/ % Reduction Larvae/ plant % Reduction Larvae/ plant % Reduction Larvae/ plant % Reduction Larvae/ plant % Reduction Larvae/ plant % Reduction
planta

Bt-SC @ 0.7 (1.0) 99.2 0.1 (0.8) 99.3 1.2 (1.3) 98.5 0.0 (0.7) 100 1.8 97.8 0.0 100
3 ml/l (1.5) (0.7)

(2020) 36:78
Bt + Nr CF@ 1.2 (1.3) 98.7 0.2 (0.8) 97.9 2.9 (1.9) 96.3 0.1 (0.8) 98.5 4.5 94.5 0.4 86.1
3 ml/l (2.2) (0.9)
Bt + Bb CF@ 7.8 (2.8) 91.8 0.5 (1.0) 95.0 7.8 (2.9) 90.1 0.9 (1.2) 81.6 6.9 91.5 0.9 67.6
3 ml/l (2.7) (1.2)
Bt-t@ 1 g/l 9.4 (3.0) 90.1 0.5 (1.0) 94.4 2.4 (1.7) 97.0 0.3 (0.9) 94.3 7.4 91.0 0.3 90.6
(2.8) (0.9)
Nr@ 1 × 1010 32.8 (5.7) 65.6 4.4 (2.2) 53.5 28.9 (5.4) 63.4 0.9 (1.2) 81.6 12.1 85.3 1.1 62.7
spores /l (3.5) (1.3)
Bb@ 1 × 1010 40.6 (6.4) 57.4 3.5 (2.0) 63.4 36.3 (6.1) 54.1 1.4 (1.4) 70.4 35.5 56.6 1.5 48.8
spores/l (5.9) (1.4)
Profenofos @ 0.6 (1.0) 99.4 0.0 (0.7) 100 1.1 (1.3) 98.6 0.0 (0.7) 100 2.6 96.8 0.0 100
1 ml /l (1.8) (0.7)
Control 95.3 (9.8) – 9.5 (3.2) – 79.1 (8.9) – 4.7 (2.3) – 81.9 – 2.9 –
(9.1) (1.8)
CD (p ≤ .05) 0.84 – 0.15 – 0.84 – 0.17 – 0.89 – 0.20 –

Values in parenthesis are square root transformed values (√ x + 0.5);

a
No. of larvae at 5 days after spray;
b
Per cent reduction over control

Page 9 of 14
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Page 10 of 14
Table 7 Field efficacy of Bt based SC formulations against major lepidopteran pests of sunflower (kharif, 2015–16)
Treatment Hyderabad Latur Nandyal
H. armigera H. armigera T. orichalcea H. armigera T. orichalcea
Larvae/ planta % Reductionb Larvae/ plant % Reduction Larvae/ plant % Reduction Larvae/ plant % Reduction Larvae/ plant % Reduction

Bt-SC @ 2 ml/l 0.0 100 0.0 100 0.0 100 0.1 95.0 0.1 93.5
(0.7) (0.7) (0.7) (0.8) (0.8)
Bt + Nr CF @ 3 ml/l 0.0 100 0.1 89.8 0.0 100 0.3 76.4 0.1 87.9
(0.7) (0.8) (0.7) (0.9) (0.8)
Bt + Bb CF @ 3 ml/l 0.2 80 0.3 76.4 0.2 70.2 0.1 90.7 0.3 74.8
(0.8) (0.9) (0.8) (0.8) (0.9)
Bt-t @ 1 g/l 0.3 73 0.4 68.5 0.1 80.6 0.1 95.0 0.1 93.5

World Journal of Microbiology and Biotechnology

(0.9) (1.0) (0.8) (0.8) (0.8)
Nr @ 1 × 1010 spores/l 0.7 33 0.4 68.5 0.3 59.7 0.5 62.1 0.3 69.2
(1.1) (1.0) (0.9) (1.0) (0.9)
Bb @ 1 × 1010 spores/l 0.5 53 0.2 84.3 0.3 50.8 0.6 57.1 0.5 50.5
(1.0) (0.8) (0.9) (1.1) (1.0)
Profenofos @ 1 ml/l 0.0 100 0.1 89.8 0.0 100 0.2 85.7 0.0 100
(0.7) (0.8) (0.7) (0.8) (0.7)
Control 1.0 – 1.3 – 0.7 – 1.4 – 1.1 –
(1.2) (1.3) (1.1) (1.4) (1.3)
CD (p ≤ .05) 0.10 – 0.19 – 0.10 – 0.24 – 0.12 –

Values in parenthesis are square root transformed values (√ x + 0.5);

a
No. of larvae at 5 days after spray;
b
Per cent reduction over control

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World Journal of Microbiology and Biotechnology (2020) 36:78 Page 11 of 14 78

and Nr. This is also corroborated with the EM observations of

H. armigera larvae treated with Bt + Bb CF. Our TEM studies
pertaining to midgut of Bt treated larvae are in tune with the
findings of El-Ghany et al. (2015), who reported degeneration
of columnar epithelial cells with loss of nuclear membrane
coupled with degeneration of chromatin, vacuolation, destruc-
tion of microvilli and loss in organization of goblet cavities
through histopathological studies of H. armigera. The SEM
studies on cuticular infection in H. armigera larvae by Bb
conidia in the CF revealed acceleration of germination prob-
ably aided by formulating in oil since the conidia are lipophilic
in nature. Formulations of the lipophilic conidia of B. bassi-
ana and M. anisopliae in oil increase the effectiveness enable
faster spread of the inoculum on the insect surface into the
intersegmental regions where the cuticle is thin thereby result-
ing in faster germination, infection and kill (Prior et al. 1988;
Bateman et al. 1993). Conidia of EPF have been formulated in
vegetable and petroleum-based oils for increasing the infectiv-
ity (Nahar et al. 2003; Moore, Bateman et al. 1993). Bateman
et al. (1993) reported superior performance of M. flavoviride
formulated in cottonseed oil to water based suspensions. Prior
et al. (1988) reported 36 times higher infectivity of B. bassiana
conidia to the cocoa weevil Pantorhytes plutus when formu-
lated in coconut oil. Studies pertaining to mode of action of
CF of Bt and B. bassiana are lacking in literature. Present
studies in this direction reveal a simultaneous infection of the
Fig. 3 Viability of (a) Bt (b) Nr and Bb in SC formulations during larva with Bb through the cuticle and degeneration of midgut
storage at room temperature columnar epithelial cells due to Bt within 24 HAT.
Our laboratory bioassays show that Bt requirement
was highest for S. litura followed by H. armigera and A.
Log CFU value of Bt in Bt + Nr CF was 16.82 at zero day janata which is indicative of susceptibility in the order A.
and remained till 18 months followed by a slight decrease to janata > H. armigera > S. litura. SC formulations of Bt sin-
16.72 and 16.62 at 21 and 24 months respectively. Similarly, gly and in combination with Bb and Nr were superior to
log CFU value of Bt in Bt + Bb CF was 16.99 at zero day either Bt or EPFs used singly rendering faster kill at lower
and remained till 18 months followed by a slight decrease doses of the active ingredients in the formulations. These
to 16.82 and 16.62 at 21 and 24 months respectively. Log studies are also indicative of a synergistic effect of Bb and
CFU value of Nr in Bt + Nr CF was 11.20 at zero day that Nr when used along with Bt. Bt might be applied in con-
remained till 12 months and decreased slightly to 10.00 by cert with other insect pathogens, including EPF. However,
24 months of storage. Log CFU value of Bb in Bt + Bb CF little is known with regard to interactions between Bt and
also followed a similar trend with a value of 14.10 at zero day EPF (Navon 2000). Increased efficacy of a combination of
that remained till 12 months and showed a slight decrease to Bt and B. bassiana when compared to Bt or B. bassiana
12.75 by 24 months of storage (Fig. 3b). In addition, neither used singly has been reported against several insects viz.,
human pathogens nor other bacterial/fungal contaminants Earias vittella, Malacosama neustria, T. ni, Tuta absoluta
were observed in the 24 month shelf-life period. etc. (Sandner and Cichy 1967; Ali et al. 2015; Wang et al.
2014; Sayed and Behle 2017; Tsoulnara and Port 2016). Our
laboratory bioassays too have revealed positive effects of
the combined use of Bt with Bb and Nr. The pesticide use
Discussion pattern in the current day situations has led to resistance
build-up by pests and pesticide residues, which necessitates
This study constitutes the first successful attempt at develop- use of safer pesticides with different modes of action. With
ment of storable CFs of Bt with Bb and Nr. The fact that the laboratory-selected resistance to Bt demonstrated in many
formulations had an extended shelf-life of 24 months at room pests and field-evolved resistance to Bt documented in the
temperature is a testimony to the compatibility of Bt with Bb diamondback moth, P. xylostella and cabbage looper, T. ni,

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 78 Page 12 of 14 World Journal of Microbiology and Biotechnology (2020) 36:78

adaptation by pests is now considered the biggest threat to It is being increasingly recognized that formulation type
the long-term success of Bt (Tabashnik et al 1998; Ferre and holds the key to effective pest management. Long shelf life
Van Rie 2002; Janmaat and Myers 2003). Laboratory and and reliable efficacy, which are affected by moisture, are the
field bio-efficacy studies revealed that Bt and Bb and Nr are two basic impediments for commercialization of microbial
compatible when used in combination. In addition, the CFs pesticides. Liquid SC formulations of Bt singly and in com-
gave higher and faster kill of the pest when compared to Bt bination with EPF were successfully developed in this study
or the entomopathogenic fungi used singly. Since the modes for management of major lepidopteran pests with efficacy
of action of the two pathogens are diverse with ingestion for on par with the chemical insecticide profenofos. Milling
Bt and infection through cuticle for B. bassiana, simultane- lowered the effective dose requirement of Bt. The CFs were
ous attack by the two pathogens through different routes more effective than Bt and EPF used singly. The formula-
could debilitate the host rapidly and thereby mitigate the tions had an extended shelf-life of 24 months at room tem-
problem of resistance development in the insect. perature. Thus the SC formulations fulfil the criteria essen-
The route of infection of B. bassiana and the primary site tial for effective pest management and commercialization.
of activity of the δ-endotoxin of Bt are spatially separate
within an insect. Few reports exist on synergistic interac- Acknowledgements The authors thank Director, ICAR-IIOR for pro-
viding the facilities to carry out the work. The authors also gratefully
tion of Bt and B. bassiana in combination as tank-mix spray acknowledge the financial support under the ICAR network project on
suspensions for management of insect pests in field viz. the “Application of Microorganisms in Agriculture and Allied Sectors”.
European corn borer and Colorado Potato beetle (Lewis and The logistic support provided by AICRP (Oilseeds) centres (Latur,
Bing 1991; Wraight and Ramos 2005). Yethapur and Palem) is also gratefully acknowledged.
Tank-mix formulations may not produce reliable results
always since effectiveness is based on parameters like host Compliance with ethical standards
range of both microbial formulations, effective dose of each
Conflict of interest No potential conflict of interest was reported by
formulation and ratios for their combined use, shelf-life of the authors.
each formulation, etc. Hence our approach has been towards
development of a ready-to-use storable CF whose effective
field dose against target pests can be determined. The CFs References
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Affiliations

P. S. Vimala Devi1 · P. Duraimurugan1 · K. S. V. Poorna Chandrika2 · V. Vineela1 · P. P. Hari1

2
* P. S. Vimala Devi Crop Production Section, ICAR-Indian Institute of Oilseeds
[email protected] Research, Rajendranagar, Hyderabad 500 030, Telangana,
India
1
Crop Protection Section, ICAR-Indian Institute of Oilseeds
Research, Rajendranagar, Hyderabad 500 030, Telangana,
India

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