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Bacillus thuringiensis as microbial biopesticide: uses and application for

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Kumar et al. Egyptian Journal of Biological Pest Control
https://doi.org/10.1186/s41938-021-00440-3
(2021) 31:95
Egyptian Journal of
Biological Pest Control

REVIEW ARTICLE Open Access

Bacillus thuringiensis as microbial

biopesticide: uses and application for
sustainable agriculture
Pradeep Kumar1* , Madhu Kamle1, Rituraj Borah1, Dipendra Kumar Mahato2 and Bharti Sharma3

Abstract
Background: Bacillus thuringiensis (Bt) has been used in agriculture for a long time because of its insecticidal
proteins which make it a valuable environment-friendly biopesticide. However, its use is not only limited to
insecticidal properties. Current and previous studies indicate its potential as a biofertilizer for promoting plant
growth, the development of transgenic plants, and others. It is the presence of δ-endotoxins, especially cry protein,
which attributes the insecticidal property to the bacteria. Besides, there are some vegetative and secreted
insecticidal proteins that exert their toxic activity towards specific species.
Main body of abstract: The present review briefly provides an overview of the Bt uses and application as a
biocontrol agent against insect pest for sustainable agriculture. Historical development of Bt as biocontrol,
classification of various cry proteins, their mechanisms of actions against different insect-pest, and incorporation of
cry genes in the plant for developing transgenic Bt plants such as Bt cotton, potato, and maize. Applications of Bt
as biofertilizer and the various bioformulations as biopesticide are also described.
Short conclusion: Uses of harmful pesticides and chemical cause various health issues and environmental
problem; therefore, the Bt served as the best alternative to overcome the above issue. Also, we aim to explore the
potential as plant growth-promoting potential and solubilization of minerals and the uses as a biofertilizer, keeping
the high specificity and environmental safety of Bt. Its various formulations are commercially available and
considered an efficient alternative to chemical pesticides.
Keywords: Bacillus thuringiensis, Biopesticide, Biofertilizer, Environment, Human health

Background results in pore formation in the cell membrane of the

Bacillus thuringiensis (Bt) is a gram-positive, spore- gut, followed by paralysis of the gut and eventually caus-
forming bacteria known for its ability to produce crystal ing the death of the larvae (Vachon et al. 2012). Cry pro-
proteins (Cry). Cry protein is believed to be toxic to teins target a diverse range of insect species primarily of
many insects and that is why Bt is used as a microbial the order Lepidoptera (butterflies and moths), Coleop-
insecticide for improved resistance in plants by genetic tera (beetles and weevils), and Diptera (flies and mosqui-
modification (Salehi Jouzani et al. 2008). When the in- toes); however, there are reports regarding their toxicity
sect larvae ingest the proteinaceous crystal, the digestive towards hymenopterans (wasps and bees) and nema-
enzymes present in the gut activates the toxin and todes (Domínguez-Arrizabalaga et al. 2020) (Fig. 1). The
use of Bt as biopesticide are efficient like chemical pesti-
cides, therefore, forms a part of the Integrated Pest Man-
* Correspondence: [email protected]
1
Applied Microbiology Laboratory, Department of Forestry, North Eastern agement strategy that prefers non-chemical pesticides
Regional Institute of Science and Technology, Nirjuli, Arunachal Pradesh for pest control (Matyjaszczyk 2018).
791109, India
Full list of author information is available at the end of the article

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Kumar et al. Egyptian Journal of Biological Pest Control (2021) 31:95 Page 2 of 7

Fig. 1 Applications of Bacillus thuringiensis for sustainable agriculture

Besides the crystal proteins, Bt also synthesizes vegeta- later it became an insecticidal agent (Berliner 1915;
tive insecticidal protein (Vip) and secreted insecticidal Mazier et al. 1997). The first commercial production of
protein (Sip), which are biodegradable and affect specific Bt was reported in 1938 in France and sold under the
targets, especially species of Coleoptera and Lepidoptera name Sporeine (Brar et al. 2006). A study by Angus
(Chakroun et al. 2016). Since the use of chemical pesti- (1956) demonstrated that the insecticidal action of Bt
cides results in the development of resistance by the in- was due to certain crystalline protein inclusions formed
sects and is harmful to both humans and the during sporulation. Later, a study by Zakharyan et al.
environment, the focus is to develop effective biopesti- (1976) noted the presence and involvement of plasmid
cides using Bt. The current research focuses on develop- in crystal formation in Bt. Then, González et al. (1982)
ing genetically engineered crops to express insecticidal used plasmid curing technique and affirmed the
property from Bt (Bravo et al. 2011). Moreover, it is a localization of genes that code for crystal proteins on
host-specific bioinsecticide; hence, there is limited transmissible plasmids. However, the first cloning and
chances of negatively affecting other organisms (Jain characterization of these crystal protein-encoding genes
et al. 2016). Further, its role as an insecticide has ex- that showed toxicity towards tobacco hornworm larvae
plored its potential in promoting plant growth (Armada were performed by Schnepf and Whiteley (1981) from
et al. 2016), bioremediation of heavy metals (Aceves- plasmid DNA of Bt subsp. kurstaki HD-1. This resulted
Diez et al. 2015), and production of polymers (Singh in rapid cloning of other cry genes, which ultimately led
et al. 2013). to the expansion of genetic engineering towards Bt
transgenic plants and finally, Bt cotton reached the mar-
Main text ket in 1996 (Shelton et al. 2002).
History
Bacillus thuringiensis (Bt) was first discovered in 1901 Bt toxin-diversity and classification
by the Japanese biologist, Shigetane Ishiwatari, before Bacillus thuringiensis strains produce 2 types of toxins,
which it was believed that an undescribed bacterium is cry and cyt proteins, which are also known as delta-
responsible for causing disease in silkworms (Ishiwata endotoxins (Bravo et al. 2007). These are synthesized by
1901). In 1911, Bt was rediscovered by Ernst Berliner, in the bacteria during its stationary phase when sporulation
Germany, as the causative agent of a disease known as occurs. They are produced as parasporal crystalline in-
Schlaffsucht in Mediterranean flour moth caterpillars, clusions which get activated by the proteolytic action in-
Ephestia kuehniella Zell. Bt was named after the prov- side the insect’s gut after ingestion. These proteins are
ince Thuringia where the infection was found. Hence, Bt not known to be toxic against invertebrates and are
was initially considered a risk for silkworm rearing but called parasporins (PS) (Ohba et al. 2009). Besides this,
Kumar et al. Egyptian Journal of Biological Pest Control (2021) 31:95 Page 3 of 7

certain toxins are produced during the vegetative phase, derived from Lysinibacillus sphaericus (Cry 35 and
termed as vegetative insecticidal proteins (Vip) and se- Cry36) (Berry 2012) and the ETX group contains toxins
creted insecticidal protein (Sip). The Sip (Sip1Aa1 ob- that show resemblance to toxins produced by Clostrid-
tained from Bt strain EG2158) is the only known protein ium epsilon (Alves et al. 2014). Their structure shows
to show toxicity against coleopteran larvae. These are se- fold like aerolysin, a toxin produced by Aeromonas
creted by the bacteria in the culture medium (Palma hydrophila and involved in pore formation (Knapp et al.
et al. 2014). 2010). The second types are the cytolytic (cyt) genes en-
The main types are the crystal (Cry) genes which en- coding non-specific cytolytic factor toxins that can aug-
code intestinal cry protein (ICP) toxins and have a wide ment the Cry toxins for enhanced insecticidal property
spectrum of insecticidal activity. There are more than especially against dipterans (Butko 2003). This helps in
800 cry genes classified under 75 families (Cry1 to Cry decreasing the resistance of insects towards cry proteins.
75), 40 cyt genes grouped in 3 families (Cyt 1, cyt2, For instance, a study by Soberón et al. (2013) observed
cyt3), and 146 Vip genes categorized under 4 families that Cyt1Aa toxin produced by Bt helped in decreasing
(Vip 1 to Vip4) (Crickmore et al. 2020). Initially, these the resistance developed by the larvae of Culex quinque-
toxins were classified into 4 classes based on their amino fasciatus against Cry 4 and Cry11Aa toxin. Moreover,
acid sequence homology and insecticidal properties. The there is evidence indicating a wider range of toxicity
CryI toxins are toxic to Lepidopterans (butterflies and (Guerchicoff et al. 2001).
moths), CryII genes are toxic to lepidopterans and dip-
terans (flies and mosquitoes), CryIII genes are toxic to Mechanism of toxicity (Bt as insecticide)
coleopterans (beetles and weevils), and CryIV genes are The most approved and widely accepted primary mode
toxic to dipterans (Höfte and Whiteley 1989). An add- of action of the Bt toxin is the lysis of epithelial cells in
itional class, CryV, was added for the nematode-active the midgut of the insect. The toxin acts from the exter-
toxins (Sick et al. 1994). Similarly, secreted toxins show ior of the cells and enters into the plasma membrane
their toxicity to a particular order of insects. Vip1 and but it does not enter the cytoplasm. The earliest effect
Vip2 are binary toxins and toxic to coleopterans and he- shows the blabbing and swelling of columnar cells and
mipterans (Sattar and Maiti 2011), Vip3, on the other subsequent lysis of the columnar cells. The effect seen
hand, is single-chain and show insecticidal activity to- on goblet cell is slower when compared (Liu et al. 2018).
wards lepidopterans (Estruch et al. 1996). The nomen- The steps involved in the action in lepidopteran are ex-
clature also determines the similarity in the amino acid plained in brief through. On the ingestion of the toxin in
sequence between the cry genes on 4 levels for their dis- lepidopteran in the first 5 min, the glucose uptake is in-
tinguished. However, this naming method did not con- creased in the gut cells and the histopathology takes
sider the biological activity. In this system, each protoxin place in the gut cells (Knowles 1994). After 5 min, the
acquired a name consisting of the mnemonic Cry (cyt, mid-gut gets paralyzed. Within 10 min, the columnar
Vip, or Sip) and divided into various ranks. For example, cells get swelled up and the columnar microvilli get bub-
at level 1, each gene in the family cry1 shares at least bled. The histopathology takes place in the goblet cells.
45% similarity and will be categorized under cry1. The Subsequently, the pH is increased in the blood and de-
secondary rank introduces a capital letter, say, Cry1A, creased in the lumen. Then, within 30 min, the activity
which indicates towards the sequences with the similar- of potassium ion is increased in the gut cells which lead
ity between 45 and 78%. This is followed by tertiary to the decrease in the blood potassium ion level. The
rank, say, Cry1Aa, which increases the sequence identity leucine and the glucose level start to decrease leading
up to 95% but not less than 78%. At the fourth level, the gut cells to metabolically break down. After 30 min,
quaternary rank, say, Cry1Aa2, clones that share at least the cells undergo lysis and start to shed from the base-
95% sequence identity are distinguished (Crickmore ment membrane. Within 1 to 7 h, general paralysis takes
1998; 2020). place. After 1 to 3 days, it dies to starvation (Liu et al.
Another method of classification by Bt toxin Nomen- 2018). Different types of insecticidal proteins are pro-
clature Committee uses the homology approach and duced by Bt. About 950 different types of toxins are
forms 3 groups based on phylogenetics-3 domain cloned from about 74 groups of cry, 3 groups of vip, and
groups, bin group, and epsilon toxin (ETX) group. The 3 groups of cyt protein (Crickmore et al. 2020).
cry toxins belonging to three-domain group differ in Li et al. (2011) studied the toxicity behavior of Cry1Ac
their amino acid sequence but share a common three- and Cry3Aa against the pea aphid, Acyrthosiphon pisum
domain structure. The structure includes a perforating (Harris). Cry1Ac was observed to be efficiently hydro-
domain, central domain, and galactose-binding domain, lyzed by aphid stomach membrane-associated cysteine
each playing a specific role during toxin activation proteases (CP) whereas Cry3Aa was processed incom-
(Pardo-Lopez et al. 2013). The bin group contains toxins pletely and degraded partially. Cry1Ac could bind to the
Kumar et al. Egyptian Journal of Biological Pest Control (2021) 31:95 Page 4 of 7

aphid gut epithelium but bioassays showed low aphid Further, Muddanuru et al. (2019) developed transgenic
toxicity. In addition, the competition assays using un- castor (Ricinus communis L.) expressing the Cry1Aa of
labelled Cry1Ac and GalNAc and in vitro binding of Bt against lepidopteran insect pests. Bt toxins of the
biotinylated-Cry1Ac to gut BBMVs (brush border mem- Cry1I class show dual specificity for insects of Coleop-
brane vesicles) confirmed binding specificity besides gly- tera and Lepidoptera. Berretta et al. (2020) evaluated the
can mediation of Cry1Ac binding. Further, Zhang et al. toxicity of a Cry1Ia protein from an Argentinian Bt
(2019) revealed Vip3Aa11 and Vip3Aa39 proteins present- strain against agricultural pests of Tenebrionidae, Curcu-
ing 39 amino acid differential sites, sharing 95.06% amino lionidae, Noctuidae, and Tortricidae. The protein was
acid sequence similarity and effective against some Lepi- toxic to Cydia pomonella, and Rachiplusia nu; however,
doptera insect larvae. They also revealed 558 midgut genes Alphitobius diaperinus, Anthonomus grandis, and Spo-
expressed differentially in Vip3Aa11-M-A and 65 midgut doptera frugiperda were not susceptible. Besides this,
genes expressed differentially in Vip3Aa39-M-A. The val- Singh et al. (2021) revealed the potential of Cry protein
idation for the sensitivity of these two Vip3Aa proteins to isolated from Bt to hamper the growth of S. litura, G.
trypsin and their binding properties to Agrotis ipsilon mid- malonella, B. cucurbitae, and C. pipens larvae, thereby,
gut BBMV based on transcriptome profiling showed simi- the potential for plant breeders to produce resistant
lar sensitivity to trypsin and both proteins could bind to crops.
Agrotis ipsilon midgut BBMV along with competitive In recent years, it has been seen that the cry genes
binding between them (Zhang et al. 2019). with resistant properties against nematodes and other
insects have not been approved at the commercial level.
Bt and genetically modified plants Along with the advantages of GM crops, there lie poten-
The recent advancements in modern biotechnology es- tial risks to both human and animal health. It is assumed
pecially in the field of agriculture are observed globally. that consumption of GM crops can cause the potential
The global average of the biotech crops has reached development of diseases that are resistant to antibodies.
191.7 million hectares in the last 22 years (ISAAA 2019). Although, any kind of threat or long-term effect has not
These are the fastest adapted crops that have now been reported (Bawa and Anilakumar 2013). Also, the
reached to 70 countries after commercialization (Pocket insecticidal proteins of Bt are utilized for formulations of
2018). Traits of Bt such as pest resistant and herbicide spore-crystal complexes along with their genes for pro-
are most extensively used in plant genetic engineering. ducing several GM crops. Despite the variability of the
Bt toxin genes are widely used in many crops for enhan- Cry proteins, it is still crucial to search for toxins with a
cing pest-resistant quality (Jouzani et al. 2017). Since the broad-spectrum application so that alternatives are avail-
commercialization of Bt crops, approximately 198 var- able to address the issues of insect resistance (Lazarte
ieties have been produced lining up 8 plants including et al. 2021).
potato, soybean, corn, maize, eggplant, cotton, rice, pop-
lar, tomato, and maize. Recently, maize has gained the Bt as a biofertilizer
largest number of approvals (137) from over 35 coun- There are several bacterial species such as Klebsiella,
tries (ISAAA 2016). During sporulation, Bt forms aggre- Pseudomonas, Rhizobium, Caulobacter, and Azotobacter
gated crystals that are cry toxins, which is an insecticidal are known for enhancing plant growth (Kumar et al.
protein with a parasporal body and crystalline structure. 2016). Bt, although known for its insecticidal activity, is
These toxins are effective on specific species like Nema- also a plant growth-promoting bacterium. The indirect
toda, Lepidoptera, Coleoptera, Diptera, and Hymenop- effect can be observed as it decreases the infection by in-
tera (Abbas 2018). Genetically modified (GM) crops sects but certain studies indicate towards its direct effect
consist of one or more than one vip or cry genes. Cot- by colonization in legumes which result in an increase in
ton, corn, and potato have 42, 115, and 30 variants, re- nodulation and thus growth of the plant (Qi et al. 2016).
spectively, and they are the most extensively approved A study by Seshadri et al. (2007) showed that species
Bt-GM crop (ISAAA 2016). There are in total seven of Bacillus (Bt and B. sphaericus) can solubilize phos-
anti-lepidopteran vip and cry genes used for enhancing phates and thereby supporting plant growth. This is
the resistance to Lepidopteran genes namely cry1A.105, beneficial since the mineral is present in a high amount
cry1Ab, cry1F, cry1Ac, cry2Ae, cry2Ab, and vip3A. in soil, but its uptake is limited because of its undis-
About 111 Bt varieties of anti-coleopteran genes are ap- solved form. Similarly, dissolution of iron is also difficult,
proved, among which some are anti-lepidopteran pests. and plant uses the siderophore-iron complex for its up-
Two genes namely cry34Ab1–cry35Ab1 and cry3A are take. Siderophores are produced by microorganisms and
used in nearly 34 and 60 GM crops, respectively, to pro- bind with iron and aid in their uptake. A study by Wil-
duce varieties that are resistant to coleopteran pests son et al. (2006) indicated the production of catecholate-
(Jouzani et al. 2017). type siderophores by Bt strain ATCC 33679, which
Kumar et al. Egyptian Journal of Biological Pest Control (2021) 31:95 Page 5 of 7

bound with high affinity to iron and improved its supply being engineered for the development of Bt-GM crops,
to plants. Improved growth in plants has been shown in which are resistant to pests and insects but without
a study by Mishra et al. (2009) where they investigated harmful effects on humans, the environment, and benefi-
that Bacillus thuringiensis-KR1 along with Bradyrhizo- cial organisms. The use of Bt formulations in form of
bium japonicum increased the number of root nodules, sprays or liquid suspensions on a commercial scale is in-
improved root volume, root weight, and shoot weight in creasing. Since the toxin is produced continuously for a
soybean. longer time, it is comparatively better than chemical pes-
Another study by Mishra et al. (2009) showed that co- ticides in terms of application and field management as
inoculation of Bt with another bacteria Rhizobium legu- well as cost and efficiency. Further research is underway
minosarum in pea plants enhanced the nodulation and for exploring other useful benefits of Bt. Besides, being
increased the dry weight of pea. The colonization of Bt used as a microbial pesticide, Bt has also been used as a
is not only observed in lentils but is also reported by plant growth-promoting bacterium for enhancing the
Pindi et al. (2014) which showed enhanced growth in productivity of crops. However, more focused and in-
cabbage and cotton respectively. Similarly, when co- depth research is still required to ensure its effectiveness
inoculated with mycorrhiza, Armada et al. (2016) re- and long-term effects on the crops, environment, and
ported an improvement in drought tolerance and an in- human health. In addition, studies at the cellular and
crease in oxidative metabolism in plants of Lavandula. A molecular levels using animal models are vital to fully
recent study by Bandopadhyay (2020) involved the use understand its mode of action for usage as medicine.
of Bt A5-BRSC as a charcoal-based biofertilizer for 2 Additionally, advanced research on the stability, quan-
years on Abelmoschus esculentus and evaluated its po- tity, and crop-specific application dosages are equally es-
tential in improving plant growth considering both nu- sential to ensure the safety of sustainable agriculture.
tritional and morphological characteristics of the plant.
Abbreviations
He observed a significant increase in shoot height, leaf Bt: Bacillus thuringiensis; Cry: Crystal; Vip: Vegetative insecticidal protein;
diameter, weight of both fruit and seed, and length of Sip: Secreted insecticidal protein; DNA: Deoxyribonucleic acid; ICP: Intestinal
roots. Along with this, nearly about 70% increase in pro- cry protein; ETX: Epsilon toxin
tein content of leaf, more than 60% increase in soluble Acknowledgements
sugar content, and about 30% increase in protein con- All authors are highly grateful to the authority of the respective department
tent of the pod were observed with the use of Bt as a and Institution for their support in conducting this research. Author (P.K.)
would like to thank the DST-SERB (ECR/2017/001143) for their financial
biofertilizer. These evident studies thus prove that using support
Bt is not only helpful as an insecticide but also has po-
tential as a biofertilizer that is also beneficial for the Authors’ contributions
P.K. conceived and designed the manuscript. M.K., D.K.M., B.S., and P.K. wrote
growth of the plant. the manuscript. R.B. helped in revision. P.K. critically reviewed the manuscript
and did the required editing. All authors have read and approved the
Bt formulations manuscript.
Though Bt serves as an excellent biopesticide, it faces Funding
some problem during storage and stability. The formula- Author, P.K., would like to thank the Early Career Research Award, DST-SERB,
tion will not only stabilize the biopesticide, but it will Government of India (file no. ECR/2017/001143), for their financial support to
carry out this research work.
also solve some other problems such as application and
handling of the product, protect from environmental ef- Availability of data and materials
fects, and enhance activities of agents like microbes in Not applicable.
the field (Derua et al. 2018). The most important object- Declarations
ive is the widespread and commercialization of these
biopesticides globally. The present day’s formulation has Ethics approval and consent to participate
Not applicable.
additional benefits of speed killing, dry and wet (mois-
ture), wind, rains, and characteristics of plants such as Consent for publication
the chemistry of leaves (Brar et al. 2006). Not applicable.

Competing interests
Conclusion The authors declare that they have no competing interests.
Bacillus thuringiensis (Bt) is used as a potential biopesti-
Author details
cide to control pests in agriculture due to its various in- 1
Applied Microbiology Laboratory, Department of Forestry, North Eastern
secticidal proteins. Thereby, the insecticidal toxins Regional Institute of Science and Technology, Nirjuli, Arunachal Pradesh
derived from Bt make it a novel organism with immense 791109, India. 2CASS Food Research Centre, School of Exercise and Nutrition
Sciences, Deakin University, Burwood, VIC 3125, Australia. 3Department of
potential for agricultural development. With the ad- Dairy Science and Food Technology, Institute of Agricultural Sciences,
vancement in technology, its genetic content is now Banaras Hindu University, Varanasi 221005, India.
Kumar et al. Egyptian Journal of Biological Pest Control (2021) 31:95 Page 6 of 7

Received: 26 March 2021 Accepted: 10 June 2021 Domínguez-Arrizabalaga M, Villanueva M, Escriche B, Ancín-Azpilicueta C,
Caballero P (2020) Insecticidal activity of Bacillus thuringiensis proteins against
Coleopteran pests. Toxins 12(7):430. https://doi.org/10.3390/toxins12070430
Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG (1996) Vip3A, a
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