Bacillus thuringiensis (Bt) is more than a special agent for biological control of pests

doi:10.15406/jabb.2022.09.00282

Journal of

eISSN: 2572-8466

Applied Biotechnology & Bioengineering

Review Article Volume 9 Issue 2

Bacillus thuringiensis (Bt) is more than a special agent for biological control of pests

Juan Manuel Sanchez–Yanez,¹

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Jose Luis Rico,² Gerard Ulíbarri^3,4

¹ Microbiología Ambiental, Instituto de Investigación en Química y Biología, México
²Catálisis, Departamento de Ingeniería Química, México
³Department of Chemistry and Biochemistry, Laurentian University, Canada
⁴Translational Research Institute, México

Correspondence: Juan Manuel Sanchez–Yanez, Microbiologia Ambiental, Instituto de Investigación en Química y Biologia, México

Received: February 08, 2022 | Published: March 23, 2022

Citation: : Sánchez–Yáñez JM, Rico JL, Ulíbarri G. Bacillus thuringiensis (Bt) is more than a special agent for biological control of pests. J Appl Biotechnol Bioeng. 2022;9(2):33-39. DOI: 10.15406/jabb.2022.09.00282

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Abstract

Bacillus thuringiensis (Bt) is a bacterial genus known mainly for its capability to synthesize, in addition to spores, a parasporal body or crystal (δ––endotoxin), comprised of proteins which are toxic to pest insects, i.e., Coleoptera, Diptera and Lepidoptera, and other orders. As well as plant pathogenic nematode and many other applications as endophytic plant growth promoting bacteria, or cleaning enviornmental form some chemicals polluting agents, and even in human medicine for cancer prevention. The Bt coexists in a close relationship with insects to which eventually infects and poisons. It is likely that these unfavorable changes occurred during the evolutionary pathway in the interactions Bacillus–insect, which resulted in the present biochemical diversity between these species. The goal of this minireview is to show role of Bt in comparation with other genus Bacillus and explain why Bt is of interest for biological control of pests in conventional, protected and organic agriculture regarding other biotechnological applications to fully exploit the beneficial potential of Bt.

Keywords: pest biological control, endophytic plant promoting bacteria, bioremediation, cancer prevention

Introduction

The genus and specie called Bacillus thuringiensis (Bt) is well known as an entomotoxic and/or entomopathogenic bacteria used against insect pests (IP) in agriculture and public health, today a key tool in Biological Control of the following orders: Coleoptera, Diptera, Hymenoptera, Homoptera, Orthoptera and Phthiraptera.^1–30 Bt is applied to biological control of important human diseases which vectors are insects (VHD) belonging to the Diptera order with following genus involved in: malaria; Anopheles, dengue; Aedes, yellow fever; Aedes and Haemogogus, chikungunya; Aedes and zika; Aedes. The spores and crystals of Bt israelensis can intoxicate the young stage of larvae of a wide variety of Diptera, especially when solar radiation is minimal, Bt it is considered an ecological option to control these vector insects, although the lack of sufficient research on the persistence of Bt israelensis spores and crystals to the various aquatic conditions where oviposit mosquitoes limits a widespread application that ensures the disappearance of these vector insects for diseases of global importance.^1,3,4,18,28 Also, there is evidence that certain varieties of Bt can intoxicate and infect arthropods classified as part of the spiders, since some genera and species attack agricultural crops of commercial value, research in this regard is promising with the expectation that new varieties of Bt will be apply for the biological control of mites in substitution of chemical pesticides.^5,20,21 One of the most interesting proprieties of Bt in human epidemiology its genetic capacity²⁹ for controlling snail of genera Milax gagates vector of the parasitic human and animal helminths such as Angiostrongglus costaricensis, Angiostoma margaretae and Cryptosporidium parvum. As well as the genus of snail Biomphalaria, vector of a trematode Schistosoma mansoni of importance in public health in the world.⁶In addition, it has been reported that Bt crystals are also an alternative for the biological control of nematodes of veterinary importance such as the ruminant genera Haemonchus, Teladorsagia, Nippostrongylus and Ancylostma through a toxic action of these Bt crystals on the juvenile forms of these nematodes.^7,8 There is evidence that certain varieties of Bt synthesize crystals that are toxic to genera and species of phytopathogenic nematodes as the well–known Meloidogyne hapla responsible for millions of losses in agriculture, especially because chemical control causes serious problems for human health and environmental pollution.^8,19 Beside this fact related to pest control. Another application of Bt for sustainable agriculture its ability to invade the root system and phylloplane²⁵ of various families of plants of commercial value as an endophyte, in which it can optimize the dose of mineral nitrogen fertilization, also is able to solubilize of phosphates to facilitate mineral absorption that allows plants to plants face soils with problems of elements necessary for agricultural production. While it can act in consortium with other genera and species of microorganisms that promote plant growth: actinomycetes, with mycorrhizal fungi and bacteria for different domestic crops.^9–12

In terms of human health dealing with preventing several types of cancer there are reports that showed that parasporal crystals of Bt have anticancer activity, as well as the well–known MOLT–4, A549 associated to human lung cancer including HeLa (human uterus cervix cancer) cells, crystal from some strains Bt showed different toxicity like A1190 and A1462) were capable to destroy leukemic and normal T–cells, now those Bt proteins are called parasporin–2, 3, and 4 (PS2, PS3, PS4) to apply in treatment for preventing several types of cancer.^13–15

Bt is regarding an ecological tool for recovering soil polluted by some pesticides, in that sense Bt is capable of synthesizing probiotics: organic substances and enzymes²¹ that induce the mineralization of environmental toxins. This is the case of an organophosphate pesticide known as chlorpyrifos used in the control of domestic and agricultural insect pest. In the soil, chlorpyrifos at a relatively high concentration of 70 mg/Kg of soil can be effectively eliminated over a period of 80 days by the application of a Bt probiotic, analyzed by high pressure liquid chromatography, which showed that this probiotic induced the mineralization of chlorpyrifos, which allowed the reuse of the soil without causing negative environmental collateral effects.¹⁶ Bt exert their toxicity by crystal proteins (δ––endotoxins) or other toxins synthesized during its growth and sporulation.^14,17–21 Such proteins are very specific and safe to mammals’ toxicity tests of Bt protein crystals for humans and animals support that the ingestion of these proteins does not allow them to solubilize, the Cry proteins rapidly degrade after being ingested. While both humans and animals lack specific recognition receptors for Cry proteins, necessary for them to be active. Consequently, no toxicity was detected when the dose for laboratory animals was oral. While there is no evidence of infection in rats, no skin damage was detected from the Cry proteins of Bt.^13,22–24

Mode of action of Bt crystals on insect pests

In contrast, consumption of the crystal proteins by insects causes disturbances and eventually their death.^{26,27,31–34} It is believed^35–39that the first step of the Bt toxin is the recognition of target receptor or molecules located within or on the membrane surface of epithelial cells of the midgut of a susceptible insect. Although, the specific binding of Cry toxin proteins to brush border membrane vesicle (BBMV) of midgut has been reported,^{11,28,39,40–44} less is known about the binding of Vip toxins. Recently, it has been suggested that the Vip3Aa toxin is bound specifically to a 48–kDa protein present at the BBMV prepared from the midgut epithelial cells of black cutworm (Agrotis ipsilon) larvae.^43,44 Biopesticides based on Bt were first commercialized in France in 1930 to destroy insects in agricultural crops as well as urban plagues, i.e., Coleoptera, Diptera and Lepidoptera.^{22,23,34,45–47} However, Bt also infects and kills beneficial insects such as: Bómbix mori, Danaus plexippus.⁴⁸ There exists a large variety of insects which are not considered IPs neither VHDs,^{18,22,23,30,41,49} Biopesticides and anti–pest crops have been developed by genetically modifying the crops with Bt genes, resulting in thousands of Bt strains isolated and studied.^{4,20,21,50–54} However, the evolution of resistance of pests to the insecticidal proteins from Bt produced by transgenic crops is drastically reducing the effectiveness of this method.^50,55–61 Indeed, in 2005 was reported that 1 out of 13 insect species were vulnerable to transgenic crops, whereas eight years later, 5 out of 13 had developed resistance to Bt modified crops.^44,62 The sporulation of Bt could happen in soil, water or air,⁶³ however, it is generally accepted that it occurs inside the guts of insects^{18,21,40,42,49,64}and inside other animals such as birds.⁶⁵ Although Bt is considered a typical bacterium, its sporulation in different environmental conditions may not occur, in contrast to Bacillus cereus.^30,66 In addition, a physiological difference between Bt and other members of the Bacillus species is the genetic stability to continuous sporulation. There are several reports in the literature which mention that Bt easily loses its sporulation capacity, under certain environmental conditions, providing strong evidence of a difference in the extrachromosomal DNA compared to other species.^67,68 Furthermore, the spores of the Bt are sensible to light and susceptible to some very specific pesticides, which is a drawback of the spore–crystal complex for foliar application.⁶⁹ However, Bt spores have been isolated from leaves of domestic and wild plants,⁷⁰albeit susceptible to UV irradiation^71–77It is still unclear whether these spores were transported by wind, insects or were merely isolated cases.⁷³In another study, Ignoffo et. al. reported in 1978⁷⁸ that the viability was lost in less than 48h after aspersion of Bt spores on young leaves of corn and bean plants⁷⁹which was attributed to solar irradiation.⁷⁸ It is believed that the Bt spores lack certain proteins that protect them against physical elements, this protection is a typical and basic characteristic of most spores of the Bacillus genus.^73,80 The lack of these proteins, partially explains the poor stability of the Bt spores to solar.^32,81 In contrast, the spores of Bacillus cereus reproduce and are resistant to solar irradiation, an advantage that is utilized to control pathogenic bacteria on leaves of commercial plants.^65,74

Ecology of Bacillus thuringiensis

The spores of Bt are also sensitive to heavy metals: aluminium (Al), cupper (Cu), lead (Pb), etc, another aspect of the physiology of the Bt is its incapacity to sporulate under nutritional stress conditions^30,80contrary to other members of the genus Bacillus that do not require mineral medium or glucose in order to induce sporulation.⁷³ Research using Bt showed that the genetically modified serotypes from Northern Mexico, as well as the Howard Raylord Deuchman strands from the U.S. Anti–doping Agency,⁸¹ were incapable of growing in a mineral media, or in soil extracts used as growing media. In contrast, when B. cereus is introduced in the same media, it rapidly sporulates as most known saprobic Bacillus species do.⁸²

It is generally accepted that Paenibacillus (before called: Bacillus) is a genus capable of atmospheric nitrogen fixation, which was expected since the species Bacillus subtilis, B. lincheniformis, and B. cereus grow and survive in soils with deficiency in.^30,80 These findings suggest that nutrients of Bt are obviously distinct to the rest of the Bacillus genus, which supports the hypothesis of its relationship and biochemical dependency on insects. As typical soil bacteria, Bt is incapable to synthetize vitamins or other compounds essential for its survival, in contrast to B. cereus or other species of this genus.^{5,30,35,83,84}

Autecology of spore and crystals of Bacillus thuringiensis

Table 1 shows some of the diversity of Bt–based pesticides used to control pest insects in agricultural crops, stored grains, apiculture, forestry and urban areas.^37,82,85 Several North American³⁰ and European companies produce a variety of Bt formulations, geared to create different environmental, natural and artificial, applications. Some of these formulations are encapsulated in order to avoid the rapid denaturalization of the δ–endotoxin, such as MVP and M–One Plus commercialized by Mycogen. These products are efficient at Lopelluis controlling insects on infected leaves, as well as in aquatic environments.⁸⁶ The aforementioned unjustified the use of Bt–transgenic seeds in agriculture due to unknown risks associated with ecological factors and human health.^{20,29,35,85,87,88} However, many reports have showed that crystal of proteins of Bt remnants from any application are binding to some physicochemical soil properties such as clay and humic acids by doing so these proteins has potential hazard to beneficial native animals like insects still not well known, in that sense research must be done in order to establish environmental rules to regulate any Bt–transgenic to prevent ecological disaster.^89–93

Commercial product	Company	*Bacillus thuringiensis* subspecies/varieties	Crop to protect
Bactospeine	Duphar	Kurstaki	Vegetables, fruits stored grains
Biobit	Novo labs
Condor	Ecogen
Cutlass	Ecogen
Dipel	Abbot labs
Javelin	Sandoz
Larvo Bt	Knoll labs
MVP		Encapsulated Kurstaki
Certain	Sandoz	Aizawai	Honey producers
Skeetal	Novo labs	Israelensis	Toxic to mosquitoes
Teknar	Sandoz
Vectorbac	Abbot labs
M-One	Sandoz	San Diego	Stored grains, potatoes
			and leaves
Trident	Sandoz	Tenebrionis	Tree leaves
Tfoil	Ecogen	Tenebrionis
M-One plus	Mycogen	Kurstaki
		San Diego encapsulated toxin

Table 1 Some commercial biopesticides based in Bacillus thuringiensis species^{20,24–26,28–30,32,35,37,38,46,51,81,82,85–93,96}

Table 2 shows the biochemical and physiological differences of Bt compared to other typical microorganisms of the genus Bacillus. Unlike other species commonly found in soils, such as Bacillus cereus⁸³ and Bacillus subtilis, Bt is unable to synthesize growth factors, explaining its incapability of growing in mineral agar, or in the absence of combined organic nitrogen.^30,94,95 In addition to the normal tolerability of the spores of the Bacillus species to radiation, chemical agents, and thermal stability, the spores of the Bacillus stearothermophilus resist temperatures higher than 100 ºC, a genetic/biochemical property absent in the spores of Bt^37,82,84,96or applied for bioremediation soil polluted by petroleum products mixing Bt with Lysinbacillus sphaericus.⁹⁷

Characteristics	Response of *B. thuringiensis*	Response of representative *Bacillus* spp
Need of a growth factor	Positive	Negative
Induction to sporulation	No required	Stress required
Resistance of spores to UV	Negative	Positive
Growing in a mineral medium	Negative	Positive
Genetic stability	Negative	Positive

Table 2 Biochemical and physiological differences of the Bacillus thuringiensis compared to other typical microorganisms of the genus Bacillus^{30,35,37,64,71,75,77,82–84,94–97,99,105,108,112–115–117}

Table 3 presents selected features of some representative species of the Bacillus genus

The interactions between insects and bacteria are well documented.⁹⁸ Natural and forced symbioses are often observed: mutualism in the case of Bacillus sp and bees^{35,48,67,80,88,99–103}parasitism is observed with Bacillus papillae, B.larvae, B.anthracis, B. sphaericus (now Lysinbacillus sphaericus), and B.cereus.^{35,46,66,104,105}

Species	Reproduction
Bacillus spp	Only in symbiosis with bees
Bacillus papillae and B. larvae	Obligate parasite in insects only
Bacillus anthracis.	Facultative parasite in animals and humans
Bacillus sphaericus and Bt	Facultative parasite in mosquitos (Diptera)
Bacillus cereus	Saprobe in soils and rhizosphere
Paenibacillus polymixa (before Bacillus polymxya)	Asymbiotic nitrogen bacteria in soils

Table 3 Selected reproduction features for representative species of the Bacillus genus ^{21,32,35,46,48,66,67,71,75–77,80,82,88,98–105,112–117}

Table 4 presents a hypothetic response of Bt to the environment. It is assumed in this representation that the spores of Bt do not germinate under physicochemical stress conditions. Under this scenario, the Bt would only survive due to the interactions with insects^27,35,38,106and plants as endophyte.¹⁰⁷ Furthermore, since the spores are unstable to the environment,^{32,71,72,75,77,94,108–111}continuous sporulation of Bt would be only possible under favorable conditions: good oxygenation, simple carbon–source provision, suitable pH and absence of life–threatening physicochemical agents.^82,100,102

Condition	Response	Consequence
Not germinating spores	lost viability	Not survival
	(due to nutritional requirements)
Crystal protein	Mineralization	Inactivation (hours)
	(except when adhered to sand)
Vegetative sporulating forms	No response to environmental stimulation	Dormancy of vegetative cells (but not for sporulating)

Table 4 Hypothetic possible responses of Bacillus thuringiensis to abiotic and biotic natural environmental conditions^{27,32,35,38,65,68,71,72,75–77,82,94,100–102,106–112}

Final comments

This short note shows that Bacillus thuringiensis behaves differently compared to other species of the Bacillus genus. Bt is considered as an entomotoxic and entomopathogenic bacteria, and its toxins are used to control insect pest, however, more research is needed to elucidate the effect of the remaining spores to continue the infection of other insects. It is also worth commenting that although Bt is genetically related to other species such as Bacillus cereus, B. subtilis, B. licheniformis and another Bacillus, their metabolism is strongly different. For instance, the B. cereus, B. subtilis, B. licheniformis species can grow in a medium with mineral nitrogen without B vitamins, or the Paenibacillus polymyxa species can even use atmospheric N₂ as a source of nitrogen, Bt can only grow in presence of organic nitrogen compounds and B vitamins. This last requirement for growth on Bt makes the interaction with the insect even stronger, ending the insect’s offspring life from within as soon as Bt sporulation conditions are optimal. Finally, characterization of other toxins produced by Bt strains will further improved the application of these metabolites in other areas, however, the side effects resulted from the use of these toxins to the environment must necessarily be addressed.^112–116

Acknowledgments

To project 2.7 of the CIC–UMSNH (2022), Morelia, Mexico, and to BIONUTRA, SA de CV, Maravatio, Mexico, for the support on this publication.

Author contributions: Conceptualization JMSY and GU.; data curation, JMSY, JL and GU; writing—original draft preparation, JMSY and GU.; writing—review and editing, JMSY and GU; Final version JMSY. All authors have read and agreed to the published version of the manuscript.