|Spores and bipyramidal crystals of Bacillus thuringiensis morrisoni strain T08025|
Bacillus thuringiensis (or Bt) is a Gram-positive, soil-dwelling bacterium, commonly used as a biological pesticide. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well on leaf surfaces, aquatic environments, animal feces, insect-rich environments, and flour mills and grain-storage facilities. It has also been observed to parasitize other moths such as Cadra calidella—in laboratory experiments working with C. calidella, many of the moths were diseased due to this parasite.
During sporulation, many Bt strains produce crystal proteins (proteinaceous inclusions), called δ-endotoxins, that have insecticidal action. This has led to their use as insecticides, and more recently to genetically modified crops using Bt genes, such as Bt corn. Many crystal-producing Bt strains, though, do not have insecticidal properties. The subspecies israelensis is commonly used for control of mosquitoes and of fungus gnats.
Taxonomy and discovery
In 1902, B. thuringiensis was first discovered in silkworms by Japanese sericultural engineer Ishiwatari Shigetane (石渡 繁胤). He named it B. sotto, using the Japanese word sottō (卒倒, 'collapse'), here referring to bacillary paralysis. In 1911, German microbiologist Ernst Berliner rediscovered it when he isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars in Thuringia (hence the specific name thuringiensis, "Thuringian"). B. sotto would later be reassigned as B. thuringiensis var. sotto.
In 1976, Robert A. Zakharyan reported the presence of a plasmid in a strain of B. thuringiensis and suggested the plasmid's involvement in endospore and crystal formation. B. thuringiensis is closely related to B. cereus, a soil bacterium, and B. anthracis, the cause of anthrax; the three organisms differ mainly in their plasmids.:34–35 Like other members of the genus, all three are aerobes capable of producing endospores.
There are several dozen recognized subspecies of B. thuringiensis. Subspecies commonly used as insecticides include B. thuringiensis subspecies kurstaki (Btk), subspecies israelensis (Bti) and subspecies aizawa.
Mechanism of insecticidal action
Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal δ-endotoxins (called crystal proteins or Cry proteins), which are encoded by cry genes. In most strains of B. thuringiensis, the cry genes are located on a plasmid (cry is not a chromosomal gene in most strains).
Cry toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles) and Hymenoptera (wasps, bees, ants and sawflies), as well as against nematodes. Thus, B. thuringiensis serves as an important reservoir of Cry toxins for production of biological insecticides and insect-resistant genetically modified crops. When insects ingest toxin crystals, their alkaline digestive tracts denature the insoluble crystals, making them soluble and thus amenable to being cut with proteases found in the insect gut, which liberate the toxin from the crystal. The Cry toxin is then inserted into the insect gut cell membrane, paralyzing the digestive tract and forming a pore. The insect stops eating and starves to death; live Bt bacteria may also colonize the insect, which can contribute to death. The midgut bacteria of susceptible larvae may be required for B. thuringiensis insecticidal activity.
It has been shown that a small RNA called BtsR1 can silence the Cry toxin when outside the host by binding to the RBS site of the Cry5Ba toxin transcript and inhibiting its expression. The silencing results in increased ingestion by C. elegans and is relieved inside the host, resulting in host death.
In 1996 another class of insecticidal proteins in Bt was discovered: the vegetative insecticidal proteins (Vip; InterPro: IPR022180). Vip proteins do not share sequence homology with Cry proteins, in general do not compete for the same receptors, and some kill different insects than do Cry proteins.
In 2000, a novel subgroup of Cry protein, designated parasporin, was discovered from non-insecticidal B. thuringiensis isolates. The proteins of parasporin group are defined as B. thuringiensis and related bacterial parasporal proteins that are not hemolytic, but capable of preferentially killing cancer cells. As of January 2013, parasporins comprise six subfamilies: PS1 to PS6.
Use of spores and proteins in pest control
Spores and crystalline insecticidal proteins produced by B. thuringiensis have been used to control insect pests since the 1920s and are often applied as liquid sprays. They are now used as specific insecticides under trade names such as DiPel and Thuricide. Because of their specificity, these pesticides are regarded as environmentally friendly, with little or no effect on humans, wildlife, pollinators, and most other beneficial insects, and are used in organic farming; however, the manuals for these products do contain many environmental and human health warnings, and a 2012 European regulatory peer review of five approved strains found, while data exist to support some claims of low toxicity to humans and the environment, the data are insufficient to justify many of these claims.
New strains of Bt are developed and introduced over time as insects develop resistance to Bt, or the desire occurs to force mutations to modify organism characteristics or to use homologous recombinant genetic engineering to improve crystal size and increase pesticidal activity or broaden the host range of Bt and obtain more effective formulations. Each new strain is given a unique number and registered with the U.S. EPA and allowances may be given for genetic modification depending on "its parental strains, the proposed pesticide use pattern, and the manner and extent to which the organism has been genetically modified". Formulations of Bt that are approved for organic farming in the US are listed at the website of the Organic Materials Review Institute (OMRI) and several university extension websites offer advice on how to use Bt spore or protein preparations in organic farming.
Use of Bt genes in genetic engineering of plants for pest control
The Belgian company Plant Genetic Systems (now part of Bayer CropScience) was the first company (in 1985) to develop genetically modified crops (tobacco) with insect tolerance by expressing cry genes from B. thuringiensis; the resulting crops contain delta endotoxin. The Bt tobacco was never commercialized; tobacco plants are used to test genetic modifications since they are easy to manipulate genetically and are not part of the food supply.
In 1995, potato plants producing CRY 3A Bt toxin were approved safe by the Environmental Protection Agency, making it the first human-modified pesticide-producing crop to be approved in the US, though many plants produce pesticides naturally, including tobacco, coffee plants, cocoa, and black walnut. This was the 'New Leaf' potato, and it was removed from the market in 2001 due to lack of interest. For current crops and their acreage under cultivation, see genetically modified crops.
In 1996, genetically modified maize producing Bt Cry protein was approved, which killed the European corn borer and related species; subsequent Bt genes were introduced that killed corn rootworm larvae.
The Bt genes engineered into crops and approved for release include, singly and stacked: Cry1A.105, CryIAb, CryIF, Cry2Ab, Cry3Bb1, Cry34Ab1, Cry35Ab1, mCry3A, and VIP, and the engineered crops include corn and cotton.:285ff
Corn genetically modified to produce VIP was first approved in the US in 2010.
In India, by 2014, more than seven million cotton farmers, occupying twenty-six million acres, had adopted Bt cotton.
Dietary risk assessment
Concerns over the safety of consumption of genetically-modified plant materials that contain Cry proteins have been addressed in extensive dietary risk assessment studies. While the target pests are exposed to the toxins primarily through leaf and stalk material, Cry proteins are also expressed in other parts of the plant, including trace amounts in maize kernels which are ultimately consumed by both humans and animals.
Animal models have been used to assess human health risk from consumption of products containing Cry proteins. The United States Environmental Protection Agency recognizes mouse acute oral feeding studies where doses as high as 5,000 mg/kg body weight resulted in no observed adverse effects. Research on other known toxic proteins suggests that toxicity occurs at much lower doses, further suggesting that Bt toxins are not toxic to mammals. The results of toxicology studies are further strengthened by the lack of observed toxicity from decades of use of B. thuringiensis and its crystalline proteins as an insecticidal spray.
Introduction of a new protein raised concerns regarding the potential for allergic responses in sensitive individuals. Bioinformatic analysis of known allergens has indicated there is no concern of allergic reactions as a result of consumption of Bt toxins. Additionally, skin prick testing using purified Bt protein resulted in no detectable production of toxin-specific IgE antibodies, even in atopic patients.
Studies have been conducted to evaluate the fate of Bt toxins that are ingested in foods. Bt toxin proteins have been shown to digest within minutes of exposure to simulated gastric fluids. The instability of the proteins in digestive fluids is an additional indication that Cry proteins are unlikely to be allergenic, since most known food allergens resist degradation and are ultimately absorbed in the small intestine.
Ecological risk assessment
Ecological risk assessment aims to ensure there is no unintended impact on non-target organisms and no contamination of natural resources as a result of the use of a new substance, such as the use of Bt in genetically-modified crops. The impact of Bt toxins on the environments where transgenic plants are grown has been evaluated to ensure no adverse effects outside of targeted crop pests.
Persistence in environment
Concerns over possible environmental impact from accumulation of Bt toxins from plant tissues, pollen dispersal, and direct secretion from roots have been investigated. Bt toxins may persist in soil for over 200 days, with half-lives between 1.6 and 22 days. Much of the toxin is initially degraded rapidly by microorganisms in the environment, while some is adsorbed by organic matter and persists longer. Some studies, in contrast, claim that the toxins do not persist in the soil. Bt toxins are less likely to accumulate in bodies of water, but pollen shed or soil runoff may deposit them in an aquatic ecosystem. Fish species are not susceptible to Bt toxins if exposed.
Impact on non-target organisms
The toxic nature of Bt proteins has an adverse impact on many major crop pests, but ecological risk assessments have been conducted to ensure safety of beneficial non-target organisms that may come into contact with the toxins. Widespread concerns over toxicity in non-target lepidopterans, such as the monarch butterfly, have been disproved through proper exposure characterization, where it was determined that non-target organisms are not exposed to high enough amounts of the Bt toxins to have an adverse effect on the population. Soil-dwelling organisms, potentially exposed to Bt toxins through root exudates, are not impacted by the growth of Bt crops.
Multiple insects have developed a resistance to B. thuringiensis. In November 2009, Monsanto scientists found the pink bollworm had become resistant to the first-generation Bt cotton in parts of Gujarat, India - that generation expresses one Bt gene, Cry1Ac. This was the first instance of Bt resistance confirmed by Monsanto anywhere in the world. Monsanto responded by introducing a second-generation cotton with multiple Bt proteins, which was rapidly adopted. Bollworm resistance to first-generation Bt cotton was also identified in Australia, China, Spain, and the United States. Additionally, the Indian mealmoth, a common grain pest, is also developing a resistance since B. thuringiensis has been extensively used as a biological control agent against the moth. Studies in the cabbage looper have suggested that a mutation in the membrane transporter ABCC2 can confer resistance to B. thuringiensis.
Several studies have documented surges in "sucking pests" (which are not affected by Bt toxins) within a few years of adoption of Bt cotton. In China, the main problem has been with mirids, which have in some cases "completely eroded all benefits from Bt cotton cultivation". The increase in sucking pests depended on local temperature and rainfall conditions and increased in half the villages studied. The increase in insecticide use for the control of these secondary insects was far smaller than the reduction in total insecticide use due to Bt cotton adoption. Another study in five provinces in China found the reduction in pesticide use in Bt cotton cultivars is significantly lower than that reported in research elsewhere, consistent with the hypothesis suggested by recent studies that more pesticide sprayings are needed over time to control emerging secondary pests, such as aphids, spider mites, and lygus bugs.
Similar problems have been reported in India, with both mealy bugs and aphids although a survey of small Indian farms between 2002 and 2008 concluded Bt cotton adoption has led to higher yields and lower pesticide use, decreasing over time.
There are controversies around GMOs on several levels, including whether making them is ethical, whether food produced with them is safe, whether such food should be labeled and if so how, whether agricultural biotech is needed to address world hunger now or in the future, and more specifically to GM crops—intellectual property and market dynamics; environmental effects of GM crops; and GM crops' role in industrial agricultural more generally. There are also issues specific to Bt transgenic crops.
The most publicised problem associated with Bt crops is the claim that pollen from Bt maize could kill the monarch butterfly. The paper produced a public uproar and demonstrations against Bt maize; however by 2001 several follow-up studies coordinated by the USDA had asserted that "the most common types of Bt maize pollen are not toxic to monarch larvae in concentrations the insects would encounter in the fields." Similarly, B. thuringiensis has been widely used for controlling Spodoptera littoralis larvae growth due to their detrimental pest activities in Africa and Southern Europe. However, S. littoralis showed resistance to many strains of B. thuriginesis and were only effectively controlled by few strains.
Wild maize genetic mixing
A study published in Nature in 2001 reported Bt-containing maize genes were found in maize in its center of origin, Oaxaca, Mexico. In 2002, paper concluded, "the evidence available is not sufficient to justify the publication of the original paper." A significant controversy happened over the paper and Nature's unprecedented notice.
A subsequent large-scale study in 2005 failed to find any evidence of genetic mixing in Oaxaca. A 2007 study found the "transgenic proteins expressed in maize were found in two (0.96%) of 208 samples from farmers' fields, located in two (8%) of 25 sampled communities." Mexico imports a substantial amount of maize from the U.S., and due to formal and informal seed networks among rural farmers, many potential routes are available for transgenic maize to enter into food and feed webs. One study found small-scale (about 1%) introduction of transgenic sequences in sampled fields in Mexico; it did not find evidence for or against this introduced genetic material being inherited by the next generation of plants. That study was immediately criticized, with the reviewer writing, "Genetically, any given plant should be either non-transgenic or transgenic, therefore for leaf tissue of a single transgenic plant, a GMO level close to 100% is expected. In their study, the authors chose to classify leaf samples as transgenic despite GMO levels of about 0.1%. We contend that results such as these are incorrectly interpreted as positive and are more likely to be indicative of contamination in the laboratory."
Colony collapse disorder
As of 2007, a new phenomenon called colony collapse disorder (CCD) began affecting bee hives all over North America. Initial speculation on possible causes included new parasites, pesticide use, and the use of Bt transgenic crops. The Mid-Atlantic Apiculture Research and Extension Consortium found no evidence that pollen from Bt crops is adversely affecting bees. According to the USDA, "Genetically modified (GM) crops, most commonly Bt corn, have been offered up as the cause of CCD. But there is no correlation between where GM crops are planted and the pattern of CCD incidents. Also, GM crops have been widely planted since the late 1990s, but CCD did not appear until 2006. In addition, CCD has been reported in countries that do not allow GM crops to be planted, such as Switzerland. German researchers have noted in one study a possible correlation between exposure to Bt pollen and compromised immunity to Nosema." The actual cause of CCD was unknown in 2007, and scientists believe it may have multiple exacerbating causes.
Some isolates of B. thuringiensis produce a class of insecticidal small molecules called beta-exotoxin, the common name for which is thuringiensin. A consensus document produced by the OECD says: "Beta-exotoxins are known to be toxic to humans and almost all other forms of life and its presence is prohibited in B. thuringiensis microbial products". Thuringiensins are nucleoside analogues. They inhibit RNA polymerase activity, a process common to all forms of life, in rats and bacteria alike.
- Madigan MT, Martinko JM, eds. (2005). Brock Biology of Microorganisms (11th ed.). Prentice Hall. ISBN 978-0-13-144329-7.[page needed]
- du Rand, Nicolette (July 2009). Isolation of Entomopathogenic Gram Positive Spore Forming Bacteria Effective Against Coleoptera (PhD Thesis). Pietermaritzburg, South Africa: University of KwaZulu-Natal. hdl:10413/1235.[page needed]
- Cox PD (1975). "The influence of photoperiod on the life-cycles of Ephestia calidella (Guenee) and Ephestia figulilella Gregson (Lepidoptera: Phycitidae)". J. Stored Prod. Res. 11 (2): 77. doi:10.1016/0022-474X(75)90043-0.
- Kumar PA, Sharma RP, Malik VS (1996). "The insecticidal proteins of Bacillus thuringiensis". Advances in Applied Microbiology. 42: 1–43. doi:10.1016/s0065-2164(08)70371-x. ISBN 9780120026425. PMID 8865583.
- Roh JY, Choi JY, Li MS, Jin BR, Je YH (April 2007). "Bacillus thuringiensis as a specific, safe, and effective tool for insect pest control". Journal of Microbiology and Biotechnology. 17 (4): 547–59. PMID 18051264.
- "Bti for Mosquito Control". EPA.gov. US EPA. 2016-07-05. Retrieved 28 June 2018.
- "Fungus Gnats Management Guidelines--UC IPM". ipm.ucanr.edu. University of California Integrated Pest Management.
- New Innovative Pesticides. EPA. 1977. p. 61.
In 1915 the bacterium was re-examined and named Bacillus sotto. [...] At about the same time, Beriner was isolating the organism
- Natural Enemies in the Pacific Area: Biological Control. Fukuoka Entomological Society. 1967. p. 99.
"Sotto" in Japanese means "sudden collapse" or "fainting", and "sotto" of Bacillus thuringiensis var. sotto derives its name from the "sotto" disease.
- Reardon RC, Dubois NR, McLane W (1994). Bacillus thuringiensis for managing gypsy moth: a review. USDA Forest Service.
Mediterranean flour moths, Ephestia (=Anagasta) kuehniella (Zeller), that were found in stored grain in Thuringia
- Steinhaus E (2012). Insect Pathology: An Advanced Treatise. Elsevier. p. 32. ISBN 978-0-323-14317-2.
Bacillus sotto Ishiwata [→] Taxonomic reassignment: Bacillus thuringiensis var. sotto Ishiwata. [Heimpel and Angus, 1960]
- Zakharyan R.A et. el. (1979). "Plasmid DNA from Bacillus thuringiensis". Microbiologiya. 48 (2): 226–9. ISSN 0026-3656.
- Cheng TC, ed. (1984). Pathogens of invertebrates: application in biological control and transmission mechanisms. p. 159. ISBN 978-0-306-41700-9.
- Økstad OA, Kolstø A (2011). "Chapter 2: Genomics of Bacillus Species". In Wiedmann M, Zhang W (eds.). Genomics of Foodborne Bacterial Pathogens, 29 Food Microbiology and Food Safety. Springer Science+Business Media, LLC. pp. 29–53. doi:10.1007/978-1-4419-7686-4_2. ISBN 978-1-4419-7685-7.
- Nogales E, Downing KH, Amos LA, Löwe J (June 1998). "Tubulin and FtsZ form a distinct family of GTPases". Nature Structural Biology. 5 (6): 451–8. doi:10.1038/nsb0698-451. PMID 9628483.
- Jenkins C, Samudrala R, Anderson I, Hedlund BP, Petroni G, Michailova N, Pinel N, Overbeek R, Rosati G, Staley JT (December 2002). "Genes for the cytoskeletal protein tubulin in the bacterial genus Prosthecobacter". Proceedings of the National Academy of Sciences of the United States of America. 99 (26): 17049–54. Bibcode:2002PNAS...9917049J. doi:10.1073/pnas.012516899. PMC 139267. PMID 12486237.
- Yutin N, Koonin EV (March 2012). "Archaeal origin of tubulin". Biology Direct. 7: 10. doi:10.1186/1745-6150-7-10. PMC 3349469. PMID 22458654.
- Larsen RA, Cusumano C, Fujioka A, Lim-Fong G, Patterson P, Pogliano J (June 2007). "Treadmilling of a prokaryotic tubulin-like protein, TubZ, required for plasmid stability in Bacillus thuringiensis". Genes & Development. 21 (11): 1340–52. doi:10.1101/gad.1546107. PMC 1877747. PMID 17510284.
- Circkmore N. "Bacillus thuringiensis toxin nomenclature". Archived from the original on 9 October 2008. Retrieved 2008-11-23.
- Dean DH (1984). "Biochemical genetics of the bacterial insect-control agent Bacillus thuringiensis: basic principles and prospects for genetic engineering" (PDF). Biotechnology & Genetic Engineering Reviews. 2: 341–63. doi:10.1080/02648725.1984.10647804. PMID 6443645.
- Beegle CC, Yamamoto T (1992). "Invitation Paper (C.p. Alexander Fund): History Of bacillus Thuringiensis berliner Research and Development". The Canadian Entomologist. 124 (4): 587–616. doi:10.4039/Ent124587-4.
- Xu J, Liu Q, Yin X, Zhu S (2006). "A review of recent development of Bacillus thuringiensis ICP genetically engineered microbes". Entomological Journal of East China. 15 (1): 53–8.
- Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (September 1998). "Bacillus thuringiensis and its pesticidal crystal proteins". Microbiology and Molecular Biology Reviews. 62 (3): 775–806. doi:10.1128/MMBR.62.3.775-806.1998. PMC 98934. PMID 9729609.
- Wei JZ, Hale K, Carta L, Platzer E, Wong C, Fang SC, Aroian RV (March 2003). "Bacillus thuringiensis crystal proteins that target nematodes". Proceedings of the National Academy of Sciences of the United States of America. 100 (5): 2760–5. Bibcode:2003PNAS..100.2760W. doi:10.1073/pnas.0538072100. PMC 151414. PMID 12598644.
- W.S. Cranshaw, Colorado State University Extension Office. Last updated March 26, 2013. Bacillus thuringiensis Fact Sheet
- Babu M, Geetha M. "DNA shuffling of Cry proteins". Retrieved 2008-11-23.
- Broderick NA, Raffa KF, Handelsman J (October 2006). "Midgut bacteria required for Bacillus thuringiensis insecticidal activity". Proceedings of the National Academy of Sciences of the United States of America. 103 (41): 15196–9. Bibcode:2006PNAS..10315196B. doi:10.1073/pnas.0604865103. JSTOR 30051525. PMC 1622799. PMID 17005725.
- Peng D, Luo X, Zhang N, Guo S, Zheng J, Chen L, Sun M (January 2018). "Small RNA-mediated Cry toxin silencing allows Bacillus thuringiensis to evade Caenorhabditis elegans avoidance behavioral defenses". Nucleic Acids Research. 46 (1): 159–173. doi:10.1093/nar/gkx959. PMC 5758910. PMID 29069426.
- Palma L, Hernández-Rodríguez CS, Maeztu M, Hernández-Martínez P, Ruiz de Escudero I, Escriche B, Muñoz D, Van Rie J, Ferré J, Caballero P (October 2012). "Vip3C, a novel class of vegetative insecticidal proteins from Bacillus thuringiensis". Applied and Environmental Microbiology. 78 (19): 7163–5. doi:10.1128/AEM.01360-12. PMC 3457495. PMID 22865065.
- Estruch JJ, Warren GW, Mullins MA, Nye GJ, Craig JA, Koziel MG (May 1996). "Vip3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects". Proceedings of the National Academy of Sciences of the United States of America. 93 (11): 5389–94. Bibcode:1996PNAS...93.5389E. doi:10.1073/pnas.93.11.5389. PMC 39256. PMID 8643585.
- Mizuki E, Park YS, Saitoh H, Yamashita S, Akao T, Higuchi K, Ohba M (July 2000). "Parasporin, a human leukemic cell-recognizing parasporal protein of Bacillus thuringiensis". Clinical and Diagnostic Laboratory Immunology. 7 (4): 625–34. doi:10.1128/CDLI.7.4.625-634.2000. PMC 95925. PMID 10882663.
- Ohba M, Mizuki E, Uemori A (January 2009). "Parasporin, a new anticancer protein group from Bacillus thuringiensis". Anticancer Research. 29 (1): 427–33. PMID 19331182.
- Official Website of the Committee of Parasporin Classification and Nomenclature Accessed Jan 4, 2013[verification needed]
- Lemaux PG (2008). "Genetically Engineered Plants and Foods: A Scientist's Analysis of the Issues (Part I)". Annual Review of Plant Biology. 59: 771–812. doi:10.1146/annurev.arplant.58.032806.103840. PMID 18284373.
- "DiPelProDf data sheet" (PDF). Archived from the original (PDF) on September 8, 2013.
- "DiPelProDf data sheet" (PDF). Archived from the original (PDF) on March 13, 2014.
- "Conclusion on the peer review of the pesticide risk assessment of the active substance Bacillus thuringiensis subsp. kurstaki (strains ABTS 351, PB 54, SA 11, SA 12, EG 2348)". EFSA Journal. 10 (2): 2540. August 8, 2012. doi:10.2903/j.efsa.2012.2540.
- Hayes' Handbook of Pesticide Toxicology, Volume 1. Academic Press, imprint of Elsevier. 2010. pp. 442–443. ISBN 9780080922010.
- Huang F, Buschman LL, Higgins RA (2001). "Larval feeding behavior of Dipel-resistant and susceptible Ostrinia nubilalis on diet containing Bacillus thuringiensis (Dipel EStm)". Entomologia Experimentalis et Applicata. 98 (2): 141–148. doi:10.1046/j.1570-7458.2001.00768.x. ISSN 0013-8703.
- "Novel Bacillus thuringiensis isolate". USPTO. 1987. US4910016.
- "Formation of and methods for the production of large bacillus thuringiensis crystals with increased pesticidal activity". USPTO. 1996. US6303382.
- US patent 5955367, Adams, Lee Fremont, "Production of bacillus thuringiensis integrants", published 1999-09-21
- Pesticides; Data Requirements for Biochemical and Microbial Pesticides. U.S. Environmental Protection Agency (EPA). Final Rule.
- 40 CFR 158.2100 - Microbial pesticides definition and applicability.
- "Search: bacillus, thuringiensis". OMRI.
- Caldwell B, Sideman E, Seaman A, Shelton A, Smart C, eds. (2013). "Material Fact Sheet: Bacillus thuringiensis (Bt)" (PDF). Resource Guide for Organic Insect and Disease Management (2nd ed.). pp. 109–12. ISBN 978-0-9676507-8-4.
- Cranshaw, W.S. (November 2, 2012). "Bacillus thuringiensis Fact Sheet". Colorado State University Extension Service.
- Höfte H, de Greve H, Seurinck J, et al. (December 1986). "Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715". European Journal of Biochemistry. 161 (2): 273–80. doi:10.1111/j.1432-1033.1986.tb10443.x. PMID 3023091.
- Vaeck M, Reynaerts A, Höfte H, et al. (1987). "Transgenic plants protected from insect attack". Nature. 328 (6125): 33–7. Bibcode:1987Natur.328...33V. doi:10.1038/328033a0.
- Staff, GMO Compass. Last updated July 29, 2010 "Tobacco" entry in GMO Compass database Archived 2013-10-02 at the Wayback Machine
- Key S, Ma JK, Drake PM (June 2008). "Genetically modified plants and human health". Journal of the Royal Society of Medicine. 101 (6): 290–8. doi:10.1258/jrsm.2008.070372. PMC 2408621. PMID 18515776.
- Jan Suszkiw (November 1999). "Tifton, Georgia: A Peanut Pest Showdown". Agricultural Research magazine. Archived from the original on 12 October 2008. Retrieved 2008-11-23.
- "Genetically Altered Potato Ok'd For Crops". Lawrence Journal-World. AP. 6 May 1995 – via Google News.
- Staff, CERA. NewLeaf Entry in CERA Archived August 12, 2014, at the Wayback Machine
- van Eijck, Paul (March 10, 2010). "The History and Future of GM Potatoes". PotatoPro Newsletter. Archived from the original on October 12, 2013. Retrieved October 5, 2013.
- Hellmich RL, Hellmich KA (2012). "Use and Impact of Bt Maize". Nature Education Knowledge. 3 (10): 4.
- Ric Bessin, Extension Entomologist, University of Kentucky College of Agriculture. May 1996, last updated November 2010. Bt-Corn for Corn Borer Control
- Castagnola AS, Jurat-Fuentes, JL. Bt Crops: Past and Future. Chapter 15 in [Bacillus thuringiensis Biotechnology], Ed. Estibaliz Sansinenea. Springer, Mar 2, 2012
- Erin Hodgson and Aaron Gassmann, Iowa State Extension, Department of Entomology. May 2010. New Corn Trait Deregulated in U.S.
- Seeds of Doubt: An activist’s controversial crusade against genetically modified crops. By Michael Specter. The New Yorker, August 25, 2014
- Staff, Monsanto. August, 2009. Application for authorization to place on the market MON 87701 × MON 89788 soybean in the European Union, according to Regulation (EC) No 1829/2003 on genetically modified food and feed Archived 2012-09-05 at the Wayback Machine Linked from the GMO Compass page on the MON87701 x MON89788 Archived 2013-11-09 at the Wayback Machine event.
- "Monsanto's Bt Roundup Ready 2 Yield Soybeans Approved for Planting in Brazil". Crop Biotech Update.
- Fearing, P.L., Brown, D., Vlachos, D., Meghji, M., L. Privalle. 1997. Quantitative analysis of Cry1A(b) expression in Bt maize plants, tissues, and silage and stability of expression over generations. Mol. Breed. 3:169-176.
- US EPA. 2001. Bt Plant-Incorporated Protectants October 15, 2001 Biopesticides Registration Action Document
- Sjoblad RD, McClintock JT, Engler R (February 1992). "Toxicological considerations for protein components of biological pesticide products". Regulatory Toxicology and Pharmacology. 15 (1): 3–9. doi:10.1016/0273-2300(92)90078-n. PMID 1553409.
- Koch MS, Ward JM, Levine SL, Baum JA, Vicini JL, Hammond BG (April 2015). "The food and environmental safety of Bt crops". Frontiers in Plant Science. 6: 283. doi:10.3389/fpls.2015.00283. PMC 4413729. PMID 25972882.
- Randhawa GJ, Singh M, Grover M (February 2011). "Bioinformatic analysis for allergenicity assessment of Bacillus thuringiensis Cry proteins expressed in insect-resistant food crops". Food and Chemical Toxicology. 49 (2): 356–62. doi:10.1016/j.fct.2010.11.008. PMID 21078358.
- Batista R, Nunes B, Carmo M, Cardoso C, José HS, de Almeida AB, Manique A, Bento L, Ricardo CP, Oliveira MM (August 2005). "Lack of detectable allergenicity of transgenic maize and soya samples" (PDF). The Journal of Allergy and Clinical Immunology. 116 (2): 403–10. doi:10.1016/j.jaci.2005.04.014. hdl:10400.18/114. PMID 16083797.
- Betz FS, Hammond BG, Fuchs RL (October 2000). "Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests". Regulatory Toxicology and Pharmacology. 32 (2): 156–73. doi:10.1006/rtph.2000.1426. PMID 11067772.
- Astwood JD, Leach JN, Fuchs RL (October 1996). "Stability of food allergens to digestion in vitro". Nature Biotechnology. 14 (10): 1269–73. doi:10.1038/nbt1096-1269. PMID 9631091.
- US EPA. 2001. Bt Plant-Incorporated Protectants Biopesticides Registration Action Document 
- Helassa N, Quiquampoix H, Staunton S (2013). "Structure, Biological Activity and Environmental Fate of Insecticidal Bt (Bacillus thuringiensis) Cry Proteins of Bacterial and Genetically Modified Plant Origin". In Xu J, Sparks D (eds.). Molecular Environmental Soil Science. Springer Netherlands. pp. 49–77. doi:10.1007/978-94-007-4177-5_3. ISBN 978-94-007-4177-5.
- Dubelman S, Ayden BR, Bader BM, Brown CR, Jiang, Vlachos D (2005). "Cry1Ab Protein Does Not Persist in Soil After 3 Years of Sustained Bt Corn Use". Environ. Entomol. 34 (4): 915–921. doi:10.1603/0046-225x-34.4.915.
- Head G, Surber JB, Watson JA, Martin JW, Duan JJ (2002). "No Detection of Cry1Ac Protein in Soil After Multiple Years of Transgenic Bt Cotton (Bollgard) Use". Environ. Entomol. 31 (1): 30–36. doi:10.1603/0046-225x-31.1.30.
- Clark BW, Phillips TA, Coats JR (June 2005). "Environmental fate and effects of Bacillus thuringiensis (Bt) proteins from transgenic crops: a review" (PDF). Journal of Agricultural and Food Chemistry. 53 (12): 4643–53. doi:10.1021/jf040442k. PMID 15941295.
- Sears MK, Hellmich RL, Stanley-Horn DE, Oberhauser KS, Pleasants JM, Mattila HR, Siegfried BD, Dively GP (October 2001). "Impact of Bt corn pollen on monarch butterfly populations: a risk assessment". Proceedings of the National Academy of Sciences of the United States of America. 98 (21): 11937–42. Bibcode:2001PNAS...9811937S. doi:10.1073/pnas.211329998. PMC 59819. PMID 11559842.
- Saxena D, Stotzky G (2000). "Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil" (PDF). Soil Biology & Biochemistry. 33 (9): 1225–1230. doi:10.1016/s0038-0717(01)00027-x.
- "Cotton in India". Monsanto.com. 2008-11-03. Retrieved 2013-07-09.
- Bagla P (March 2010). "India. Hardy cotton-munching pests are latest blow to GM crops". Science. 327 (5972): 1439. Bibcode:2010Sci...327.1439B. doi:10.1126/science.327.5972.1439. PMID 20299559.
- Tabashnik BE, Gassmann AJ, Crowder DW, Carriére Y (February 2008). "Insect resistance to Bt crops: evidence versus theory". Nature Biotechnology. 26 (2): 199–202. doi:10.1038/nbt1382. PMID 18259177.
- Baxter SW, Badenes-Pérez FR, Morrison A, Vogel H, Crickmore N, Kain W, Wang P, Heckel DG, Jiggins CD (October 2011). "Parallel evolution of Bacillus thuringiensis toxin resistance in lepidoptera". Genetics. 189 (2): 675–9. doi:10.1534/genetics.111.130971. PMC 3189815. PMID 21840855.
- Lu Y, Wu K, Jiang Y, Xia B, Li P, Feng H, Wyckhuys KA, Guo Y (May 2010). "Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China". Science. 328 (5982): 1151–4. Bibcode:2010Sci...328.1151L. doi:10.1126/science.1187881. PMID 20466880.
- Just DR, Wang S, Pinstrup-Andersen P (2006). Tarnishing Silver Bullets: Bt Technology Adoption, Bounded Rationality and the Outbreak of Secondary Pest Infestations in China. American Agricultural Economics Association Annual Meeting. Long Beach, CA. Lay summary – Cornell Chronicle (July 25, 2006).
- Wang S, Just DR, Pinstrup-Andersen P (2008). "Bt-cotton and secondary pests". International Journal of Biotechnology. 10 (2/3): 113–21. doi:10.1504/IJBT.2008.018348.
- Wang Z, Lin H, Huang J, Hu R, Rozelle S, Pray C (2009). "Bt Cotton in China: Are Secondary Insect Infestations Offsetting the Benefits in Farmer Fields?". Agricultural Sciences in China. 8: 83–90. doi:10.1016/S1671-2927(09)60012-2.
- Zhao JH, Ho P, Azadi H (February 2011). "Benefits of Bt cotton counterbalanced by secondary pests? Perceptions of ecological change in China". Environmental Monitoring and Assessment. 173 (1–4): 985–94. doi:10.1007/s10661-010-1439-y. PMID 20437270.; Erratum published 2012 Aug 5: Zhao JH, Ho P, Azadi H (2012). "Erratum to: Benefits of Bt cotton counterbalanced by secondary pests? Perceptions of ecological change in China". Environmental Monitoring and Assessment. 184 (11): 7079. doi:10.1007/s10661-012-2699-5.
- Bhaskar Goswami, InfoChange "Making a meal of Bt cotton" Retrieved on 2009-4-6
- "Bug makes meal of Punjab cotton, whither Bt magic?". The Economic Times. 4 September 2007. Retrieved 14 March 2018.
- Stone, Glenn Davis (2011). "Field versus Farm in Warangal: Bt Cotton, Higher Yields, and Larger Questions". World Development. 39 (3): 387–98. doi:10.1016/j.worlddev.2010.09.008.
- Krishna VV, Qaim M (2012). "Bt cotton and sustainability of pesticide reductions in India". Agricultural Systems. 107: 47–55. doi:10.1016/j.agsy.2011.11.005.
- Staff, Frontline. Harvest of Fear: Viewpoints
- Losey JE, Rayor LS, Carter ME (May 1999). "Transgenic pollen harms monarch larvae". Nature. 399 (6733): 214. Bibcode:1999Natur.399..214L. doi:10.1038/20338. PMID 10353241.
- Emily Waltz for Nature News. September 2, 2009 GM crops: Battlefield Nature 461, 27-32 (2009)
- Mendelsohn M, Kough J, Vaituzis Z, Matthews K (September 2003). "Are Bt crops safe?". Nature Biotechnology. 21 (9): 1003–9. doi:10.1038/nbt0903-1003. PMID 12949561.
- Hellmich RL, Siegfried BD, Sears MK, et al. (October 2001). "Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen". Proceedings of the National Academy of Sciences of the United States of America. 98 (21): 11925–30. Bibcode:2001PNAS...9811925H. doi:10.1073/pnas.211297698. JSTOR 3056825. PMC 59744. PMID 11559841.
- "Bt Corn and Monarch Butterflies". USDA Agricultural Research Service. 2004-03-29. Archived from the original on 6 November 2008. Retrieved 2008-11-23.
- Salama HS, Foda MS, Sharaby A (1989). "A proposed new biological standard for bioassay of bacterial insecticides vs. Spodoptera spp". Tropical Pest Management. 35 (3): 326–330. doi:10.1080/09670878909371391.
- Quist D, Chapela IH (November 2001). "Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico". Nature. 414 (6863): 541–3. Bibcode:2001Natur.414..541Q. doi:10.1038/35107068. PMID 11734853.
- Kaplinsky N, Braun D, Lisch D, Hay A, Hake S, Freeling M (April 2002). "Biodiversity (Communications arising): maize transgene results in Mexico are artefacts". Nature. 416 (6881): 601–2, discussion 600, 602. Bibcode:2002Natur.416..601K. doi:10.1038/nature739. PMID 11935145.
- "NOW with Bill Moyers. Science & Health. Seeds of Conflict - NATURE Article Debate | PBS".
- Ortiz-García S, Ezcurra E, Schoel B, Acevedo F, Soberón J, Snow AA (August 2005). "Absence of detectable transgenes in local landraces of maize in Oaxaca, Mexico (2003-2004)". Proceedings of the National Academy of Sciences of the United States of America. 102 (35): 12338–43. Bibcode:2005PNAS..10212338O. doi:10.1073/pnas.0503356102. JSTOR 3376579. PMC 1184035. PMID 16093316.
- Serratos-Hernández J, Gómez-Olivares J, Salinas-Arreortua N, Buendía-Rodríguez E, Islas-Gutiérrez F, De-Ita A (2007). "Transgenic proteins in maize in the Soil Conservation area of Federal District, Mexico". Frontiers in Ecology and the Environment. 5 (5): 247–52. doi:10.1890/1540-9295(2007)5[247:TPIMIT]2.0.CO;2. ISSN 1540-9295.
- Piñeyro-Nelson A, Van Heerwaarden J, Perales HR, et al. (February 2009). "Transgenes in Mexican maize: molecular evidence and methodological considerations for GMO detection in landrace populations". Molecular Ecology. 18 (4): 750–61. doi:10.1111/j.1365-294X.2008.03993.x. PMC 3001031. PMID 19143938.
- Dalton R (November 2008). "Modified genes spread to local maize". Nature. 456 (7219): 149. doi:10.1038/456149a. PMID 19005518.
- Schoel B, Fagan J (October 2009). "Insufficient evidence for the discovery of transgenes in Mexican landraces". Molecular Ecology. 18 (20): 4143–4, discussion 4145–50. doi:10.1111/j.1365-294X.2009.04368.x. PMID 19793201.
- "ARS : Questions and Answers: Colony Collapse Disorder". ARS News. 2008-05-29. Archived from the original on 5 November 2008. Retrieved 2008-11-23.
- Latsch, Gunther (March 22, 2007). "Are GM Crops Killing Bees?". Spiegel Online.
- Rose R, Dively GP, Pettis J (2007). "Effects of Bt corn pollen on honey bees: Emphasis on protocol development". Apidologie. 38 (4): 368–77. doi:10.1051/apido:2007022.
- USDA. Colony Collapse Disorder: An Incomplete Puzzle Agricultural Research Magazine. July 2012
- McGrath, Matt (5 March 2009). "'No proof' of bee killer theory". BBC News.
- "EPA pesticide database". Ofmpub.epa.gov. 2010-11-17. Archived from the original on 2013-04-09. Retrieved 2013-07-09.
- Staff Environment Directorate, Organisation for Economic Co-operation and Development. Paris 26. Jul 2007. OECD Environment, Health and Safety Publications, Series on Harmonisation of Regulatory Oversight in Biotechnology No. 42. . Published by the U.S. Environmental Protection Agency
- Yin, Ruiheng (2016). Structural basis of transcription inhibition by the nucleoside-analog inhibitor thuringiensin (Thesis). Rutgers University - Graduate School - New Brunswick. doi:10.7282/T3S75JHW.
- de Maagd RA, Bravo A, Crickmore N (April 2001). "How Bacillus thuringiensis has evolved specific toxins to colonize the insect world". Trends in Genetics. 17 (4): 193–9. doi:10.1016/S0168-9525(01)02237-5. PMID 11275324.
- Bravo A, Gill SS, Soberón M (March 2007). "Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control". Toxicon. 49 (4): 423–35. doi:10.1016/j.toxicon.2006.11.022. PMC 1857359. PMID 17198720.
- Pigott CR, Ellar DJ (June 2007). "Role of receptors in Bacillus thuringiensis crystal toxin activity". Microbiology and Molecular Biology Reviews. 71 (2): 255–81. doi:10.1128/MMBR.00034-06. PMC 1899880. PMID 17554045.
- Tabashnik BE, Van Rensburg JB, Carrière Y (December 2009). "Field-evolved insect resistance to Bt crops: definition, theory, and data". Journal of Economic Entomology. 102 (6): 2011–25. doi:10.1603/029.102.0601. PMID 20069826.
- Bacillus thuringiensis General Fact Sheet (National Pesticide Information Center)
- Bacillus thuringiensis Technical Fact Sheet (National Pesticide Information Center)
- Breakdown of the Bt toxin and effects on the soil quality Research project and results
- The Bacillus thuringiensis Toxin Specificity Database at Natural Resources Canada
- Bacillus thuringiensis Taxonomy (NIH)
- Bacillus thuringiensis genomes and related information at PATRIC, a Bioinformatics Resource Center funded by NIAID
- bEcon - Economics literature about the impacts of genetically engineered (GE) crops in developing economies
- Type strain of Bacillus thuringiensis at BacDive - the Bacterial Diversity Metadatabase