Pesticidal protein-producing bacteria
Historically, the bacterium Bacillus thuringiensis has been the main focus of studies in the field of pesticidal proteins, with limited studies on other species. However, a range of other bacteria have been found to be sources of such proteins, particularly in recent years as the scope of species examined has expanded. A non-exhaustive selection of bacteria from which invertebrate-active proteins have been derived are briefly described below.
Bacillus mycoides
Phylum Firmicutes; Class Bacilli; Order Bacillales; Family Bacillaceae; Genus Bacillus
A widespread bacterium with spores that can be isolated from garden soil and whose spores are found in air, foods etc (1).
B. mycoides is a gram positive bacterium with non-motile, spore producing cells (2). When cultured on agar it forms 'hairy looking' Rhizoid colonies with a creamy color. These grow via end to end cell linkages, resulting in filaments with a genetically predetermined clockwise or counterclockwise curvature. The bacteria will readily colonize the entire agar plate. The favored growing temperature range is 10°C - 35°C, outside of which filament curvature will be lost. This bacterium can be grown in a nutrient broth, though it forms a suspension rather than rhizoid colony (1)(2). Genomic studies have shown that B. mycoides can encode members of pesticidal protein families.
Some strains of the bacterium have been shown to be a pathogen in channel catfish, causing lesions and muscle necrosis (4) but no mammalian toxicity has been reported. No insecticidal activity has been reported to date.
Current Applications: The B. mycoides isolate: BacJ/BmJ has been found to reduce Cersospora Leaf Spot disease by 38-91% in field & greenhouse sugar beet growth trials. These results are attributed to the ability of the isolate to induce systemic resistance to the disease (3). This isolate has also been found to reduce the incidence of bacterial vascular necrosis in sugar beet; bacterial spot (Xanthomonas campestris pv. vesicatoria) on pepper and tomato crops and angular leaf spot (Pseudomonas syringae pv. lachrymans) in cucumbers (5).
References:
1. http://www.tgw1916.net/Bacillus/mycoides.html
2. Di Franco, C., Beccari, E., Santini, T., Pisaneschi, G. and Tecce, G. (2002). Colony shape as a genetic trait in the pattern-forming Bacillus mycoides. BMC Microbiology, 2(1), p.33.
3. Bargabus, R., Zidack, N., Sherwood, J. and Jacobsen, B. (2002). Characterisation of systemic resistance in sugar beet elicited by a non-pathogenic, phyllosphere-colonizing Bacillus mycoides, biological control agent. Physiological and Molecular Plant Pathology, 61(5), pp.289-298.
4. Neher, O.T., Johnston, M.R., Zidack, N.K. and Jacobsen, B.J. (2009). Evaluation of Bacillus mycoides isolate BmJ and B. mojavensis isolate 203-7 for the control of anthracnose of cucurbits caused by Glomerella cingulata var. orbiculare. Biological Control, 48(2), pp.140–146.
5. Neher, O.T., Johnston, M.R., Zidack, N.K. and Jacobsen, B.J. (2009). Evaluation of Bacillus mycoides isolate BmJ and B. mojavensis isolate 203-7 for the control of anthracnose of cucurbits caused by Glomerella cingulata var. orbiculare. Biological Control, 48(2), pp.140–146.
Bacillus thuringiensis
Phylum Firmicutes; Class Bacilli; Order Bacillales; Family Bacillaceae; Genus Bacillus
Bt is a widespread bacterium, with spores having been isolated from soil (amongst other environments) in locations across the world, including Antarctica (1). A defining characteristic of this bacterium is its ability to produce parasporal protein crystals during sporulation, many of which have been found to have pesticidal activity (called δ-endotoxins) (2).
This gram positive bacterium has motile, rod shaped cells which grow aerobically. They can be plated on agar, giving a variety of colony appearances, though they are generally creamy coloured. They may be circular or irregular in shape and tend to have a rough or grainy appearance, though smooth/moist cultures do occur. They may also be grown in nutrient broth. Bt will grow between 10°C-45°C and at pH 5.7-7 (1).
There are a wide variety of Bacillus thuringiensis subspecies, many of which have been of significant interest to Bt toxin researchers including: aizawai, israelensis and kurstaki.
*For an exhaustive list of Bt subspecies please click here.
Bt strains may show pathogenicity towards mosquitoes, butterflies, moths, flatworms, mites, nematodes and protozoa via ∂-endotoxin synthesis (3). Bt toxins have been shown to have little to no impact on non-target species, making them useful biopesticides (1) (4).
Current Applications: Bt is the most widely used bio-pesticide in the world, with 90% of all biopesticide sales involving products related to Bacillus thuringiensis. It has a rapid and sustained effect on target larvae, whilst having negligible effect on non-target species. This has made it popular with biotechnology companies and Bt genes were first introduced into GM crops (including cotton and corn) in the 1980s. Cultivation of Bt GM crops has increased year on year and reached 32 million hectares world wide in 2006 (5). Agrichemical companies currently have a range of Bt GMO crops on the market and today: Bt maize and Bt cotton are cultivated globally. In 2006, these transgenic crops covered an area of 32.1 million ha; Insect-resistant crops covered 19 million ha and crops with a combination of transgenic traits (pest and herbicide resistance) covered 13.1 million ha. In 2005 alone Bt cotton represented 66% of China's cotton crop (5).
Additionally: 2 Bt subspecies: aizawai and kurstaki have been developed into sprayable products to control lepidopteran pests. While the subspecies israelensis is very toxic to mosquito and black-fly larvae and thus is used extensively in products for the urban control of these pests, its use being promoted via the World Health Organization (WHO) Onchocerciasis Control Programme.
References:
1. https://tgw1916.net/Bacillus/thuringiensis.html.
2. Kumar PA, Sharma RP, Malik VS (1996). "The insecticidal proteins of Bacillus thuringiensis" (PDF). Advances in Applied Microbiology. 42: 1–43. PMID 8865583
3. Palma, L., Muñoz, D., Berry, C., Murillo, J., and Caballero, P. (2014) Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins 6, 3296-3325.
4. National Pesticide Information Centre (2015). How might I be exposed to Bacillus thuringiensis (Bt). [online] Available at: [Accessed 14 Oct. 2019].
5. Sanchis, V. and Bourguet, D. (2008). Bacillus thuringiensis: applications in agriculture and insect resistance management. A review. Agronomy for Sustainable Development, [online] 28(1), pp.11–20. Available at: [Accessed 14 Oct. 2019].
Brevibacillus laterosporus
Phylum: Firmicutes; Class: Bacilli; Order: Bacillales; Family: Paenibacillaceae; Genus: Brevibacillus
Brevibacillus laterosporus has been isolated from a wide array of sources including: marine environments; dead honey bee larvae previously infected with European Foulbrood Disease (EFD); healthy worker bee intestines and paper (1). It is an aerobic, gram variable bacterium, characterised by its ability to produce a canoe-shaped parasporal inclusion, next to the spore itself (1)(2).
B. laterosporus cells are motile and cultures grow easily between 15°C-50°C (30°C preferred), on TSR or nutrient agar, at pH 6.8.
Interestingly, researchers from Saudi Arabia (4) and from Argentina (3) have found that B. laterosporus may be beneficial to honey bee colonies via secretion of antimicrobial agents which aid in the resistance to the bacterium Paenibacillus larvae: the cause of American and European Foul Brood disease in honey bees (3) (4).
B. laterosporus strains are able to produce a range of toxins, many with similarities to those from Bacillus thuringiensis, Lysinibacillus sphaericus and other insect pathogenic bacteria. These include Cry8, Cry43, Vpa2 and Vpb1 family proteins.
References:
1. https://tgw1916.net/Bacillus/laterosporus.html
2. de Oliveira, E.J., Rabinovitch, L., Monnerat, R.G., Passos, L.K.J. and Zahner, V. (2004). Molecular Characterization of Brevibacillus laterosporus and Its Potential Use in Biological Control. Applied and Environmental Microbiology, 70(11), pp.6657–6664.
3. Alippi, A.M. and Reynaldi, F.J. (2006). Inhibition of the growth of Paenibacillus larvae, the causal agent of American foulbrood of honeybees, by selected strains of aerobic spore-forming bacteria isolated from apiarian sources. Journal of Invertebrate Pathology, 91(3), pp.141–146.
4. Khaled, J.M., Al-Mekhlafi, F.A., Mothana, R.A., Alharbi, N.S., Alzaharni, K.E., Sharafaddin, A.H., Kadaikunnan, S., Alobaidi, A.S., Bayaqoob, N.I., Govindarajan, M. and Benelli, G. (2017). Brevibacillus laterosporus isolated from the digestive tract of honeybees has high antimicrobial activity and promotes growth and productivity of honeybee’s colonies. Environmental Science and Pollution Research, 25(11), pp.10447–10455.
5 Glare, T. R., Durrant, A., Berry, C., Palma, L., Ormskirk, M. M., and Cox, M. (2020) Phylogenetic determinants of toxin gene distribution in genomes of Brevibacillus laterosporus Genomics 112, 1042-1053
Dickeya dadantii
Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Enterobacteriales; Family: Pectobacteriaceae; Genus: Dickeya
Dickeya dadantii (1)(formerly Erwinia chrysanthemi) has been shown to produce pesticidal proteins active against the pea aphid Acyrthosiphon pisum and that are structurally-related to the Cyt pesticidal proteins of Bacillus thuringiensis (2). There are no current field applications of this bacterium.
References
1 https://tgw1916.net/Enterobacteria/Dickeya.html
2 Loth, K., Costechareyre, D., Effantin, G., Rahbe, Y., Condemine, G., Landon, C., and da Silva, P. (2015) New Cyt-like delta-endotoxins from Dickeya dadantii: structure and aphicidal activity. Scientific reports 5, 8791
Lysinibacillus sphaericus
Phylum: Firmicutes; Class: Bacilli; Order: Bacillales; Family: Bacillaceae; Genus: Lysinibacillus
Formerly known as Bacillus sphaericus, Lysinibacillus sphaericus (1) may consist of several actual bacterial species but these are only distinguishable by DNA homology analyses. Those strains in homology group IIA may produce a range of pesticidal proteins. Highly active strains produce the Tpp1/Tpp2 binary pesticidal combination (formerly known as BinA and BinB), and may also produce a further two part pesticidal combination of Cry48/Tpp49 (formerly Cry49). These strains and those with lower insecticidal activity may produce Mtx1 proteins and Mpp2, Mpp3 and Mpp4 (formerly Mtx2, Mtx3 and Mtx4) proteins. The principle targets of all of these agents are mosquito larvae. Strains throughout the Lysinibacillus sphaericus group may also produce the pesticidal protein sphaericolysin (2,3).
Current Applications: Several commercial formulations of L. sphaericus strains are available for the control of mosquito larvae, particularly Culex and Anopheles species.
References:
1 https://tgw1916.net/Bacillus/sphaericus.html
2 Berry, C. (2012) The bacterium, Lysinibacillus sphaericus, as an insect pathogen. J. Invertebr. Pathol. 109, 1-10
3 Silva-Filha, M. H. N. L., Berry, C., and Regis, L. (2014) Lysinibacillus sphaericus: toxins and mode of action, applications for mosquito control and resistance management. in Advances in Insect Physiology: Insect midgut and insecticidal proteins (Dhadialla, T. S., and Gill, S. S. eds.), Elsevier, Oxford. pp 89-176
Paenibacillus spp.
Phylum: Firmicutes; Class: Bacilli; Order: Bacillales; Family: Paenibacillaceae; Genus: Paenibacillus
Paenibacillus popilliae (formerly Bacillus popilliae (1)) and Paenibacillus lentimorbus (formerly Bacillus lentimorbus) are the causative agent of milky disease in the Japanese beetle Popillia japonica. There are no media for the growth of these bacteria which must be cultured in larvae. P. lentimorbus has been shown to encode pesticidal proteins from the Cry43 family.
Paenibacillus larvae is a pathogen of honey bee larvae and causes American foul brood disease.
Current Applications: Preparations of P. popilliae have been produced for biocontrol. After several years’ of use, a self inoculating control in soil can be established.
References:
1 http://www.tgw1916.net/Bacillus/popilliae.html
Paraclostridium bifermentans
Phylum: Firmicutes; Class: Clostridia; Order: Paraclostridiales; Family: Peptostreptococcaceae; Genus: Paraclostridium
Previously referred to as Clostridium bifermentans (1), this anaerobic bacterium is known to produce Cry16 and Cry17 and other pesticidal proteins that are active against mosquito larvae (2).
References :
1 https://tgw1916.net/Clostridium/bifermentans.html
2 Qureshi, N., Chawla, S., Likitvivatanavong, S., Lee, H. L., and Gill, S. S. (2014) The Cry toxin operon of Clostridium bifermentans subsp. malaysia is highly toxic to Aedes larval mosquitoes. Appl Environ Microbiol 80, 5689-5697
Photorhabdus luminescens
Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Enterobacteriales; Family: Morganellaceae; Genus: Photorhabdus
Photorhabdus luminescens (1) is an endosymbiotic bacterium of Heterorhabditis insect pathogenic nematodes. Released into the insect haemocoel after nematode invasion, the bacteria rapidly establish a monoculture through the production of a cocktail of antibiotics and kill the host insect through the production of a range of pesticidal proteins including the Mcf1 protein, the two part Pra/Prb (formerly PirA/PirB) and App1A/App2A (formerly PaxA/PaxB) combinations. P. luminescens also produces Tc (Toxin complex) three-part pesticidal protein complexes. Other Photorhabdus species (e.g. Photorhabdus asymbiotica) produce a similar range of pesticidal proteins. Photorhabdus is the only genus of terrestrial bioluminescent bacteria.
Current Applications: Field applications are via the use of the entomopathogenic nematodes.
References:
1 https://tgw1916.net/Enterobacteria/Photorhabdus.html
Pseudomonas protegens
Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Pseudomonales; Family: Pseudomonadaceae; Genus: Pseudomonas
Pseudomonas protegens strains were formerly classified as Pseudomonas fluorescens strains. In addition to the known activity of some strains in controlling plant pathogenic fungi, the production of Mcf family insecticidal proteins in some strains has been demonstrated.
References:
1 https://tgw1916.net/Pseudomonas/fluorescens.html
2 Paulsen, I. T., Press, C. M., Ravel, J., Kobayashi, D. Y., Myers, G. S., Mavrodi, D. V., DeBoy, R. T., Seshadri, R., Ren, Q., Madupu, R., Dodson, R. J., Durkin, A. S., Brinkac, L. M., Daugherty, S. C., Sullivan, S. A., Rosovitz, M. J., Gwinn, M. L., Zhou, L., Schneider, D. J., Cartinhour, S. W., Nelson, W. C., Weidman, J., Watkins, K., Tran, K., Khouri, H., Pierson, E. A., Pierson, L. S., 3rd, Thomashow, L. S., and Loper, J. E. (2005) Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat Biotechnol 23, 873-878
Serratia entomophila & Serratia proteamaculans
Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Enterobacteriales; Family: Yersiniaceae; Genus: Serratia
These bacteria are causative agents of amber disease in the grass grub Costelytra zealandica. These bacteria are able to produce pesticidal proteins related to those from P. luminescens and also produce anti-feedant proteins.
References:
1 Hurst, M. R., Glare, T. R., and Jackson, T. A. (2004) Cloning Serratia entomophila antifeeding genes--a putative defective prophage active against the grass grub Costelytra zealandica. J. Bacteriol. 186, 5116-5128
Xenorhabdus spp.
Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Enterobacteriales; Family: Morganellaceae; Genus: Xenorhabdus
Xenorhabdus spp (1) are endosymbiotic bacteria of Steinernema insect pathogenic nematodes. Released into the insect haemocoel after nematode invasion, the bacteria rapidly establish a monoculture through the production of a cocktail of antibiotics and kill the host insect through the production of a range of pesticidal proteins similar to those of Photorhabdusstrains, including the App1B/App2B (formerly XaxA/XaxB) two part pesticidal combination.
References:
1) http://www.tgw1916.net/Enterobacteria/Xenorhabdus.html
Yersinia spp.
Phylum: Proteobacteria; Class: Gammaproteobacteria; Order: Enterobacteriales; Family: Yersiniaceae; Genus: Yersinia
Some Yersinia spp (1) such as Yersinia enterocolitica strains can produce a range of pesticidal proteins similar to those from Photorhabdus spp, including the App1C and App3A (formerly YaxA/YaxB) two part pesticidal combination
References:
1 https://www.tgw1916.net/Enterobacteria/Yersinia.html