Biopesticides - Bacterial

Biocontrol is the use of living organisms or biological agents to control pests or to maintain  at a harmless levels. The practice or process by which an undesirable organism is controlled  by means of another (beneficial) organism is 

• eco-friendly  

• easy to use 

Biopesticides are commercial preparations of microorganisms (microbial pesticides) that  control pests e.g. bacteria, entomopathogenic fungi/ viruses/nematodes/protozoa. They are  applied as and when required, to control pests / diseases either selectively or with broad  spectrum approach. Biopesticides are generally target specific and affect only the target pest  and closely related organisms. Biopesticides are less toxic than conventional pesticides.  When used as a component of Integrated Pest Management (IPM) programs these  biopesticides can greatly decrease the use of conventional pesticides, while crop yields  remain high.  

Microbial biopesticides  

∙ Bacteria 

⮚ Obligate spore formers eg., Bacillus thuringiensis, Bacillus subtilis, Bacillus  cereus, B. popilliae, B. lentimorbus 

⮚ Non-spore formers eg., Pseudomonas fluorescens, P. cepacia 

∙ Entomopathogenic fungi (e.g. Beauveria bassiana, Metarhizium spp.) 

∙ Entomopathogenic viruses - Baculoviruses  

- Polyhedrosis viruses - NPV & CPV 

- Granulosis viruses - NGV & CGV  

Advantages 

• less toxic than conventional pesticides 

• affect only the target pest and closely related organisms 

• cheaper when produced locally 

• effective in very small quantities and often decompose quickly  

• lower exposures and no pollution problems 

Disadvantages 

• High specificity – require exact identification of the pest/pathogen and may require  multiple pesticides to be used  

• Often slow speed of action (thus making them unsuitable if a pest outbreak is an  immediate threat to a crop)  

• Variable efficacy - biotic and abiotic factors (should multiply within the target insect  pest/pathogen) 

Bacillus thuringiensis 

• Most widely used biopesticides – many subspecies and strains of Bacillus thuringiensis,  or Bt.  

• produces a different mix of proteins, and specifically kills one or a few related species of  insect larvae  

• Bacillus thuringiensis- Gram positive, aerobic, motile, spore-forming, soil bacterium 

• During sporulation, synthesize protein crystals – inclusion bodies/parasporal bodies;  insecticidal activity against Lepidoptera (butterflies and moths), Coleoptera (beetles) or  Diptera (small flies, mosquitoes) 

• The toxins produced have a basic structure but show different degrees of binding affinity  to the toxin receptors in the insect gut and hence differ in insect host range  

• Bacillus thuringiensis subsp. aizawai, Bt subsp. kurstaki, Bt subsp. israelensis, Bt subsp.  sphaericus, and Bt subsp. tenebrionis are effectively used for controlling different groups  of target insects.  

• Bt subsp. kurstaki are effective against caterpillars, Bt subsp. israelensis and Bt subsp.  sphaericus target mosquito larvae, and Bt subsp. tenebrionis is effective against some  coleopterans. 

• Little effect on other organisms, more environment friendly than synthetic pesticides  

• Vegetative cells contain endospores and crystals of an insecticidal protein toxin (δ - endotoxin) 

∙ Lysis of cells – release of spores and toxin crystals (bipyramidal shape).  

  

4 types of toxins 

 α exotoxin – heat labile (insect toxin) 

 β exotoxin – heat stable (fly factor) 

 - both are water soluble 

 γ exotoxin  

 δ endotoxin – parasporal body/ crystalline toxin – during sporulation; limited  spectrum of activity 

• δ endotoxin – consists of 

 Cry (crystal) toxins, encoded by different cry genes, the cry genes are located on  plasmid - basis of Bt classification 

 Cry toxins - encoded by different toxin genes on plasmids of B. thuringiensis - 5 or 6  different plasmids in a single Bt strain. 

B. thuringiensis serves as an important reservoir of Cry toxins for production of  biological insecticides and insect-resistant genetically modified crops. 

 Cyt (cytolytic) toxins - which can enhance the activity of Cry toxins – enhance the  effectiveness of insect control  

 Vip (vegetative insecticidal proteins) –Another class of insecticidal proteins in Bt. Vip  proteins do not share sequence homology with Cry proteins, and do not compete for the  same receptors. Some kill different insects than do Cry proteins. 

 β-exotoxins - Some isolates of B. thuringiensis produce a class of insecticidal small  molecules called β-exotoxin, the common name for which is thuringiensin. β-exotoxin and  the other Bacillus toxins may contribute to the insecticidal toxicity of the bacterium to  lepidopteran, dipteran, and coleopteran insects. β-exotoxin is known to be toxic to humans  and almost all other forms of life and its presence is prohibited in B. thuringiensis microbial  products.  

Engineering of plants to contain and express only the genes for δ-endotoxins avoids the  problem of assessing the risks posed by these other toxins that may be produced in microbial  preparations. 

∙ Mechanism of insecticidal action 

Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal δ-endotoxins (called crystal proteins or Cry proteins). When insects ingest these crystals, their alkaline  digestive tracts denature the insoluble crystals, making them soluble.  

 Crystals are aggregates of a large protein - protoxin - must be activated 

 Crystal protein is highly insoluble in normal conditions; entirely safe to humans, higher  animals and most insects.  

 Solubilised in reducing conditions of high pH (above about pH 9.5) - mid-gut of  lepidopteran larvae; highly specific insecticidal agent  

 Solubilisation in the insect gut – protoxin cleaved by a gut protease to produce an active  toxin -δ-endotoxin. 

 Binding of toxin to specific receptors called cadherins on the brush border epithelial  membrane of the gut cells - pore formation, osmotic cell shock  

 Lysis of gut epithelial cells; the larva stops feeding, and the gut pH lowered  This lower pH - bacterial spores germinate, and the bacterium invades the host, causing a  lethal septicemia. Live Bt bacteria colonize the insect which can contribute to death.  Death in 30 mnts to 3 days 

 Plasmid exchange between Bt strains - conjugation-like process; wide variety of strains  with different combinations of Cry toxins 

Presence of transposons in Bt - increased variety of toxins produced naturally by Bt  strains; basis for genetically engineered strains with novel toxin combinations 

Commercial Bt products - powders containing a mixture of dried spores and toxin crystals  Trade names such as Dipel, Doom and Thuricide  

 Applied to leaves or other environments where the insect larvae feed - as liquid sprays on  crop plants; must be ingested to be effective 

 Inactivated by UV radiation – applied on undersides of leaves 

Transgenic Crops 

 Bt toxin - incorporated directly into plants through the use of genetic engineering - Bt  cotton, Bt brinjal etc 

∙ 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 USA- called 'New Leaf' potato 

∙ 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. 

∙ Later, engineered crops included corn and cotton. Corn genetically modified to produce  VIP was first approved in the US in 2010.  

∙ Monsanto developed a soybean expressing Cry1Ac and the glyphosate-resistance gene  for the Brazilian market, which completed the Brazilian regulatory process in 2010.  ∙ Insect-resistant transgenic Bt crops -high specificity, reduction in chemical pesticide  applications, increased crop yield 

Insect resistance 

∙ 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. 

∙ Monsanto immediately 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.  

∙ Two different kinds of Bt genes expressed – to prevent development of resistance to Bt  toxin proteins 

Other bacterial biopesticides 

Bacillus sphaericus is an obligate aerobe bacterium used as a larvicide against mosquitoes  including Culex spp., some Aedes spp., and Anopheles spp. It is a gram positive bacterium, with  rod shaped cells that form spherical endospores. In the course of sporulation, Bacillus sphaericus produces an inclusion body which is toxic to mosquito larvae. The larvicide of B. sphaericus consists of two proteins of 51 and 42 kDa, which are processed in the midgut of the larval host  and are required for toxicity to mosquito larvae. These two proteins,  differ in their  sequences from all the other known insecticidal proteins of Bacillus thuringiensis.  

 B. popilliae is a Gram positive spore-forming rod which causes milky disease in Japanese beetle  larvae. Two types of bacterium were isolated from two types of milky disease. Type A disease  was characterized by a pure white appearance of the grubs/larvae due to a large number of  refractile bacterial spores in the haemolymph (insect blood) and is caused by B. popilliae. Type  B disease is caused by B. lentimorbus and the grubs showed a transition from white to brown  colour. Commercial "milky spore" powders are marketed under several names, by several  companies under the trade names "Milky Spore", "Grub Attack" and "Grub Killer. The milky  disease bacteria are highly pathogenic and also highly persistent in the environment so they can  be used for mass release to achieve lasting control.  

Nonspore formers 

Pseudomonas fluorescens is a common gram negative, rod-shaped non spore forming bacterium  that colonize

soil, water and plant surfaces. It secretes a soluble greenish fluorescent pigment  called fluorescein,

particularly under conditions of low iron availability. They enhance plant growth promotion and protect them against many fungal diseases by the production of a number  of secondary metabolites including antibiotics, siderophores and hydrogen cyanide  

Other non-spore-forming ones include Serratia, Yersinia, Photorhabdus, and Xenorhabdus.  which cause infection when ingested by susceptible insect hosts.  

Several species of naturally occurring bacteria infect a variety of arthropod pests and play  an important role in their management. Such entomopathogens are mass-produced in vitro  (bacteria, fungi, and nematodes) and sold commercially. Using entomopathogens as  biopesticides in pest management is called microbial control, which is a critical part of integrated  pest management (IPM) against several pests. Understanding the mode of action, ecological  adaptations and host range is essential for successfully utilizing entomopathogen-based  biopesticides for pest management in agriculture.