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;  which has insecticidal activity against Lepidoptera (butterflies and moths), Coleoptera (beetles) or  Diptera (small flies, mosquitoes) 

• The toxins produced by inclusion bodies/parasporal bodies show different degrees of binding affinity  to the toxin receptors in the insect gut and  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. 

• Toxins are specific in action- has little effect on other organisms, more environment friendly than synthetic pesticides  

• Vegetative cells contain endospores and crystals of an insecticidal protein toxin (δ - endotoxin), which cause lysis of cells finally resulting in release of spores and toxin crystals (bipyramidal shape).    

∙ 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.

Bt  produces different 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, Cyt (cytolytic) toxins and Vip (vegetative insecticidal proteins)

 Cry (crystal) toxins, are encoded by different cry genes located  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  

Cry and Cyt toxins produced by Bacillus thuringiensis affect insect larvae by damaging the mid-gut epithelium, leading to feeding inhibition and death. Their actions are similar in outcome but differ in specificity and mechanism.

Effect of Cry toxins on insect larvae: Larvae ingest Cry toxin crystals along with Bt spores. In the alkaline mid-gut, Cry protoxins are activated by digestive enzymes. Activated toxins bind to specific receptors on the mid-gut epithelial cells. This binding causes pore formation in the gut membrane. Gut cells lyse resulting in paralysis of the digestive system. Larva stops feeding and dies due to starvation and septicemia.
Effect of Cyt toxins on insect larvae: Cyt toxins are also ingested by larvae, mainly dipteran larvae. They directly interact with membrane lipids of gut epithelial cells, causing membrane disruption and cell lysis. Leads to rapid destruction of gut cells. Often acts synergistically with Cry toxins, enhancing larval mortality.

 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. 

 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 

Bacillus thuringiensis is an eco-friendly and widely used biopesticide, but it has several limitations that affect its field performance.

1. Narrow Spectrum of Activity - Bt is highly pest-specific- Effective mainly against certain insect groups (especially lepidopteran larvae)

2. Slow Action -Bt does not cause immediate death-Insects stop feeding first and die after 2–5 days-Not suitable when quick result is required.

3. Effectiveness Depends on Pest Stage- Bt is most effective only on early larval stages-Older larvae are less susceptible.

4. Environmental Sensitivity -Bt spores and toxins are degraded by sunlight (UV radiation)- High temperatures and rainfall reduce persistence- Repeated applications may be necessary.

5. Requires Ingestion-Bt must be ingested by the insect to be effective-Not effective against non-feeding or hidden pests.

6. Possibility of Resistance Development- Continuous and improper use of Bt can lead to development of resistance in target pests-This reduces long-term effectiveness.

Thus, while Bt is a safe and eco-friendly biopesticide, its slow action, narrow target range, environmental sensitivity, and dependence on pest stage and ingestion limit its effectiveness. Therefore, Bt is best used as a component of Integrated Pest Management (IPM) rather than as a sole control measure.

Bt Crops 

    Bt crops are genetically modified (GM) plants that are engineered to produce insecticidal proteins (Cry toxins) from Bacillus thuringiensis (Bt). These proteins protect the plants from insect pests.  Bt toxin is incorporated directly into plants through the use of genetic engineering 

eg., Bt  cotton, Bt brinjal, Bt corn, Bt rice, Bt soybean etc. 

Advantages: Bt crops reduces chemical pesticide use, provides continuous pest protection throughout plant growth, is environmentally safer, reduces pest damage, increasing yield and quality. They can be integrated into IPM programs for sustainable agriculture.

Limitations include possibility of resistance development in pests, regulatory and public concerns regarding GM crops, not being effective against all insect types etc.

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 

 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. 

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. 

Grubs ingest the bacterial spores while feeding. Spores germinate inside the gut. The bacterium multiplies, causing the grub’s body to turn milky white. Infected grubs die after a few days. Commercial "milky spore" powders are marketed under several names, by several  companies under the trade names "Milky Spore", "Grub Attack" and "Grub Killer. 

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, which are processed in the midgut of the larval host  and are required for toxicity to mosquito larvae.

Nonspore formers 

Pseudomonas fluorescens is a common gram negative, rod-shaped non spore forming bacterium thatcolonize plant surfaces. It secretes a soluble greenish fluorescent pigment  called fluorescein, particularlyunder conditions of low iron availability. They enhance plant growth promotion, stimulates the plant’s defense system. It protects plants against many fungal diseases by the production of secondary metabolites including antibiotics (inhibit the growth of pathogens), hydrogen cyanide, siderophores (bind iron strongly in the soil and reduces iron availability for pathogens) and hydrolytic enzymes such as chitinase, cellulase, protease (degrade pathogen cell walls).  

Other non-spore-forming bacteria 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.