Microbial control of insect pests
- 1. Microbial Control of Insect Pests Vivek Kumar Department of Biosciences Swami Rama Himalayan University Jolly Grant, Dehradun
- 2. Introduction • Microorganisms include bacteria, viruses, and small eukaryotes (e.g. protists, fungi, and nematodes). • Some are pathogenic, usually killing insects, and of these many are host-specific to a particular insect genus or family. • Infection is from spores, viral particles, or organisms that persist in the insect’s environment, often in the soil. • These pathogens enter insects by several routes. • Entry via the mouth is common for viruses, bacteria and nematodes.
- 3. • Cuticular and/or wound entry occurs in fungi and nematodes; the spiracles and anus are other sites of entry. • Viruses and protists also can infect insects via the female ovipositor or during the egg stage. • The microorganisms then multiply within the living insect, disease is common in dense insect populations (pest or non-pest) and under environmental conditions suitable to the microorganisms. • At low host density, however, disease incidence is often low as a result of lack of contact between the pathogens and their insect hosts.
- 4. Bacillus thuringiensis • Bacillus thuringiensis (Bt) is a natural occurring, soil- borne bacteria that has been used since the 1950s for natural insect control. • It consists of a spore, which gives it persistence, and a protein crystal within the spore, which is toxic.
- 5. • When the bacteria is consumed by certain insects, the toxic crystal is released in the insects highly alkaline gut, blocking the system which protects the pest's stomach from its own digestive juices. • The stomach is penetrated, and the insect dies . The toxic protein differs, depending on the subspecies of Bt producing it, yielding a variance of Bt toxic to different insect species (or none at all).
- 6. • The stomach is penetrated, and the insect dies by poisoning from the stomach contents and the spores themselves. • This same mechanism is what makes Bt harmless to birds, fish and mammals whose acidic gut conditions negate the bacteria’s effect. • This bacterium differs from other species belonging to this genus by the presence of a parasporal inclusion body (crystal) of protein origin, formed during sporulation. • This crystal is composed of Cry proteins which are encoded by Cry genes.
- 7. Discovery • Bt was first discovered in 1901 by a Japanese scientist investigating the decline in silkworm moth populations, which he attributed to the rod-shaped, Gram-positive bacterium. • In 1911 it was rediscovered by a German scientist, and a solution of crystallized Bt toxins was found to be highly effective against certain crop pests. • Including the corn borer, corn rootworm, corn earworm, and bollworms.
- 9. • In the United States the product was first used commercially as an insecticide spray in 1958. • Several different strains of the bacterium are currently used to control for a number of agricultural insect pests and their larvae. • Bt toxin can be applied to crops, including potatoes, corn, and cotton, as a spray or, less commonly, in granular form.
- 10. Mechanism: • Cry toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles), Hymenoptera (wasps, bees, ants and sawflies) and 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.
- 11. Mechanism of Bt
- 12. • 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 B. thuringiensis parasporal body contains protein toxins that kill over 100 species of moths by dissolving in the alkaline gut of caterpillars and destroying the epithelium.
- 14. Bt toxins present in peanut leaves (right dish) protect it from extensive damage caused to unprotected peanut leaves by lesser cornstalk borer larvae
- 15. Bt kurstaki (Bt-k) – Bacillus thuringiensis var kurstaki • Bt-k is most effective when applied to caterpillars during their 1st and 2nd instars, when they are still small. • It must be ingested by the insect, as it is a stomach toxin. • Harmless to humans, animals and beneficial insects. • Bt-k biodegrades quickly in sunlight and may require reapplication under heavy insect pressure.
- 16. Bt-k is a naturally occurring soil bacteria ideal for controlling : tent caterpillars, gypsy moth, cabbage looper, tomato hornworm and other leaf eating caterpillars on trees, shrubs, tomatoes and other vegetables.
- 17. Bt israelensis (Bt-i) – Bacillus thuringiensis var israelensis • Bt-i is a highly specific biological pesticide for use against mosquito, black fly and fungus gnat larvae. • It may be applied safely to irrigation and roadside ditches, pastures, marshes and ponds, water gardens, flower pots, bird baths, rain gutters…any place there is standing water! • Once ingested, Bt-i kills 95-100% of mosquito larvae within 24 hours. • Highly effective because it kills these pests before they become biting adults. Will not harm people, pets, wildlife or fish.
- 18. Pseudomonas syringae • It is a rod-shaped, Gram-negative bacterium with polar flagella. • As a plant pathogen, it can infect a wide range of species, and exists as over 50 different pathovars. • P. syringae is named after the lilac tree (Syringa vulgaris), from which it was first isolated. • Pseudomonas syringae strain ESC-10 and Pseudomonas syringae strain ESC-11 are natural strains of bacteria that occur on many kinds of plants throughout the world.
- 19. • They were originally isolated and identified from apples. • They are applied to certain fruits before storage in order to protect the fruits from several small insects. • At least one P. syringae strain is highly virulent to insects, causing death to most individuals within as few as 4 days and growing to high population densities within insect hosts. • Two species of Serratia are responsible for amber disease in the scarab Costelytra zealandica, a pest of pastures in New Zealand, and have been developed for scarab control. • Bacillus sphaericus has a toxin that kills mosquito larvae.
- 20. Nematodes • Mermithid nematodes are large and infect their host singly, eventually killing it as they break through the cuticle. • They kill a wide range of insects, but aquatic larvae of black flies and mosquitoes are prime targets for biological control by mermithids. • A major obstacle to their use is the requirement for in vivo production, and their environmental sensitivity (e.g. to temperature, pollution, and salinity).
- 22. • Heterorhabditids and steinernematids are small, soil-dwelling nematodes, associated with symbiotic gut bacteria (of the genera Photorhabdus and Xenorhabdus) that are pathogenic to host insects, killing them by septicemia. • In conjunction with their respective bacteria, nematodes of Heterorhabditis and Steinernema can kill their hosts within two days of infection. • They can be mass-produced easily and cheaply and applied with conventional equipment, and have the advantage of being able to search for their hosts. • The infective stage is the third-stage juvenile.
- 23. • They can be mass-produced easily and cheaply and applied with conventional equipment, and have the advantage of being able to search for their hosts. • The infective stage is the third-stage juvenile. • The Neotylenchidae contains the parasitic Deladenus siricidicola, which is one of the biological control agents of the sirex wood wasp, Sirex noctilio — a serious pest of forestry plantations of Pinus radiata in Australia. • The juvenile nematodes infect larvae of S. noctilio, leading to sterilization of the resulting adult female wasp.
- 24. Fungi • Fungal spores that contact and adhere to an insect germinate and send out hyphae. • These penetrate the cuticle, invade the hemocoel and cause death either rapidly owing to release of toxins. • Or more slowly owing to massive hyphal proliferation that disrupts insect body functions. • The fungus then sporulates, releasing spores that can establish infections in other insects; and thus the fungal disease may spread through the insect population.
- 25. • A novel application method uses felt bands containing living fungal cultures applied to the tree trunks or branches, as is done in Japan using a strain of Beauveria brongniartii against longhorn beetle borers in citrus and mulberry. • Useful species of entomopathogenic fungi belong to genera such as Beauveria, Entomophthora, Hirsutella, Metarhizium, Nomuraea, and Verticillium. • Many of these fungi overcome their hosts after very little growth in the insect hemocoel, in which case toxins are believed to cause death.
- 26. • Verticillium lecanii is used commercially to control aphids and scale insects in European glasshouses. • Entomophthora species also are useful for aphid control in glasshouses. • Species of Beauveria and Metarhizium, known as white and green muscardines, respectively (depending on the color of the spores), are pathogens of soil pests, such as termites and beetle larvae, and can affect other insects, such as spittle bugs of sugarcane and certain moths that live in moist microhabitats. • One Metarhizium species, M. anisopliae, has been developed as a successful myco-insecticide for locusts and other grasshoppers in Africa.
- 27. Safety studies • The use of Bt toxins as plant-incorporated protectants prompted the need for extensive evaluation of their safety for use in foods and potential unintended impacts on the environment. • 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
- 28. Toxicology studies • 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
- 29. Allergenicity studies • 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.
- 30. Digestibility studies • 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.
- 31. 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.
- 32. 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.
- 33. • 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.
- 34. 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.
- 35. 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.
- 36. Insect resistance • 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.
- 37. • 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.
- 38. • classical biological control (i.e. an introduction of an exotic pathogen such as the bacterium Paenibacillus popilliae established in the USA for the control of the Japanese beetle Popillia japonica. • Inoculation (e.g. a single treatment that provides season-long control, as in the fungus Verticillium lecanii used against Myzus persicae aphids in glass- houses).