Microbial Insecticides 1

R. Weinzierl, T. Henn, P. G. Koehler and C. L. Tucker

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Synthetic chemical insecticides provide many benefits to food production and human health, but they also pose some hazards. In many instances, alternative methods of insect management offer adequate levels of pest control and pose fewer hazards. One such alternative is the use of microbial insecticides- -insecticides that contain microorganisms or their by-products. Microbial insecticides are especially valuable because their toxicity to nontarget animals and humans is extremely low. Compared to other commonly used insecticides, they are safe for both the pesticide user and consumers of treated crops. Microbial insecticides also are known as biological pathogens, and biological control agents (Table 1). Microbial insecticides are comprised of microscopic living organisms (viruses, bacteria, fungi, protozoa, or nematodes) or the toxins produced by these organisms. They are formulated to be applied as conventional insecticidal sprays, dusts, liquid drenches, liquid concentrates, wettable powders, or granules. Each product's specific properties determine the ways in which it can be used most effectively. This publication has been developed to help readers make the most effective use of microbial insecticides. It summarizes the strengths and weaknesses of products available commercially (and a few still under development), describes the types of microorganisms formulated for insect control, and lists the most promising uses for microbial insecticides. With the exception of insecticidal products containing nematodes, all the microbial insecticides discussed in this publication are regulated by the United States Environmental Protection Agency (US EPA). (Nematodes used for insect control are multicellular parasites, and parasites and predators are not regulated by the US EPA.) Although this publication provides background information on microbial insecticides, readers should consult product labels for specific application directions. All insecticides should be use only in the manner specified on the product d label.

Advantages of Microbial Insecticides

Individual products differ in important ways, but the following list of beneficial characteristics applies to microbial insecticides in general. The organisms used in microbial insecticides are essentially nontoxic and nonpathogenic to wildlife, humans, and other organisms not closely related to the target pest. The safety offered by microbial insecticides is their greatest strength. The toxic action of microbial insecticides is often specific to a single group or species of insects, and this specificity means that most microbial insecticides do not directly affect beneficial insects (including predators or parasites of pests) in treated areas. If necessary, most microbial insecticides can be used in conjunction with synthetic chemical insecticides because in most cases the microbial product is not deactivated or damaged by residues of conventional insecticides. (Follow label directions concerning any limitations.) Because their residues present no hazards to humans or other animals, microbial insecticides can be applied even when a crop is almost ready for harvest. In some cases, the pathogenic microorganisms can become established in a pest population or its habitat and provide control during subsequent pest generations or seasons.

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Disadvantages of Microbial Insecticides

The limitations or disadvantages listed below do not prevent the successful use of microbial insecticides. Understanding how these limitations affect specific microorganisms will help users to choose effective products and take necessary steps to achieve successful results. Because a single microbial insecticide is toxic to only a specific species or group of insects, each application may control only a portion of the pests present in a field, garden, or lawn. If other types of pests are present in the treated area, they will survive and may continue to cause damage. Conventional insecticides are subject to similar limitations because they too are not equally effective against all pests. Nonetheless, the negative aspect of selectivity is often more noticeable for microbials. Heat, desiccation (drying out), or exposure to ultraviolet radiation reduces the effectiveness of several types of microbial insecticides. Consequently, proper timing and application procedures are especially important for some products. Special formulation and storage procedures are necessary for some microbial pesticides. Although these procedures may complicate the production and distribution of certain products, storage requirements do not seriously limit the handling of microbial insecticides that are widely available. (Store all pesticides, including microbial insecticides, according to label directions.) Because several microbial insecticides are pest-specific, the potential market for these products may be limited. Their development, registration, and production costs cannot be spread over a wide range of pest control sales. Consequently, some products are not widely available or are relatively expensive (several insect viruses, for example).

Bacteria
Bacterial pathogens used for insect control are spore-forming, rod-shaped bacteria in the genus Bacillus. They occur commonly in soils, and most insecticidal strains have been isolated from soil samples. Bacterial insecticides must be eaten to be effective; they are not contact poisons. Insecticidal products comprised of a single Bacillus species may be active against an entire order of insects, or they may be effective against only one or a few species. For example, products containing Bacillus thuringiensis var. kurstaki kill the caterpillar stage of a wide array of butterflies and moths (Figure 1). In contrast, Bacillus popillae (milky spore disease) kills Japanese beetle larvae but is not effective against the closely related annual white grubs (masked chafers in the genus Cyclocephala ) that commonly infest lawns.

Figure 1. Action of Bacllus thuringiensis var. kurstaki on caterpillars. (1) Caterpillar consumes foliage treated with Bt (spores and crystalline toxin). (2) Within minutes, the toxin binds to specific receptors in the gut wall, and the caterpillar STOPS FEEDING. (3) Within hours, the gut wall breaks down, allowing spores and normal gut bacteria

to enter the body cavity; the toxin dissolves. (4) In 1-2 days, the caterpillar dies from septicemia as spores and gut bacteria proliferate in its blood. (Adapted from Abbot Laboratories, "Mosquito Control: Taking the Bite Out of Summer.") The microbial insecticides most widely used in the United States since the 1960s are preparations of the bacterium Bacillus thuringiensis (abbreviated as Bt). Bt products are produced commercially in large industrial fermentation tanks. As the bacteria live and multiply in the right conditions, each cell produces (internally) a spore and a crystalline protein toxin called an endotoxin. Most commercial Bt products contain the protein toxin and spores, but some are cultured in a manner that yields only the toxin component. When Bt is ingested by a susceptible insect, the protein toxin is activated by alkaline conditions and enzyme activity in the insect's gut. The toxicity of the activated toxin is dependent on the presence of specific receptor sites on the insect's gut wall. This necessary match between toxin and receptor sites determines the range of insect species killed by each Bt subspecies and isolate. If the activated toxin attaches to receptor sites, it paralyzes and destroys the cells of the insect's gut wall, allowing the gut contents to enter the insect's body cavity and bloodstream. Poisoned insects may die quickly from the activity of the toxin or may die within 2 or 3 days from the effects of septicemia (blood poisoning). Although a few days may elapse before the insect dies, it stops feeding (and therefore stops damaging crops) soon after ingesting Bt. Bt does not colonize or cycle (reproduce and persist to infect subsequent generations of the pest) in the environment in the magnitude necessary to provide continuing control of target pests. The bacteria may multiply in the infected host, but bacterial multiplication in the insect does not result in production of abundant spores or crystalline toxins. The usual result is that few or no infective units are released into the environment when a poisoned insect dies. Consequently, Bt products are applied much like synthetic insecticides. Bt treatments are inactivated fairly rapidly (within one to a few days) in many outdoor situations, and repeated applications may be necessary for some crops and pests. Until the early 1980s, commercial Bt products were effective only against caterpillars. In recent years, however, additional isolates that kill other types of pests have been identified and developed for insecticidal use. The nature of the crystalline protein endotoxin differs among Bt subspecies and isolates, and it is the characteristics of these specific endotoxins that determine what insects will be poisoned by each Bt product. Bt formulations that are now commercially available fall into one of the following broad categories. Bt formulations that kill caterpillars. The best-known and most widely used Bt insecticides are formulated from Bacillus thuringiensis var. kurstaki isolates that are pathogenic and toxic only to larvae of the butterflies and moths. Many such Bt products have been registered with the United States Environmental Protection Agency. The most common trade names for these commercial products include Dipel, Javelin, Thuricide, Worm Attack, Caterpillar Killer, Bactospeine, and SOK-Bt, but many small companies sell similar products under a variety of trade names. These products are commercially successful and widely available as liquid concentrates, wettable powders, and ready-to-use dusts and granules. They are used to control many common leaf-feeding caterpillars, including caterpillar pests on vegetables (especially the "worms" that attack cabbage, broccoli, cauliflower, and Brussels sprouts), bagworms and tent caterpillars on trees and shrubs, larvae of the gypsy moth and other forest caterpillars, and European corn borer larvae in field corn. Several of these products are used to control Indianmeal moth larvae in stored grain. One product with a very specific target is Certan,

formulated from Bacillus thuringiensis var. aizawai , and used exclusively for the control of wax moth larvae in honeybee hives. Bt products that kill caterpillars are NOT effective against other types of pests; they will not control aphids, beetles, flies, or additional pests other than caterpillars. Even certain caterpillars are not effectively controlled by Bt, especially those that live in the soil or bore into plant tissues without consuming a significant amount of the Bt applied to plant surfaces. (Again, Bt is a stomach poison that must be ingested to be effective.) The peach tree borer in stone fruits, corn earworm in corn, and the cutworms that clip off field crops or garden plants are examples of caterpillars seldom controlled by Bt treatments. Bt is not registered for the control of codling moth larvae that attack apples and pears because these larvae do not feed much (if at all) on treated surfaces. Common caterpillar pests that are controlled effectively with Bacillus thuringiensis var. kurstaki (Bt) include: European corn borer in corn Indianmeal moth in stored grain cabbage looper imported cabbageworm diamondback moth tomato/tobacco hornworm gypsy moth spruce budworm tent caterpillars fall webworm mimosa webworm bagworms spring and fall cankerworm Common caterpillar pests that are NOT controlled by normal applications of Bt include: corn earworm (on corn) codling moth peach tree borer squash vine borer

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Bt formulations that kill mosquito, black fly, and fungus gnat larvae. Production of a second group of Bt insecticides began in the early 1980s. These products utilize Bacillus thuringiensis var. israelensis (Bti), a subspecies that kills the larvae of certain Diptera (the insect order containing the flies and mosquitoes). The main targets for Bti are the larval stages of mosquitoes, black flies, and fungus gnats; it does not control larval stages of "higher" flies such as the house fly, stable fly, or blow flies. Mosquitoes that are most susceptible to Bti include species in the genera Aedes and Psorophora. Anopheles and Culex species are controlled only when higher than normal rates of Bti are applied. Bti products that are available commercially include Vectobac, Teknar, Bactimos, Skeetal, and Mosquito Attack. Bti is most effective for mosquito or black fly control when it is used on a community-wide basis by mosquito abatement district personnel. In general, any attempts to reduce the prevalence of mosquito-breeding sites and treat remaining sites are most effective when undertaken on such a scale. For most homeowners or farmers,

eliminating sites that periodically serve as sources of standing water (such as tires and empty containers) and controlling weeds around ponds or drainage lagoons is more effective than applying Bti . Bti products are formulated for spray or granular applications. Bti formulated on corn cob granules is effective against mosquito larvae developing in tires and other artificial containers. (The "Asian tiger mosquito," Aedes albopictus, develops in these containers.) The corn cob granules can be blown into tire piles to provide good penetration and uniform treatment; residual control is also greater when corn cob granules are used. Bti is not very effective for the control of mosquito larvae in turbid water or waters containing high levels of organic pollutants. Bti has also been used effectively for the control of fungus gnat larvae in greenhouses and in mushroom culture beds. For these uses, Bti is applied as a drench to potting soils or culture media. Not all Bti products are labeled for fungus gnat control. Although not a Bt , another bacterium that is pathogenic to certain mosquitoes is Bacillus sphaericus. Bacillus sphaericus is especially active against larvae of mosquitoes in the genera Culex, Psorophora, and Culiseta. Effectiveness against larvae of Aedes species varies. Aedes vexans is very susceptible, but Aedes aegypti (the yellow fever mosquito) and Aedes albopictus are not. Bacillus sphaericus kills Anopheles larvae in laboratory tests, but field results have not been promising. Because Anopheles mosquitoes are true surface feeders, the bacterium would have to remain on the water surface for an extended period to be effective. Bacillus sphaericus formulations tested up to now do not remain at the surface long enough to be effective. In addition to infecting a different group of mosquito species than Bti, Bacillus sphaericus is a potentially valuable insecticide because it remains effective in stagnant or turbid water. Although Bacillus sphaericus, as it occurs naturally, does cycle and maintain itself in the environment, insecticidal formulations currently under development do not cycle in water to infect subsequent generations of mosquito larvae. Bt formulations that kill beetles. Another group of Bt isolates, including those from Bacillus thuringiensis var. san diego and Bacillus thuringiensis var. tenebrionis, are toxic to certain beetles. Within the order Coleoptera (the beetles), species exhibit great differences in susceptibility to these isolates, presumably because of differences in the insects' gut wall receptor sites where the bacterial toxins must attach. Consequently, the range of susceptible hosts for the beetle-targeted Bt formulations does not include all beetles, or even all of the species within a beetle family or subfamily. In 1988, Bacillus thurigiensis var. san diego, sold under the trade name M-One, was first registered for use against larvae of the Colorado potato beetle. This product also kills elm leaf beetle adults and larvae, but it is not pathogenic or toxic to some other key beetle pests, such as the corn rootworms and other related species. Registration of Trident, a very similar product containing Bacillus thuringiensis var. tenebrionis, is pending. Considerable research effort is now directed to identifying and developing additional Bt isolates that are active against more or different beetle species. Although entomologists and consumers alike will need to consider the specific target insect when judging the potential for these new products, Bt formulations effective against beetles seem to offer great promise. Using Bt insecticides. Insecticides containing Bt can be very effective for insect control in a variety of situations. Reviewing a few key facts about these products can help users obtain the best results possible. Each Bt

insecticide controls only certain types of insects; therefore, it is essential to identify the target pest and to confirm that the Bt insecticide controls only certain types of insects; therefore, it is essential to identify the target pest and to confirm that the Bt product label states that the insecticide is effective against that pest. Separate stages of insects differ in their susceptibility to Bt; isolates that are effective against larval stages of butterflies, moths, or mosquitos will not kill adults. Because susceptible inse must consume Bt to be poisoned, treatments must be cts directed to the plant parts or other material that the target pest will eat. Where this is not possible (for example, where pests bore into plant tissue without feeding much on the surface foliage or fruits), Bt is usually not very effective. Bt does not kill susceptible insects immediately. Poisoned insects normally remain on plants for a day or two after treatment, but they do not continue feeding and will die soon. Where Bt is applied to plant surfaces or other sites exposed to sunlight, it is deactivated rapidly by direct ultraviolet radiation. To maximize the effectiveness of Bt treatments, sprays should thoroughly cover all plant surfaces, including the undersides of leaves. Treating in the late afternoon or evening can be helpful because the insecticide remains effective on foliage overnight before being inactivated by exposure to intense sunlight the following day. Treating on cloudy (but not rainy) days provides a similar result. Production processes that encapsulate Bt spores or toxins in a granular matrix (such as starch) or within killed cells of other bacteria also provide protection from ultraviolet radiation. Registration and sale of products containing encapsulated Bt are forthcoming. Some Bt isolates (not those used in currently available insecticides) produce significant amounts of an additional toxin called thuringiensin, an exotoxin that is released outside the bacterial cell wall. Research is underway to develop commercial insecticides containing this toxin. Although thuringiensin might be lauded as "natural" because it is produced by living organisms, it is nonetheless toxic to a wide range of animal species and humans. As thuringiensin insecticides are registered and become available users should recognize the difference , between thuringiensin and other Bt products. Thuringiensin is much more toxic and should be handled much more cautiously. Although the issue of thuringiensin's toxicity to mammals is a unique characteristic that does not detract from the overall safety of registered microbial insecticides, users are advised to handle all microbial insecticides cautiously. Bacterial spores, mold spores, and virus particles become "foreign proteins" if they are inhaled or rubbed into the skin. As such, they can cause serious allergic reactions. The dusts or liquids used to dilute and carry these microorganisms also can act as allergens or irritants. These problems do not prevent the safe use of microbial insecticides, but they do mean that users should not breathe dusts or mists of microbial insecticides. Users should wear gloves, long sleeves, and long trousers during application and wash thoroughly after completing the application. These are common sense precautions that will help to prevent unexpected reactions and minimize any effects from unknown toxicity. Recent advances in biotechnology have resulted not only in improved prospects for developing new Bt insecticides but also in an ability to place Bt toxins within crop plants in a variety of ways. For example, genes directing the production of Bt toxins can be incorporated into certain plant-dwelling bacteria. When these altered bacteria grow and multiply within an inoculated host plant, the Bt toxin is produced within the plant. Efforts are underway to test this type of Bt "application" in corn to control the European corn borer. Although the development of this technology may seem ideal, the season-long, high-level control it would provide would also pose a great risk for the development of insect resistance to the Bt toxin. As genes for production of insecticidal

compounds are added to crop plants, developers must also devise methods of preventing or managing insecticide resistance in target pests. Other bacterial insecticides. Insecticides sold under the trade names Doom, Japidemic, Grub Attack, and the generic name "milky spore disease" contain the bacteria Bacillus popillae and Bacillus lentimorbus. These bacteria cannot be grown in fermentation tanks; instead, they are "cultivated" in laboratory-reared insect larvae. Products containing Bacillus popillae and Bacillus lentimorbus can be applied to turf and "watered in" to the soil below to control the larval (grub) stage of the Japanese beetle and, less effectively, some other beetle grubs. When a susceptible grub consumes spores of these bacteria, they proliferate within it, and the grub's internal organs are liquefied and turned milky white (hence the name, milky spore disease). These symptoms develop slowly, often over a period of three to four weeks following initial infection. Bacillus popillae and Bacillus lentimorbus, unlike Bt, do cycle in the environment if a substantial grub population is present at the time of application. When grubs killed by these bacteria break apart, a new batch of spores is released into the soil. These spores can survive (waiting to infect another grub) beneath undisturbed sod for a period of 15 to 20 years. Consequently, lawn applications of milky spore disease bacteria might not have to be repeated each year. Unfortunately, Bacillus popillae and Bacillus lentimorbus offer limited usefulness in Illinois and other Midwestern states because the predominant lawn grubs in this region are annual white grubs, which are larvae of beetles called chafers (genus Cyclocephala). These larvae are not susceptible (or are only slightly susceptible) to milky spore disease

Bacterial Insecticides: Bacillus thuringiensis - April 15, 2009 Jeff Schalau, Associate Agent, Agriculture & Natural Resources University of Arizona Cooperative Extension, Yavapai County

Most farmers and home gardeners are aware of the biological insecticide Bacillus thuringiensis: or Bt for short. Bt is often used to control pl ant damaging insect larvae and is preferred because it has a minimal effect on many beneficial insects and is non-toxic to humans, pets, wildlife, and other non -target organisms. Bt products are available at garden centers or can also be ordered from catal ogs and Internet suppliers. It is sold in various formulations (spray, dust, and granule) and strains (tenebrionis, kurstaki, israelensis, aizawai, san diego). Bt is a naturally occurring soil bacterium and was discovered in diseased flour moth caterpillars in 1915. Work began on it during the 1950s and it was first sold commercially in 1961. Bt kills susceptible insects with proteins called insecticidal delta-endotoxins. After feeding on these Bt -proteins, the digestive system becomes paralyzed and the infected insect stops feeding within hours. Bt -affected insects generally die from starvation: a process which can take several days. The most commonly used strain of Bt (kurstaki strain) will kill only leaf and needle feeding caterpillars. Some of the spec ific crop pests controlled are European corn

borer, cabbage looper, tomato hornworm, alfalfa caterpillar, tent caterpillar, fall webworm and leafrollers. In the past decade, additional Bt strains have been developed that control certain types of fly larvae (israelensis strain also known as Bti). These are used to control larvae of mosquitoes, black flies and fungus gnats. Bti has become one of the standard mosquito control tools in the battle against West Nile Virus. More recently, strains have been deve loped with activity against some leaf beetle larvae, such as the Colorado potato beetle and elm leaf beetle (san diego strain and tenebrionis strain). Among the various Bt strains, insecticidal activity is quite specific (i.e., Bt strains developed for mosquito larvae do not affect caterpillars). Bt is susceptible to degradation by sunlight and most formulations persist on foliage less than a week following application. Even shorter persistence occurs with some of the newer strains developed for leaf beetl e control, which may become ineffective in about 24 hours. As with any pesticide, users should always read and follow label directions . Most Bt formulations persist on foliage less than a week following application and some of the newer strains developed for leaf beetle control become ineffective in about 24 hours. This could be viewed as a disadvantage; on the other hand, it may be desirable for protecting non -target organisms. Manufacturers are experimenting with techniques to increase its persistence. O ne involves inserting Bt delta -endotoxin genes into other species of bacteria that can better survive on leaf surfaces. As strictly a stomach poison insecticide, Bt must be eaten by an insect larva to be effective. Coverage of vulnerable plant parts must be thorough. This limits Bts usefulness against pests that are susceptible but rarely have an opportunity to eat it in field use, such as codling moth or corn earworm that tunnel into plants. Additives (sticking or wetting agents) can be used with Bt appl ications to improve coverage and resist washing. Bt users will likely see insect larvae that are still alive a day or two after treatment, and may assume the pesticide was ineffective. This is a common misperception because Bt-affected insects eat little or nothing before they die yet it takes a few days for them to expire. Bt-based products also tend to have a shorter shelf life than other insecticides. Manufacturers generally indicate reduced effectiveness after two to three years of storage. Liquid formulations are more perishable than dry formulations. Shelf life is greatest when the product is stored under cool, dry conditions and out of direct sunlight.

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Bacillus thuringiensis
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Bacillus thuringiensis

Spores and bipyramidal crystals ofBacillus thuringiensis morrisoni strain T08025

Scientific classification Kingdom: Eubacteria

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Bacillus thuringiensis (or Bt) is a Gram-positive, soil-dwelling bacterium, commonly used as a biological pesticide; alternatively, the Cry toxin may be extracted and used as a pesticide. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well as on the dark surface of plants.[1] During sporulation many Bt strains produce crystal proteins (proteinaceous inclusions), called -endotoxins, that have insecticidal action. This has led to their use as insecticides, and more recently to genetically modified crops using Bt genes. There are however many crystalproducing Bt strains that do not have insecticidal properties.[2]

Contents
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1 Disc ve y and s dy 2 Use in pes c ntrol 3 Genetic engineering for pest control o 3.1 Usage o 3.2 Advantages o 3.3 Health and safety o 3.4 Limitations of Bt crops  3.4.1 Insect resistance  3.4.2 Secondary pests 4 Possible problems o 4.1 Lepidopteran toxicity o 4.2 Wild maize genetic contamination o 4.3 Possible link to Colony Collapse Disorder

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[edit] Discovery and study

B. thuringiensis was first discovered in 1901 by Japanese biologist Shigetane Ishiwatari. In 1911, B. thuringiensis was rediscovered in Germany by Ernst Berliner, who isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars. In 1976, Zakharyan reported the presence of a plasmid in a strain of B. thuringiensis and suggested the plasmid's involvement in endospore and crystal formation.[3][4] 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. Like other members of the genus, all three are aerobes capable of producing endospores.[1] Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal -endotoxins (called crystal proteins or Cry proteins), which are encoded by cry genes.[5] In most strains of B. thuringiensis the cry genes are located on the plasmid.[6][7][8] 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 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, the alkaline pH of their digestive tract activates the toxin. Cry toxin gets inserted into the insect gut cell membrane, forming a pore. The pore results in cell lysis and eventual death of the insect.[9][10]

[edit] Use in pest control

Spores and crystalline insecticidal proteins produced by B. thuringiensis have been used to control insect pests since the 1920s. [11] 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. The Belgian company Plant Genetic Systems was the first company (in 1985) to develop genetically engineered (tobacco) plants with insect tolerance by expressing cry genes from B. thuringiensis.[12][13] B. thuringiensis-based insecticides are often applied as liquid sprays on crop plants, where the insecticide must be ingested to be effective. It is thought that the solubilized toxins form pores in the midgut epithelium of susceptible larvae. Recent research has suggested that the midgut bacteria of susceptible larvae are required for B. thuringiensis insecticidal activity.[14] Bacillus thuringiensis serovar israelensis, a strain of B. thuringiensis is widely used as a larvicide against mosquito larvae, where it is also considered an environmentally friendly method of mosquito control.

[edit] Genetic engineering for pest control

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5 See also 6 References 7 F rther reading 8 External links

Bt-toxins present in peanut leaves (bottom image) protect it from extensive damage caused by European corn borer larvae (top image).[15]

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In 1995, pot to pl nt producing Bt toxin were approved safe by t eEnvironmental Protection Agency, making it t e first pesticide producing crop to be approved in t e USA.[16] By 1996, Bt Mai e, Bt Potato and Bt cotton were being grown by farmers in t e USA.[17] Bt crops (in corn and cotton) were planted on 281,500 km in 2006 (165,600 km of Bt corn and 115900 km of Bt cotton). This was equivalent to 11.1% and 33.6% respectively of global plantings of corn and cotton in 2006.[18] Claims of major benefits to farmers, including poor farmers in developing countries, have been made by advocates of the technology, and have been challenged by opponents. The task of isolating impacts of the technology is complicated by the prevalence of biased observers, and by the rarity of controlled comparisons (such as identical seeds, differing only in the presence or absence of the Bt trait, being grown in identical situations). The main Bt crop being grown by small farmers in developing countries is cotton, and a recent exhaustive review of findings on Bt coton by t respected and unbiased agricultural economists concluded that "the overall balance sheet, though promising, is mixed. Economic returns are highly variable over years, farm type, and geographical location" .[19] Environmental impacts appear to be positive during the first ten years of Bt crop use (1996 2005). One study concluded that insecticide use on cotton and corn during this period fell by 35.6 million kg of insecticide active ingredient which is roughly equal to the amount of

pesticide applied to arable crops in the EU in one year. Using the Environmental Impact Quotient (EIQ) measure of the impact of pesticide use on the environment,[20] the adoption of Bt technology over this ten year period resulted in 24.3% and 4.6% reduction respectively in the environmental impact associated with insecticide use on the cotton and corn area using the technology.[18]

[edit] Advantages
There are several advantages in expressing Bt toxins in transgenic Bt crops:
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The level of toxin expression can be very high thus delivering sufficient dosage to the pest. The toxin expression is contained within the plant system and hence only those insects that feed on the crop perish. The toxin expression can be modulated by using tissue-specific promoters and replaces the use of synthetic pesticides in the environment. The latter observation has been well documented worldwide.[18]
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[edit] Health and safety

Overall, Bt-modified crops appear to be environmentally safe.[21] The proteins produced by Bt have been used in sprays for agricultural weed control in France since 1938 and the USA since 1958 with seemingly no ill effects on the environment. [22] Bt toxins are considered environmentally friendly by many farmers[who?] and may be a potential alternative to broad spectrum insecticides. The toxicity of each Bt type is limited to one or two insect orders, and is nontoxic to vertebrates and many beneficial arthropods. The reason is that Bt works by binding to the appropriate receptor on the surface of midgut epithelial cells. Any organism that lacks the appropriate receptors in its gut cannot be affected by Bt.[23][24] There is clear evidence from laboratory settings that Bt toxins can affect non-target organisms. Usually, but not always, affected organisms are closely related to intended targets.[25] Typically, exposure occurs through the consumption of plant parts such as pollen or plant debris, or through Bt ingested by their predatory food choices. The methodology used by these researchers has been called into question.[26] Due to significant data gaps, the real-world consequences of Bt transgenics remains unclear. Not all scientific reports on Bt safety have been positive. A 2007 study funded by the European arm of Greenpeace, suggested the possibility of a slight but statistically meaningful risk of liver damage in rats.[27] While small statistically significant changes may have been observed, statistical differences are both probable and predictable in animal studies of this kind,(known as Type I errors), that is, the probability of finding a false-positive due to chance alone. In this case, the number of positive results was within the statistically predicted range for Type I errors.[citation needed ] The observed changes have been found to be of no biological significance by the European Food Safety Authority.[28]

[edit] Limitations of Bt crops

Kenyans examining insect-resistant transgenic Bt corn.

Constant exposure to a toxin creates evolutionary pressure for pests resistant to that toxin. Already, a Diamondback moth population is known to have acquired resistance to Bt in spray [29] form (i.e., not engineered) when used in organic agriculture. The same researcher has now [30][31] reported the first documented case of pest resistance to biotech cotton. One method of reducing resistance is the creation of non crop refuges to allow some non-Bt resistant insects to survive and maintain a susceptible population. To reduce the chance that an insect would become resistant to a Bt crop, the commerciali ation of transgenic cotton and mai e in 1996 was accompanied with a management strategy to prevent insects from becoming resistant to Bt crops, and insect resistance management plans are mandatory for Bt crops planted in the USA and other countries. The aim is to encourage a large population of pests so that any genes for resistance are greatly diluted. This technique is based on the assumption that resistance genes will be recessive. This means that with sufficiently high levels of transgene expression, nearly allof the heterozygotes (S/s), the largest segment of the pest population carrying a resistance allele, will be killed before they reach maturity, thus preventing transmission of the resistance gene to their progenies.[32] The planting of refuges (i. e., fields of non-transgenic plants) adjacent to fields of transgenic plants increases the likelihood that homozygous resistant (s/s) individuals and any surviving heterozygotes will mate with susceptible (S/S) individuals from the refuge, instead of with other individuals carrying the resistance allele. As a result, the resistance gene frequency in the population would remain low. Nevertheless, there are limitations that can affect the success of the high-dose/refuge strategy. For example, expression of the Bt gene can vary. For instance, if the temperature is not ideal this stress can lower the toxin production and make the plant more susceptible. More importantly, reduced late-season expression of toxin has been documented, possibly resulting from DNA methylation of the promoter.[33] So, while the high-dose/refuge strategy has been successful at prolonging the durability of Bt crops, this success has also had much to do with key factors independent of management strategy, including low initial resistance allele frequencies, fitness costs associated with resistance, and the abundance of non host plants -Bt that have supplemented the refuges planted as part of the resistance management stra tegy.[34]

[ed ] I ec e
E B DC B H

In November 2009, Monsanto scientists found that the pink bollworm had become resistant to Bt cotton in parts of Gujarat, India. In four regions, Amreli, Bhavnagar, Junagarh and Rajkot the crop is no longer effective at killing the pests. This was the first instance of Bt resistance that was confirmed by Monsanto anywhere in the world.[35] Monsanto confirmed field resistance of the worm to the Cry1Ac first generation Bollgard cotton, which expresses a single Bt gene. [36]
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[ed ] Seco d y pe
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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, [37][38] which have in some cases "completely eroded all benefits from Bt cotton cultivation [39]. Similar problems have been reported in India, with both mealy bugs [40] [41] and aphids[42].

[edit] Possible problems

[edit] Lepidopteran toxicity
The most publicised problem associated with Bt crops is the claim that pollen from Bt maize could kill the monarch butterfly.[43] This report was puzzling because the pollen from most maize hybrids contains much lower levels of Bt than the rest of the plant[44] and led to multiple follow-up studies. It appears that the initial study was flawed by faulty pollen-collection procedure; researchers fed non-toxic pollen mixed with anther walls containing Bt toxin.[45] The weight of the evidence is that Bt crops do not pose a risk to the monarch butterfly.[46] Monarch butterflies have no innate relationship to maize crops in the wild, and are not believed to consume maize pollen (or pollen of related plants) in either life stage.
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info mation: G n tica y modifi d food controv rsi s

[edit] Wild maize genetic contamination

A study in Nature reported that Bt-containing maize genes was contaminating maize in its center of origin.[47] Nature later "concluded that the evidence available is not sufficient to justify the publication of the original paper."[48] However, there still remains a controversy over the highly unorthodox retraction on the part of Nature.[49][50] In 1998, Chapela, one of the original paper's authors spoke out against Berkeley accepting a multi-million dollar research grant from the Swiss pharmaceutical company, Novartis.[49] A subsequent large-scale study, in 2005, failed to find any evidence of contamination in Oaxaca.[51] However, further research confirmed initial findings concerning contamination of natural maize by transgenic maize.[52] However, further studies, such as that published in Molecular Ecology in 2008, have shown some small-scale (about 1%) genetic contamination (by the 35S promoter) in sampled fields in Mexico.[53][54] One meta-study has found evidence for and against Bt contamination of

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maize, concluding that the preponderance of evidence points to Bt maize contamination in Mexico.[55]

[edit] Possible link to Colony Collapse Disorder

As of 2007, a new phenomenon called Colony Collapse Disorder (CCD) is affecting bee hives all over North America. Initial speculation on possible causes ranged from new parasites to pesticide use[56] to the use of Bt resistant transgenic crops.[57] The Mid-Atlantic Apiculture Research and Extension Consortium published a report in March 2007 that found no evidence that pollen from Bt crops is adversely affecting bees.[58] The actual cause of CCD remains unknown, and scientists believe that it may have multiple causes.[59]

Bacteria are also useful in providing a degree of resistance to plants. An example is the use of Bacillus thuring nsis to supply a protein that is lethal to insect when they consume it. The use of bacterial insecticides has reduced the use of chemical insecticides which is both a cost savings to the producer and less stressful on the environment
Bioflash
In 1986, from dead Anopheles Stephensis larvae in a larval habitat, Dr. Moazami (of Nature Biotechnology Company) isolated a sporulated bacterium that demonstrates rapid and high larvicidal power against the mosquitoes such as Anopheles and Culexes species. This bacterium was identified to be in genus of Bacillus. Further typing confirmed the isolated strain is a new stereotype of Bacillus Thuringiensis H-14 and named B. Thuringiensis M-H-14. In the year 1989, a pilot plant was installed on the basis of a joint project between UNESCO, IROST and UNDP. (IRA/89/035) The pilot plant production of B. Thuringiensis M-H-14 has been tested (Field evaluation) in different provinces of Iran, in order to find out the probable side effects. After this discovery, Nature biotechnology company decided to build a 500 ton plant that would be able to produce an anti malaria bioinsecticide from the B.Thuringiensis M-H-14. Commercial preparations of Bacillus Thuringiensis are used worldwide for biological control of pest insects. The advantages of these bacterial insecticides are that they are highly selective for a very limited range of target insects and are biodegradable. After years of testing and seeking a proper release mechanism, the product has finally been completed by the name of . Muddy water conditions or the suns ultraviolet radiation does, not affect unlike most other bioinsecticides. Therefore an outstanding advantage of the formulation is ts durability. When compared to other i comparative commercial products, the quantity of required per hectare is only 1.7 Kg, as against 2.5 to as much as 19 Kg for other bio-insecticides. has a slow release formulation that allows it to stay active and effective for a minimum period of 20 days, other competing products have a maximum life of 72 hours. The slow release formulation, together with the relatively small quantity required per hectare makes Bioflash highly cost effective.
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Bacillus thuringiensis - Bacterial Insecticide

Microbiologists in Europe have played a major role in developing the Bacillus thuringiensis story, as they have in many areas of research.

FEMS, The Federation of European Microbiological Societies, is now embarking on a series of major European Congresses bringing together scientists from all parts of

Europe and providing a forum for the promotion of microbiology as a crucial resource for the development of a sustainable and healthy society in the twenty -first century. The first of the series will be held in Ljubljana, Slovenia, June 29 - July 3, 2003. Bacillus thuringiensis - or Bt as it is usually called - was one of the earliest bacteria to be seen and recognised for what it is: a potent pathogen causing a rapidly spreading and fatal infection in caterpillars. It was about 1850 that Louis Pasteur, the father of microbiology, saw the rodshaped, spore -bearing cells in the bodies of silkworms suffering from a disease called flacherie and correctly described them as being the cause of the infection. As time passed it was recognised that different strains of the bacterium would infect insects other than silkworms, some of them important pests of food and agricultural crops. The idea of using a bacterial disease to control harmful p ests was an attractive one and the first bacterial insecticide was marketed in France just before the Second World War. It was a suspension of spores of Bt and it was fairly effective in killing the European Corn Borer in the field. A German bacteriologist , Berliner, had isolated Bt in 1915 from infected Flour Moth larvae - he worked in the province of Thuringia and gave the bacterium its name. Berliner noticed that alongside the spore a second, dense body grew in the bacterial cell. Soon after the end of t he Second World War this so-called parasporal body was shown to be a beautiful crystal of protein - a protoxin that, when activated by the digestive juices of the caterpillar, released a potent toxin that attacked the wall of the gut and initiated changes that killed the host. During the latter half of the 20th Century many products appeared on the market and found some application as sprays or dusts to treat crops and garden plants. They had two attractive features; they proved to be completely safe in use - the receptor molecules to which the toxin attached are not found in mammals or other beneficial life-forms - and they were judged to be "organic". The genes controlling the production of the protein toxin were isolated during the 1990s and a major development took place in 1 998 when a Bt toxin gene was inserted into the cells of a tobacco plant, which proved to be resistant to insect attack; it had its own built -in insecticide. Today much of our corn is produced from plants containing toxin genes and several other important crop plants, such as cotton, are protected from insect attack in the same way. Naturally not everyone is happy about the use of genetically modified plants in this way and Bt, once the darling of the Organic lobby, now finds itself at the centre of a vigorous social and political debate. That debate and the science behind it provide typical examples of the themes that will be explored in depth at the 1st First Congress of European Microbiologists to be organised by FEMS, in Slovenia, June 29 - July 3, 2003.

Microbial Insecticide Based on Bacillus Thuringiensis

Cultures of this species nowadays serve as the basis for large-scale production of microbial insecticides. World-wide production now constitutes a few thousand tons annually. The cultures of B. thuringiensis are closely related to those of B. cereus, which are widely distributed in nature. They differ from the former only by the formation of crystalline inclusions. For the purpose of systematics and identification of B. thuringiensis cultures, an efficient method is serotyping by H-antigen along with some biochemical properties of the cultures. Up to now 23 serotypes have also been differentiated within some of the species. From a practical point of view it is essential that there exist certain correlations in the spectrum and activity of entomocide effect between the various subspecies as well as subserotypes (Krieg, 1986). There are three basic questions that can be asked in any study of production of toxin by B. thuringiensis. What toxins are produced? In what quality? How reproducible is the fermentation? In this discussion of our program which follows we will direct most of our attention to ask what toxins are produced. However, our interpretation must not be confused by the quantities of toxin present in product powders. This is important to remember. We will use several concepts, ratios, distributions of toxicities, crystal-types, H-types, etc. It is impossible to eliminate quantity as a variable. The quantity of toxin produced in a fermentation can be influenced by the isolate of B. thuringiensis used and by medium on which it is grown. Thus two powders may differ manyfold in toxicity yet still contain the same toxin. All analyses will be made in the light of this factor and will attempt to pinpoint type as differentiated from quantity of toxin. The basic active agent, called d-endotoxin, is produced in the form of crystalline parasporal inclusions during sporulation and liberated in the medium after its completion. Principally important especially for production purposes is that the level of endotoxin insecticide activity is not correlated with its size and quantity but is defined by subspecies and the strain. For example: the strain HD-1 belonging to the subserotype kurstaki of the subspecies alesti and having the same mass of crystalline inclusions reveals 100 times more insecticide activity than these of other strains. Industrial production of more than 30 preparations has been developed in different countries and the products have been introduced in the market under different trade names since the sixties. The main sources for the production of B.t. preparations are strains of the subspecies kurstaki, galeriae and dendrolimus. The cultures of serotype-I berliner, thuringiensis and some other subspecies also produce soluble termostabile -exotoxin of a nucleotide nature, beside crystalline inclusions. A number of preparations manufactured from exotoxin producing strains are characterized by a broad spectrum of entomopathogenous action. Some producers exclude exotoxin in ready-made preparative forms because of possible side effects.

2.2.1 Morphology of Bacillus thuringiensis Bacillus thuringiensis (B.t.) is a gramm-positive bacterium forming elliptical spores, contained in unswollen sporangia, and a parasporal body (or crystal) which appears mainly as a bipyramidal shape. B. thuringiensis is a complex species divisible into subspecies and Hserotypes by serological and biochemical tests. These produce several insecticidal toxins, two of which are used in agriculture. The relative activity of each isolate against different insect species "spectrum activity" arrives partly from the combined effects of the potencies of the varying concentrations of the different insecticides that it produces. The d-endotoxin of different isolates of B.t. can kill different insect species or differ in the degree of their activity toward them. This variation of activity spectrum according to the B.t. isolate is very important. Failure to control a pest insect with a particular B.t. preparation does not mean that all preparations will fail; it mean just that the wrong isolate was selected for use against the target insect. Similarly, even though a pest species may be satisfactorily controlled by the toxins in the present commercial preparations, it is possible that a different isolate may be more effective and thus cheaper to use. The picture is further complicated because different isolates can produce more or less of the same d-endotoxin than other (Dulmage 1970). Also maximum toxin production can be achieved only by careful attention to the interaction of fermentation conditions, media and the isolates involved - there is, for example, no one medium best suited to all isolates (Dulmage 1981). 2.2.1.1 Insecticidal Toxins Produced by Bacillus thuringiensis The b-exotoxin, "heat-stable exotoxin", is a water soluble toxin highly toxic to larvae of several species of flies, while alfa-exotoxin "heat-unstable" is toxic per os to mice and to the diamond moth, Plutella xylostella (maculipennis). -exotoxin has been defined chemically as an adenine nucleotide and ATP analogue and given the name "thuringiensin". Many regulatory authorities opted to prevent its use in agriculture, because it has a terratogenic effect in insects and has mutagenic activity. The d-endotoxin in crystals of B.t. has a limited infectivity spectrum limited, so far as we know, to certain Lepidoptera, mosquitoes, chiromonids and blackflies. The crystalline glycoprotein is formed during sporulation, it is variously called the crystal, parasporal body or d-endotoxin. There is evidence that plasmids are related to crystal formation. Different numbers of plasmids are found in most serotypes of B.t. The plasmids DNA (CCC covalently closed circular DNA) has different values from 2 to 32 Mda and depend on subspecies and serotype. The strains which loss the large plasmid > 32 Mda, loss insecticide activity. There are two categories of plasmids: 36. < 15 Mda. 37. > 15 Mda. 2.2.1.2 The Role of Bacteriophages

The role of phages in B.t. genetics is most important. They provide a mechanism for a specialized and generalized transduction which constitute functional genetic transmission systems. The discovery of lysogeny in B.t. has also opened the possibility that one of the toxins is coded by a prophage. Three types of phages lyse B.t., and these are virulent, pseudolysogenic and temperate. They respectively lyse cells immediately, or form temporary or permanent associations with mitomycin C or UV light, but none have formed a lysogenic relationship with the host. 2.2.1.3 Mode of Action The crystals from various subspecies are composed of up to four proteins with molecular weights ranging from 26 to 140 kDa. In the case of the lepidopteran-specific subspecies, the major components of this crystal is a 130 - 140 kDa protein referred to as the protoxin. Upon dissolution at alkaline pH in the mitgut of targeted larvae, the protoxin is further "activated" by specific proteolysis and the toxic moiety released. Upon its attachment to specific receptors of the columnar cells in the midgut epithelium, the cells swell and are released from the basement membrane and finally burst. The insect (larvae) stops feeding, becomes rapidly dehydrated and generally dies within the next 48 hours. 2.2.1.4 Taxonomy Many strains of B.t. have now been isolated and classified upon biochemical, enzymatic and serological criteria. The generally accepted key for the taxonomic division of the species of B. thuringiensis is based on the antigenic properties of the flagella as developed by de Barjac and Bonnefoi (1962). However, B.t. strains can also be allocated to different subspecies or varieties based on their pathotypes or insecticidal activity for different insects. For the purpose of the systematics and identification of B.t. cultures, an efficient method is serotyping by H-antigen along with determining some biochemical properties of the cultures. Up to now 23 serotypes have been described. Subspecies and a number of serotypes have also been differentiated within some of the species. From a practical point of view it is essential that there exist certain correlations in the spectrum and activity of insecticide effect between the various subspecies as well as subserotypes. The cultures of a majority of serotypes of these species B.t. var. kurstaki are characterized by strong entomocide activity to Lepidoptera, products DIPEL (Abbott), Bactospeine (Philips Duphar), THURICIDE, JAVELIN (Sandoz). Some differences in the spectrum have also been observed. Thus, the cultures of serotype 5 (galleriae) are very active to Galleria mellonella, while representatives of serotype 10 (darmstadiensis-caucasicus) do not reveal any insecticide activity towards this insect. The cultures of serotype 4 are very active to Siberian silkworm and comparatively less virulent to silkworm Bombyx mori. Of great interest was serotype 14 that was describes in 1978 as B.t. subsp. israelensis. Cultures of this serotype thus produce crystalline parasporal toxin with strong larvicide activity to mosquitoes, black fly and other insect of the Simallidae. It is worth mentioning that they are characterized by specific larvicide pathogenicity and are practically harmless to mammals, plants and useful hydrobionts.

A number of countries have organized industrial production of larvicide preparations on the basis of B.t. subsp. israelensis cultures like TECNAR, VECTOBAC, BACTIMOS and others. Of special practical interest was the description of cultures named as B.t. var. tenebrionis with activity to Coleoptera. Other pathotype of B.t. var. san diego with insecticide activity to Coleoptera products like TRIDENT (Sandoz) and M-ONE (Mycogen) and pathotype of B.t. var. aizawai with insecticide activity to Lepidoptera and Diptera products like CERTAN (Sandoz). In the case of B.t. , the subspecies (varieties) and H-serotypes as well as biotypes with different enzymatic features are defined on a biological and serological basis. This is the case with the serotypes H4a4b and H6, these biotypes have a different spectrum of pathogenic properties, the discovery of antigenic subfactors (fractions) in five of the serotypes of H3, H4, H5, and H11 allows for further differentiation of various subtypes or biotypes. The first three letters of the subspecies name can be used as an abbreviation of the strain name. For instance, KUR H-3a3b (serotype) kurstaki isolated from Ephestia kuhniella (Kurstak 1962); KUR H-3a3b HD-1 kurstaki isolated from Pectinophora gossipiella (Dulmage 1970); ISR H14- israelensis. Lepidoptera B.t. var. aizawai Certan (Sandoz) Coleoptera thuringiensis var. kurstaki (mainly HD1 and HD12 isolates) has been used for some years on a limited scale in both agriculture and forestry. It is well known that B.t. is very selective in its biological action, and kills only a limited range of insects; birds, mammals and fish are not affected. This selectivity is a key to marketable product; very favorable toxicology and environmental profile, zero pre-harvest interval on vegetable crops, "bio-rational" registration like in North America. Table 2.1. Pathotypes of Bacillus: thuringiensis Pathotype Example Commercial products A, B and C subspecies Specific to Lepidoptera B.t. var. kurstaki Dipel (Abbott) Bactospeine (Philips Duphar) Thuricide, Javelin (Sandoz) Bathurin 82 (Slu1ovice) Diptera B.t. var. israelensis Vectobac (Abbott) Bactimos (Philips Duphar) Teknar (Sandoz) Moskitur (Slu1ovice) Coleoptera B.t. var. san diego Trident (Sandoz) M-One (Mycogen) However B. thuringiensis var. kurstaki has drawbacks including speed and mode of action, and solar radiation sensitivity which have limited its usage. Combining these problems, B.t. application on foliage remains a challenge in most agricultural situations. Anti-feeding effects

observed in various B.t. formulations combined with non-optimised application rates and spray technology especially in agriculture have decreased significantly its attractiveness to farmers. 2.2.1.5 Isolation of b-Exotoxin Exotoxin formulations of sufficient purity can be obtained by rechromatography on DEAEcellulose or silicagel. The purification process which does not use any adsorption on carbon makes use of the supernatant of the B.t. cultivation medium which is thickened to 1/10 of its original volume by boiling. The inactive material is then precipitated out by stepwise ethanol additions. At 90 vol. % of ethanol an active precipitate is obtained which can be further purified on cellulose or ion exchange columns. The b-exotoxin, called thuringiensin, is a nucleotide composed of adenine, ribose, glucose, and phosphorylated allaric acid. Mode of action of b-exotoxin It inhibits the synthesis of ribonucleic acid by stopping off the polymeration catalyzed by DNA-dependent RNA-polymerase. The toxicity of b-exotoxin to caterpillars. Galleria mellonella is LD50 = 0.5 ug/g, that to mice is LD50 = 18 ug/g. The spectrum of effectiveness of b-exotoxin is much broader than that of delta-endotoxin and it is lethal to insects of Lepidoptera, Coleoptera, Isoptera, and Orthoptera groups. Effectiveness varies depending on dose, time, and mode of application. Application of b-exotoxin in practice Autoclaved preparation was effective against Two-spotted mite (Tetranychus urticae), citrus mite (Panonychus citri), and Nematode Meloidogyne). Formulations containing b-exotoxin are prohibited in Europe and the U.S. A formulation called Bitoxibacillin containing 0.5-0.8 % exotoxin is in use in the USSR. In production where the bacterial mass is separated from the liquid of the cultivation medium by centrifugation, the exotoxin is removed. Growth cycle Living cycle of B. thuringiensis has two phases: vegetative and sporogenic. Growth 100 % max.

Fig. 2.1. Diagram of sporulation in B.thuringiensis l M, mesosome; CW, cell wall; PM, plasma membrane; AF, axial filament; FS, forespore

septum; IF, incipient forespore; Ol, ovoid inclusion; PC, parasporal crystal; F, forespore; IM, inner membrane; OM, outer membrane; PW, primordial cell wall, E, exosporium, LC, lamella spore coat; OC, outer spore coat; C, cortex; IMC, incorporated mother cell cytoplasm; S, mature spore in an unlysed sporangium. (Bechtel and Bulla et al. 1980). I. phase, 7 hours AF (axial filament) II. phase, 7-8 hours FS (forespore septum) III. phase, 8-9 hours F (forespore) IV.- VI phase, 9-12 hours E, LC, OC, C and IMC VII. phase, after 12 hours S and lyse sporangium

2.2.1.6 Recovery of Bacillus thuringiensis Using the lactose-acetone technique (after Dulmage et al., 1970)

2.2.1.7 Bioassay Procedures In all assays, the powder is administrated to the insects by mixing it into their diet, with larvae being allowed to feed ad libitum on the powder-diet mixtures. 2) The effect of this exposure is judged by a single criterion: death. A severely retarded or moribund larva is considered alive if it could move. The assays measured only per cent dead. 3) Preliminary assays determined kill at two levels, usually 500 ug and 50 ug powder/unit of diet. If activity is sufficient, repeat assays with an appropriate series of dilutions to determine the LC50.

LC50 and International Unit The first generally accepted standard was prepared in France from a fermentation of H -type thuringiensis and called "E-61". E-61 was assigned a potency of 1000 IU/mg and recommended as an international standard in 1966 (Burges, 1967). When the HD strain of -1 H-type kurstaki was selected for commercial production of B.t. in the USA, a formulation of HD-1, labelled HD-l-S-1971 was assigned a potency of 18,000 IU/mg on the basis of assays against E-61, using Trichoplusia ni as test insect (see ANNEX 5). How oes Bt sed i sect esist ce work?

e of t e ost widel sed enetic engineering strategies is t e development of i , a common soil Bt ased resistance to insects. Bt is short for B ill t i i acteri m that produces an insect toxin. Applications of the Bt acteria in powder form have een used to kill insects in agriculture for many years, an d Bt acterial insecticides have een a particularly important insect control tool to organic farmers. In the last few years, several crops have een genetically engineered to produce their own Bt toxins, making them resistant to specific groups of insects. Genetically engineered GE plants with Bt ased insect resistance produce an insect toxin in all of their tissues. Bacillus thuringiensis acteria produce proteins called delta -endotoxins that are toxic to insects when ingested. hen an insect consumes the protein, protease enzymes in the insect s digestive system cut the normally non-toxic protein into a smaller piece that is highly toxic to insects. he smaller, activated form of the delta -endotoxin inds to a specific receptor on the surface of cells lining the insect s gut, causing a disruption of electrolyte alance, leading to death. Bt -toxins have een considered very safe for human consumption ecause the intestinal walls of mammals do not have the endotoxin receptor necessary for the toxic effect , and the proteins are degraded uickly in the stomach. here are actually many variants of Bt-toxins found in nature. ne of the uni ue features of this family of insect toxins is that different toxins affect different groups of insects. or example, the Bt-toxin most commonly used in genetic engineering, YIA , kills only moths and utterflies epidoptera), ut not insects in named other insect families. he various delta -endotoxins are given names that egin with Y ecause the endotoxins normally e xist in a crystalline form. All Bt-plants to date have only one version of endotoxin each. Eventually, combining multiple versions of Bt-toxins in the same plant could provide resistance to several different families of insects at the same time. his strategy could also be used to prevent insects from developing their own resistance to Bt: it is much more difficult for insect populations to evolve resistance to or different Bt -toxins at the same time.
b beg e dac f ba `Y

Back

Consider Microbial Insecticides

Microbial insecticides battle damaging insects by enlisting the aid of microscopic, living organisms viruses, bacteria, fungi, protozoa, or nematodes. They are unconventional insecticides, but they can be applied in conventional ways as sprays, dusts, or granules. Microbial insecticides are essentially nontoxic. They also do not pose a disease risk to wildlife, humans, and other organisms not closely related to the target insect. In fact, they can be applied when a fruit or vegetable is almost ready for harvest. Most microbial insecticides are toxic to a single species or group of insects, so you can often target a pest without the risk of killing beneficial insects in the process. Also, most microbial insecticides can be used in conjunction with conventional insecticides.

In a few cases, the microorganisms used in these products can become established in an insect population or its habitat. This means the insecticide can provide control for several weeks or seasons.

On the other hand, if several types of insects are causing damage to your lawn or garden, a single microbial insecticide will not be able to handle them all. As a result, there is a limited market for some microbial insecticides, so these products are not widely available or are relatively expensive.

Another disadvantage of microbial insecticides is that heat, drying out, or exposure to sunlight reduces the effectiveness of several types of microbial insecticides. Therefore, proper timing and application are especially important for some products.

The most popular microbial insecticides in the United States are preparations of the bacterium known as Bt. Bacterial insecticides must be eaten by the pest to do their job. The most widely used Bt products are pathogenic and toxic only to caterpillars the larvae of butterflies and moths. Other varieties of Bt are available to control Colorado potato beetle larvae and certain mosquito larvae

Bt-based bacterial insecticides More than 30 Bt formulations are on the market in the United States, and most of these are complex mixtures based on sporulated cells containing the spores and array of toxins found in either B.t. kurstaki, which is used to control a wide range of moth pests, or B.t. israelensis, used to control the larvae of numerous mosquito and blackfly species. To produce the insecticides, companies grow up the bacteria, then harvest the spores and parasporal bodies, the latter which are crystalline inclusions that contain the toxins. Both subspecies owe their success to their broad spectrum of activity and the absence of any widespread insect resistance after many years of use. These characteristics are due to the composition of the specific Bt bacterial strain used in the products, especially to the toxin complexity of the parasporal body, which in each case contains four major proteins, and to the moderate frequency of use. The first commercial formulations of Bt marketed over 30 years ago, such as Dipel and Thuricide, and still commonly used today, are those based on the HD1 isolate of B.t. kurstaki. The parasporal body of HD1 consists of four Cry proteins, Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A (fig. 1). These vary in their insect spectrum and specific toxicity (table 2), and in combination give this isolate a broad spectrum of activity against a wide range of caterpillar species attacking field (cotton, corn, soybeans), vegetable (tomatoes, broccoli, lettuce, cabbage), fruit (strawberries, grapes, peaches) and forests (deciduous and fir trees). This protein complexity probably also accounts for the lack of economically important resistance after more than 30 years of use in all but a very few species of lepidopterans, the notable exception being larvae of the diamondback moth, P. xylostella. Toxin complexity can delay resistance because there is usually at least one different receptor for each of the Cry proteins, even though some can share the same receptor. Whereas insects in a target population are unlikely to be sensitive to all four Cry proteins, moderate to high sensitivity to two or three reduces the probability that a substantial number of insects within the population will be resistant to all toxins. Under conditions of moderate Bt usage, that is, low selection pressure, the frequency of individuals containing a full set of resistance genes remains low, which translates into little or no Bt resistance. In addition to the parasporal body toxins, B.t. kurstaki and many Bts produce several other components that improve the efficacy of the Cry proteins, increasing its insect

spectrum, and impeding resistance. These include the antibiotic zwittermicin, which increases the toxicity of Cry proteins by an unknown mechanism, and the spore. The spore has its greatest effect against insects with moderate or low sensitivity to the Cry toxins, such as larvae of gypsy moth (Lymantria dispar), the diamondback moth (Plutella xylostella) and the beet armyworm (Spodoptera exigua). In larvae of these species, after an initial intoxication resulting from activation of the Cry toxins in the midgut, the spore germinates and produces enzymes (phospholipases and proteases). These contribute to the lysis of gut cells by degrading cell membranes. With respect to B.t. israelensis, strains derived from the ONR60A isolate used in commercial products are the most successful. The parasporal body of this isolate is highly toxic to mature larvae of Aedes and Culex mosquitoes as well as to the larvae of numerous species of blackflies. This high toxicity and broad spectrum among mosquitoes, blackflies, and related dipterans are due a complex of four major proteins, Cry4A, Cry4B, Cry11A and Cyt1A (fig. 1). Aside from the number of toxins, the Cry and Cyt proteins of B.t. israelensis contribute to its effectiveness in two ways. First, combinations of Cyt and Cry proteins as well as combinations of Cry proteins interact synergistically increasing the toxicity of each other from three-to fivefold. Second, laboratory studies suggest that the Cyt1A protein delays the development of resistance to the Cry4 and Cry11A proteins. In experiments with larvae of the mosquito Cx. quinquefasciatus, toxin combinations containing Cyt1A led to only a 3.2-fold level of resistance after 28 generations, whereas high levels of resistance, from 90 to greater than 100-fold, were obtained in combinations of Cry toxins lacking Cyt1A. The mechanism by which the CytA protein enhances toxicity and delays resistance is not known. In the field, products based on B.t. israelensis have been in use for well over a decade, yet no resistance has reported in mosquito or blackfly populations. This lack of resistance may be due not only to the complex protein composition of the B.t. israelensis parasporal body, but to treatment of only limited breeding areas, which permits seasonal and year-to-year mixing of treated and untreated mosquito populations, thereby reducing the buildup of resistance genes in the treated populations.