Published on March 13, 2014
Presented By M.Z.Arifeen M.Phil Scholar Department of Biotech University of Malakand. Date of Presentation 10/02/2014
•Bacillus thuringiensis (Bt) is a Gram-positive, spore forming, soil bacterium. •When resources are limited, vegetative Bt cells undergo sporulation, synthesizing a protein crystal, the insecticidal crystal proteins (ICPs) or Cry Proteins. •These Cry proteins are coded by genes (cry genes) harbored in megaplasmids • For over 50 years, Bt has been applied to crops in spray form as an insecticide, containing a mixture of spores and the associated protein crystals • The development and commercialization of insect-resistant transgenic Bt crops expressing Cry toxins revolutionized the history of agriculture •High specificity and potency, •Reduction in chemical pesticide applications, • Increased crop yield.
• The Cry protein is made as an inactive protoxin. • Cry protein has to be eaten to cause mortality. • Conversion of the protoxin (e.g., 130 kDa) into the active toxin (e.g., 68 kDa) called delta endotoxin requires the combination of a slightly alkaline pH (7.5-8) and the action of a specific protease(s) found in the insect gut • The active toxin binds to protein receptors on the insect gut epithelial cell membrane. • The toxin forms pores in the insect gut.
B. thuringienis parasporal crystal composed of Cry1 protoxin protein. Conversion of the 130-kDa protoxin into an active 68-kDa toxin requires an alkaline environment (pH 7.5 to 8) and the action of a specific protease, both of which are found in the insect gut. The activated toxin binds to protein receptors on the insect gut epithelial cells.
CADR= Cadherin receptor ALP=Alkaline phosphatase GCR =Glyco-conjugate receptor APN=Amino peptidase - N Receptors for Bt Toxin
BT crystalline Toxin 200px. Normal gut bacteria BT SPORES
• There are three domains in this delta endotoxin. • Domain I and Domain II are very conservative. • Domain III is highly variable to attach to different receptors. • There are over 200 known variants for this cry or cryt protein • Domain I is the prime candidate for formation of the transmembrane lytic pore. • Domain II is believed to have a major role in receptor binding and thus in specificity determination • domain III of the Bt toxin structure may play some role in protecting the toxins against gut proteolysis.
Bt action is very specific. Different strains of Bt are specific to different receptors in insect gut wall. Bt toxicity depends on recognizing receptors, damage to the gut by the toxin occurs upon binding to a receptor. Each insect species possesses different types of receptors that will match only certain toxin proteins, like a lock to a key. The crystal proteins bind specifically to certain receptors in the insect's intestine. Not all insects carry the same receptors allowing for high species specificity. Humans and other vertabrates do not have these receptors in their bodies, so the toxin is unable to affect us. It is because of this that farmers have to be careful to match the target pest species with a particular Bt toxin protein which is specific for that insect. This also helps the beneficial insects because they will usually not be harmed by that particular strain of Bt.
Japanese biologist, Shigetane Ishiwatari was investigating the cause of the sotto disease (sudden- collapse disease) that was killing large populations of silkworms when he first isolated the bacterium Bacillus thuringiensis (Bt) as the cause of the disease in 1901. Ernst Berliner isolated a bacteria that had killed a Mediterranean flour moth in 1911, and rediscovered Bt. He named it Bacillus thuringiensis, after the German town Thuringia where the moth was found. In 1915, Berliner reported the existence of a crystal protein within Bt.
Farmers started to use Bt as a pesticide in 1920. France soon started to make commercialized spore based formulations called Sporine in 1938 These Bt pesticide were used in the form of spray.
Bt products such as sprays are rapidly washed away by rain, and degrade under the sun's UV rays. there were many insects that are not susceptible to any of the limited number of Bt strains known at the time. All the Bt strains known at the time were toxic to lepidopteran (moth) larvae only. There were also some insects that live within the plant or underground where the Bt sprays could not reach.
• Bt plants have genes for the Bt toxins engineered to produce ICP toxic to the pest species of concern. • As the insect feeds on the Bt plant, it ingests the ICP and suffers the same fate as if it ingested leaf tissue sprayed with Bt. • At the end of 2010, an estimated 26.3 million hectares of land were planted with crops containing the Bt gene globally(James 2011). The chief advantages to Bt plants: • The pests hiding inside plant parts controlled effectively; • Multiple sprays are not needed; • Give opportunity to get rid of chemical pesticide like DDT, Organophosphate etc • A disadvantage of Bt plants is that insect-specific ICPs cannot be changed during a growing season.
•As it turns out, nature has its own biotechnologist called Agrobacterium tumefaciens which induces the growth of tumours on woody plants. These tumours are engineered by A.tumefaciens to produce a special food for the bacteria (opines) that plants normally cannot make. •These tumours arise from a unique bacterial transformation mechanism involving the Ti-plasmid which coordinates the random insertion of a subset of its DNA (t-DNA) containing opine synthase genes into a plant chromosome. By replacing portions of the t-DNA sequence with genes of interest (such as Cry). •Researchers have been able to use this transformational mechanism and confer new traits to many flowering plants including grasses such as corn and rice etc.
In March 1995, the first Bt crop deregulated in the U.S. were seven lines of Colorado Potato Beetle Resistant Bt Potato by Monsanto. Since then, many more Bt crops have been deregulated, engineered to produce a variety of different Bt proteins from various subspecies of Bt. Bt crops include: Corn: European Corn Borer Resistant Corn (first deregulated in the U.S. in May 1995) Corn Rootworm Resistant Corn (first deregulated in the U.S. in October 2002) Cotton: Lepidopteran Resistant Cotton (first deregulated in the U.S. in June 1995) Potato: Colorado Potato Beetle Resistant Bt Potato (first deregulated in the U.S. in March 1995) Potato Tuber Moth Resistant Bt Potato (being developed in South Africa) Soybean: Bt Soybean (first deregulated in the U.S. in October 2011, not yet sold commercially) Tomato: Lepidopteran Resistant Tomato (first deregulated in the U.S. in March 1998, not yet sold commercially)
Kurstaki strain (Biobit, Dipel, MVP, Steward, Thuricide, etc.): Vegetable insects Cabbage worm (cabbage looper, imported cabbageworm, diamondback moth, etc.). Tomato and tobacco hornworm. Field and forage crop insects European corn borer (granular formulations have given good control of first generation corn borers). Alfalfa caterpillar, alfalfa webworm. Fruit crop insects Leafroller. Achemon sphinx. .
Tree and shrub insects Tent caterpillar. Fall webworm. Spiny elm caterpillar. Western spruce budworm. Israelensis strains (Vectobac, Mosquito Dunks, Gnatrol, Bactimos, etc.) Mosquito. Black fly. Fungus gnat. San diego/tenebrionis strains (Trident, M-One, M-Trak, Foil, Novodor, etc.) Colorado potato beetle. Elm leaf beetle. Cottonwood leaf beetle
• The specific activity of Bt generally is considered highly beneficial. Unlike most insecticides, Bt insecticides do not have a broad spectrum of activity, so they do not kill beneficial insects. This includes the natural enemies of insects (predators and parasites), as well as beneficial pollinators, such as honeybees • Perhaps the major advantage is that Bt is essentially nontoxic to people, pets and wildlife. This high margin of safety recommends its use on food Crops or in other sensitive sites where pesticide use can cause adverse effects. • Used as alternative to DDT and organophosphates since 1920s
• Together with the reduction of pesticide application and cost reduction, Bt crops have brought tremendous benefit to both the environment and farmers . • Expanded use of transgenic crops for insect control will likely include more varieties with combinations of two or more Bt toxins, novel Bt toxins • Modified Bt toxins that have been genetically engineered to kill insects resistant to standard Bt toxins.
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