Commercial Production of Biopesticides with reference to

Bacillus Thurigiensis

What are biopesticide?

Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals. For example, canola oil and baking soda have pesticidal applications and are considered biopesticides.

Biopesticides fall into three major classes pesticide - consist of a microorganism (e.g., a 1. Microbial
bacterium, fungus, virus or protozoan's .

2. Plant-Incorporated-Protectants (PIPs)-pesticidal substances that plants produce from genetic material that has been added to the plant. 3. Biochemical pesticides-naturally occurring substances that control pests by non-toxic mechanisms.

What are the advantages of using biopesticides?

Biopesticides are usually inherently less toxic than conventional pesticides. Biopesticides generally affect only the target pest and closely related organisms, in contrast to broad spectrum, conventional pesticides that may affect organisms as different as birds, insects, and mammals.

Biopesticides often are effective in very small quantities and often decompose quickly, thereby resulting in lower exposures and largely avoiding the pollution problems caused by conventional pesticides.

BACILLUS THURIGIENSIS AS BIOPESTICIDE

The most widely used microbial pesticides are subspecies and strains of Bacillus thuringiensis, or Bt. Each strain of this bacterium produces a different mix of proteins, and specifically kills one or a few related species of insect larvae. some Bt's control moth larvae found on plants, other Bt's are specific for larvae of flies and mosquitoes. The target insect species are determined by whether the particular Bt produces a protein that can bind to a larval gut receptor, thereby causing the insect larvae to starve.

WHAT IS NATURAL BACILLUS THURIGIENSIS? Gram Positive

Spore Forming Bacteria During speculation produce protein crystal (cry) called endotoxins, that have insecticidal action In most strains of B. thuringiensis, the cry genes are located on a plasmid (in other words, cry is not a chromosomal gene in most strains).

Bacillus thurigiensis toxin

Cry toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles), Hymenoptera (wasps, be es, antsand sawflies) and nematodes. Bt has to be eaten to cause mortality. The Bt toxin dissolve in the high pH insect gut and become active. The toxins then attack the gut cells of the insect, punching holes in the lining. The Bt spores spills out of the gut and germinate in the insect causing death within a couple days.

How BT Works?

1. Insect eats Bt crystals and spores. 2. The toxin binds to specific receptors in the gut and the insects stops eating. 3. The crystals cause the gut wall to break down, allowing spores and normal gut bacteria to enter the body.
4.

The insect dies as spores and gut bacteria proliferate in the body.

Application of BT
Bt is used in agriculture as a liquid applied through overhead irrigation systems or in a granular form for control of European corn borer. The treatments funnel down the corn whorl to where the feeding larvae occur. Bt useful in applications where pesticide drift onto Gardens is likely to occur, such as treating trees and shrubs. To control mosquito larvae, formulations containing the israelensis strain are placed into the standing water of mosquito breeding sites. Use of Bt (israelensis) for control of fungus gnat larvae involves drenching the soil.

Insects Controlled by Bt
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. Fruit crop insects Tent caterpillar. Pine budworm Western spruce budworm.

Insects Controlled by Bt continue..

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.

Disadvantages
Bt is susceptible to degradation by sunlight The highly specific activity of Bt insecticides might limit their use on Crops where problems with several pests occur, including nonsusceptible insects (aphids, grasshoppers, etc.). Since Bt does not kill rapidly, users may incorrectly assume that it is ineffective a day or two after treatment.

Bt-based products tend to have a shorter shelf life than other insecticides.

Advantages
The specific activity of Bt generally is considered highly beneficial 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.

Perhaps the major advantage is that Bt is essentially nontoxic to people, pets and wildlife.

Plants genetically engineered with the Bt gene

Since 1996, a wide range of crop plants have been genetically engineered to contain the delta-endotoxin gene from Bacillus thuringiensis.

These "Bt crops" are now available commercially in the USA. They include "Bt corn", "Bt potato", "Bt cotton" and "Bt soybean". Such plants have been genetically engineered to express part of the active Cry toxin in their tissues, so they kill insects that feed on the crops. In some respects, this is an important technological and practical development, because it ensures that only those insects that attack the crop will be exposed to Bt toxins - there is no risk to other types of insect. However, there is also a "downside", because the target insects are perpetually exposed to toxins and this creates a very strong selection pressure for the development of resistance to the toxins. Various cropmanagement strategies are being developed to try to minimise this risk.

Biopesticide Production from Bacillus thuringiensis: An Environmentally Friendly Alternative

Ninfa M. Rosas Garca*
Laboratorio de Biotecnologa Ambiental. Centro de Biotecnologa . Genmica-IPN. Blvd. del Maestro s/n. Reynosa, Tamp. CP 88710 Mxico

Received: October 29, 2008; Accepted: November 26, 2008; Revised: November 28, 2008

Introduction
The bacterium Bacillus thuringiensis was discovered by Shigetane Ishiwata in 1901.

The bacterium was isolated from diseased larvae of Anagasta kuehniella, and this finding led to the establishment of B. thuringiensis as microbial insecticide.
The first record of its application to control insects was in Hungary at the end of 1920, and in Yugoslavia at the beginning of 1930s, it was applied to control the European corn borer. During the following two decades, several field tests were conducted to evaluate its effectiveness against lepidopterans, both in Europe and in the United States.

results favored the development of formulations against on this pathogen. Subsequently, the first commercial product was produced in 1938 by Libec I France. serious environmental and health issues began to be recognized by the presence of chemical residues in food, water, and air during 1950. To counteract this contamination, attention and efforts were directed to the use of biological control agents including insect pathogens. As an entomopathogenic organism, B.thuringiensis fulfills all these requirements.

MODE OF ACTION
The C-terminal extension found in the long protoxins is necessary for toxicity and is believed to play a role in the formation of the crystal within the bacterium. During proteolytic activation, peptides from the N terminus and C terminus are cleaved from the full protein. During proteolytic activation, peptides from the N terminus and C terminus are cleaved from the full protein.

Activated toxin binds to receptors located on the apical microvillus membranes of epithelial midgut cells.

For Cry1A toxins, at least four different binding sites have been described in different lepidopteran insects: 1. a cadherin-like protein(CADR), 2. a glycosylphosphatidyl-inositol (GPI)-anchored aminopeptidase-N (APN), 3. a GPI-anchored alkaline 4. phosphatase (ALP) and a 270 kDa glycoconjugate .

After binding, toxin adopts a conformation allowing its insertion into the cell membrane. Subsequently, oligomerization occurs, and this oligomer forms a ion channel induced by an increase in cationic permeability within the functional receptors contained on the brush borders membranes
This causing disruption of membrane transport and cell lysis, and leading to insect death

Photograph shows lepidopteran larvae susceptible to B.thuringiensis toxic activity.

A) Nine day-old, healthy larvae, notexposed to B. thuringiensis

B) Nine-day old larvae exposed to sublethal concentration of B. thuringiensis. Larvae exhibit a smaller size due to their poor feeding.

C) Larvae exposed to lethalconcentration of B. thuringiensis.

BACILLUS THURINGIENSIS-BASE FORMULATIONS

The active ingredient in commercial formulations is the sporecrystal complex. It is more effective to use and cheaper to obtain than the crystals alone, which are frequently used in experimental tests. A great variety of ingredients have been employed to prepare formulations, including liquid or solid carriers, surfactants, coadjuvants, fluidity agents, adherents, dispersants, stabilizers, moisturizers, attractants, and protective agents among others

An interesting and recent example of these kind of inert ingredients is the superabsorbent starch graft copolymer, which combined with a B. thuringiensis strain among many other pesticides constitutes a novel formulation that could be applied in an agricultural environment.
The biological activity of one particular bioinsecticide was enhanced when the antibiotic zwittermycin was added. This combination was also successful in pest control.

Bacillus thuringiensis-based products are classified according to their formulation. first-generation products- All products containing a blend of spores and crystals from a native strain. Second generation products -based on spores and crystals from a B. thuringiensis strain bearing artificially introduced genes coding for delta-endotoxins from several strains, in order to increase the activity spectrum against other insect pests. Third generation products- Formulations containing dead recombinant Pseudomonas fluorescens cells transformed with genes coding for deltaendotoxins

Types of Bacillus thuringiensis Formulations and Their Applications for Insect Pest Control
Emulsions Encapsulations Wettable powders Granules Powders Briquettes

Formulation

Application

Agriculture and forestry Agriculture and forestry Gardens and agriculture Agriculture and forestry Forestry Aquatic systems

Most Common Commercial Bacillus thuringiensis-based Bioinsecticides

Company Commercial Name Active Ingredient Target Pest

CHIMERIC CRYSTAL PROTEINS

In recent years, hybrid delta-endotoxins have arisen as proteins with potential for enhanced toxic activity or improved properties. Recent advances in molecular methodologies have allowed gene fusions and chimeric protein construction. This construction can include alteration of amino acid sequences, fusion of portions of two or more proteins together into a single recombinant protein, or alteration of the genetic sequences encoding for proteins with commercial application.

CURRENT & FUTURE DEVELOPMENTS

There is a great amount of scientific research on the bacterium B. thuringiensis, involving aspects ranging from its molecular biology to its activity in a bioinsecticide. The development of formulations with biodegradable ingredients is a favored approach for the reduction of chemical insecticide use, which can threaten the environment.

REFERENCES
[1] Aizawa K. Shigetane Ishiwata: His discovery of sottokin (Bacillus thuringiensis) in 1901 and subsequent investigations in Japan. Proceedings of a Centennial Symposium Commemorating Ishiwatas Discovery of Bacillus thuringiensis. Japan 2001. [2] Lord JC. From Metchnikoff to Monsanto and beyond: The path of microbial control. J Invertebr Pathol 2005; 89 (1): 19-29. [3] Cern JA. Productos comerciales nativos y recombinantes a base de Bacillus thuringiensis. Bioinsecticidas: Fundamentos y aplicaciones de Bacillus thuringiensis en el control integrado de plagas. In: Caballero P, Ferr J. Eds, Phytoma-Espaa 2001; 153-168. [4] Aronson A, Beckman W, Dunn P. Bacillus thuringiensis and related insect pathogens. Microbiol Rev 1986; 50 (1): 1-24. [5] Nester EW, Thomashow LS, Metz M, Gordon M. 100 years of Bacillus thuringiensis: A critical scientific assessment. American Academy of Microbiology. Washington DC 2002. [6] King E. Control biolgico de insectos y caros plaga. Avances recientes en la biotecnologa en Bacillus thuringiensis. In: Galn-Wong LJ, Rodrguez-Padilla C, Luna-Olvera HA, Eds. Universidad Autnoma de Nuevo Len 1996; 13-19. [7] Margalith Y, Ben-Dov E. Biological control by Bacillus thuringiensis subp. israelensis. Insect pest management, techniques for environmental protection. In: Rechcigl JE, Rechcigl NA, Eds.Lewis Publishers 2000; 243-301. [8] Aizawa K. Selection and utilization of Bacillus thuringiensis strains for microbial

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