BIOPESTICIDE

Introduction

·        Bacillus thuringiensis is a gram negative, rod shaped, spore forming bacterium that forms parasporal, crystalline inclusion bodies during sporulation period.

·        The proteinaceous inclusions are called crystal proteins or δ-endotoxins , which are toxic to class insecta.

·        The Bt preparations are used as biopesticide against Lepidoptera, Diptera and Coleoptera.

·        The δ-endotoxins of certain Bacillus thuringiensis strains are toxic against other insect orders such as Hymenoptera, Homoptera, Orthoptera and Mallophaga and against certain nemotodes, mites and protozoa.

·        Hence, BT is well accepted as eco-friendly alternative to chemical pesticides.

Ecology and Occurrence

·        Bacillus thuringiensis is ubiquitous in nature.

·        Various workers isolated Bacillus thuringiensis from several sources like Soil, Water, Saw dust, Grain dusts, Dead insects, Dried tobacco leaves, Warehouses, Stored products, Phylloplane, Compost, Mammal faeces etc.

·        Among these soil supports maximum number of Bacillus thuringiensis isolates.

Diversity

·        Besides biochemical and morphological characteristics, the diversity in Bt is studied using flagellar H-antigen agglutination reactions.

·        The diversity is also studied by techniques such as polymerase chain reaction, ELISA, western blotting of total proteins, plasmid analyses, restriction patterns and bioassays.

·        Classification based on cry genes of Bacillus thuringiensis is the acceptable one. It accepts newer submission of novel cry genes and position them phylogenetically, based on amino acid homology.

·        SDS-PAGE and PCR are routinely used to characterize cry genes because they are highly sensitive and relatively fast than other methods.

Insecticidal crystal proteins

·        The δ-endotoxins are most potent toxins encoded in cry genes of Bacillus thuringiensis and are also called as cry proteins.

·        The molecular weight of cry proteins is between 130-140 kDa, depending on the subtype.

Classification of cry proteins

Hofte and whitely (1989) classification:

·        The cry genes were classified in to four major classes based on their protein toxicity.

·        Cry I: lepidopteran specific.

·        Cry II: Lepidopteran and Dipteran specific.

·        Cry III: Coleopteran specific.

·        Cry IV: Dipteran specific.

·        Several workers reported Bt toxins with toxicity overlapping with classes. So, another classification scheme was introduced by Crickmore et al. (1998).

Crickmore et al. (1998) classification:

·        The cry genes are now assigned with four letter code based on their sequence similarity.

·        The four letter code start with an Arabic number (Primary level of 45% identity),

·        followed by an uppercase letter (Secondary: 75% identity),

·        lowercase letter (Tertiary: 95% identity),

·        and Arabic number, if necessary (Quaternary: further relatedness)

·        ex: cry 1Aa and cry 1Ab are 95% identical

cry 1Aa and cry 1Ba are 75% identical

cry 1Aa and cry 7Aa are 45% identical

·        Other virulent genes of Bacillus thuringiensis like cyt genes and vip genes are grouped as different classes.

·        An advisory committee on cry gene nomenclature has been set up.

The official website address is given below;

 http://www.biols.susx.ac.uk/home/Neil_Crickmore/B.thuringiensis/

Structure of cry toxins

·        The structure of the cry toxin protein consists of three domains

·        It also contains eight conserved amino acid blocks among these first five blocks are highly conserved and are concentrated mainly in the centre of the domains and at the junctions between the domains.

·        Domain I is made up of seven α helices, which is responsible for inserting in to the gut membrane and creating a pore.

·        Domain II appears as a triangular column of 3 anti-parallel β-sheets, which is responsible for binding to the receptors on the epithelial linings of the insect midgut.

·        Domain III contains antiparallel β-strand in a β-sandwich form. Its function remains unknown.

STRUCTURE OF CRY TOXIN

Mechanism of Action

The mechanism of action of Bacillus thuringiensis cry proteins involves

·        Upon ingestion of the endotoxin, crystalline inclusions are dissolved in alkaline pH of the insect gut juices to yield the protoxin.

·        The major proteases of the insect midgut are trypsin-like and chymotrypsin like proteases. These proteases act on protoxin and cleave a considerable stretch of C-terminal. The trimmed N-terminal is the functional toxin.

·        The Domain II of the toxin binds reversibly to specific receptors on the apical brush border of the midgut microvillae.

·        Domain I of toxin inserts itself in to apical membrane to create ion channels or pores in the membrane. This stage is irreversible and renders the toxin insensitive to further protease attack.

·        The pores formed are permeable to K+ ions, other cations, and solutes such as sucrose. This leads to disruption of gut integrity resulting in death of the insect through starvation and/or septicemia.

Large Scale Production of Bacillus thuringiensis by Fermentation

·        During fermentation, Bacillus thuringiensis vegetatively multiplies until it reaches early stationary phase.

·        When a critical nutrient is depleted, it commits sporulation and production of crystalline inclusion bodies, which harbours δ-endotoxin.

·        After complete sporulation, cells lyse to release spores and crystal proteins in the medium.

·        Carbon sources: Carbon is provided by mono, di and poly saccharides such as glucose, starch, molasses, etc. In large scale production, cheap carbon sources like jaggery, corn, black strap molasses, whey, etc. are used to reduce the cost. Since most of the industrially useful strains are not able to utilize high levels of sugars such as sucrose, attempts have been made to use industrial by products like fish meal, molasses, sago, paddy straw and milk whey. In batch fermentation the carbon source is the major limiting factor for growth of cells.

·         Nitrogen Sources: Several workers reported that inorganic ammonium components such as ammonium sulphate  did not support the growth of Bacillus thuringiensis. However, organic nitrogen sources such as meat preparation, fish meal and soybean flour were reported to support luxurious growth. In continuous fermentation, nitrogen sources instead of carbon sources was found to be the major limiting factors for the growth of Bacillus thuringiensis.

·        Metal ions such as Ca²⁺ and Mn²⁺ are critical in the medium for the growth of Bacillus thuringiensis. PO₄^{3- ­} is essential for the glucose utilization through EMP pathway.

·        An O₂ uptake rate of 150 mM/h/L was reported.

·        The high O₂ uptake is coupled with generation of large amount of heat during vegetative growth. Hence, adequate cooling capacity is essential.

·        Harvesting Bt from submerged fermentation is often difficult due to low concentration of the products. Bt toxins are usually concentrated centrifugation and filteration prior to drying. In a continuous centrifuge the product form 2-15% suspended solid. Alternatively foam floatation is also used. Spray drying the solid at 175⁰C is advocated.

Formulations

The aim of the formulation is to protect the biopesticide from agents that denature the δ-endotoxin and if possible to increase the activity of biopesticide. Generally, there are two types of formulations.

·        Cells / spores of Bacillus thuringiensis are as such sprayed. Since, one organism (Bacillus thuringiensis) is used to control another organism (insects), it is called biological control.

·        The crystalline inclusion bodies / extracted toxins are sprayed. This is called biopesticide.

·        Both are available in various forms such as wettable powder, disposable granules, dusts, microgranules, aqueous based liquid, oil based liquid and stickers with uniform coating of biopesticide. In any case, the formulation should include suitable agent to protect the biopesticide from UV light and physical factors such as rain etc.

Demerits of Bacillus thuringiensis

·        The greatest backlog is the development of resistance by insect species.

·        The loss against UV light, rain etc.