ENTOMOPATHOGENIC BACTERIA - Bacillus thuringiensis

ENTOMOPATHOGENIC BACTERIA - Bacillus thuringiensis

General Characteristics of Bacillus thuringiensis

• Gram’s classification – Gram positive
• Shape – Rod shape
• Arrangement - Single, pair or chains
• Size – Measures 1.0 – 1.2 μm wide and 3.0 – 5.0 μm in length
• Family – Bacillaceae
• Motility – Motile due to Peritrichous flagella
• Capsule – Absent
• Endospores – Cylindrical endospores (central to sub-terminal)
• Respiration – Facultative anaerobic soil dwelling bacteria.
• Optimum Temperature – A rise of temperature from 20 °C to 35 °C doubled the titre of spores and increased their thermoresistance. When the temperature of cultivation was increased to 40 °C, the process of spore formation was inhibited.
• Optimum pH – 9.5.
• Cell inclusion - Contains a parasporal inclusion body called Crystal proteins (Cry proteins) or δ-endotoxin (Delta endotoxin).
• Habitat - Bacillus thuringiensis can be found in soil, insects and their habitats, stored products, plants, forest, and aquatic environments
• Bacillus thuringiensis grows at body temperature and produces a diamond-shaped crystal from its crystal proteins (Cry proteins) and uses it to fend off insects, predators, and other pathogens.
• Bacillus thuringiensis has a circular chromosome and a GC - content of approximately 32 % - 35 %. It has a genome size of between 5.2 – 5.8 Megabases. It has many plasmids and Bacillus thuringiensis strains harbors a diverse range of plasmids that vary in number and in size (2 kb – 200 kb).

 

History of Bacillus thuringiensis

• Bacillus thuringiensis (Bt) was first discovered in 1901 by a Japanese scientist Shigetane Ishiwata from dead silkworm larvae while he was investigating the cause of the “sotto disease” (sudden-collapse disease). He named it Bacillus sotto using the Japanese word sottō ('collapse'), referring to Bacillary paralysis.
• In 1911 it was rediscovered by a German scientist Ernst Berliner, and a solution of crystallized Bacillus thuringiensis (Bt) toxins was found to be highly effective against certain crop pests, including the corn borer, corn rootworm, corn earworm, and bollworms.
• The first commercial insecticide based on Bacillus thuringiensis, Sporine, was produced in France in 1938 and used primarily to control flour moths.
• In the United States the product was first used commercially as an insecticide spray in 1958, and several different strains of the bacterium are currently used to control for a number of agricultural insect pests and their larvae.
• In 1976, Robert A. Zakharyan reported the presence of a plasmid in a strain of Bacillus thuringiensis and suggested the plasmid's involvement in Endospore and Crystal protein formation.

Toxins of Bacillus thuringiensis

• The toxin produced by Bacillus thuringiensis (Bt) has been used as an insecticide spray since the 1920s and is commonly used in Organic farming.
• Cyt and Cry proteins that form the crystal, with toxic properties to insects, are soluble in water and belong to the δ-endotoxin class of bacterial proteins. Besides these, Bacillus thuringiensis also produces several other toxins such as α-exotoxin, β-exotoxin, hemolysins and enterotoxins.
• The δ-endotoxins form two classes of toxins,

a)     The first group: α-helix toxins or α-helical (group of proteins that includes the Cry proteins containing three domains) to which the α-helix region of the protein forms a pore in the membrane and

b)     The second group: β-barrel toxins (includes Cyt proteins), these are inserted into the membrane to form a β-barrel composed of β-sheet hairpins from each monomer

•     Five types of Cry proteins are reported (Cry1Aa, Cry2Aa, Cry1Ab, Cry1Ac and Cry2Aa) with molecular weight between 30 kDa and 140 kDa, which are coded by cry genes.
•      The toxin produced by Bacillus thuringiensis (Bt) is lethal to several orders of insects, including Lepidoptera (Butterflies, Moths and Skippers), Diptera (Flies) and Coleoptera (Beetles), and they are more target-specific.
•       With Bt pesticides, routine testing is required to ensure that unwanted toxins and microbes are not present. Bt has been registered for use in pesticides by the US Environmental Protection Agency (EPA) since 1961.
•       Bacillus thuringiensis (Bt) toxin can be applied to crops including potatoes, corn, and cotton, as a spray or, less commonly, in granular form.

Target Insects

• Lepidoptera
• Diptera
• Coleoptera  

Mode of action of Bacillus thuringiensis

• The bacterium Bacillus thuringiensis (Bt) produces a toxic Insecticidal Crystalline Protein (ICP)
• ICP is also called as Protoxin and its Molecular weight is 130 to 140 kDa.
• The mode of action of Bt involves the following stages:

a)     Ingestion of sporulated Bt and ICP by an insect larva.

b)   Solubilization of the crystalline ICP in the midgut – The ICP is highly insoluble in normal conditions, so it is entirely safe to humans, higher animals and most insects.  It is solubilized in reducing conditions of high pH (above about pH 9.5) - the conditions commonly found in the mid-gut of Lepidopteran larvae. On getting solubilized in the midgut, the crystalline bodies release the protein called Delta Endotoxins. For this reason, Bacillus thuringiensis (Bt) is a highly specific insecticidal agent.

c)     Activation of the ICP by midgut proteases - The crystalline Protoxins are inactive, until they are hydrolyzed by the gut proteases. The proteases cleave amino acids from both C-terminus and N-terminus of the protoxin and thus forms the active toxin.

d)     Binding of the activated ICP to specific receptors in the midgut cell membrane - Brush border membrane vesicles (BBMVs) is the primary binding site for several insect species. The active toxins initially bind reversibly to the specific receptors located on the apical brush border membrane of the columnar cells.

e)     Insertion of the toxin in the cell membrane and formation of pores and channels in the gut cell membrane, followed by destruction of the epithelial cells - After binding to the receptor, the toxin inserts irreversibly into the plasma membrane of the cell. The formation of toxin induced pores in the columnar cell of apical membranes allows rapid fluxes of ions. The disruption of the gut integerity leads to the death of the insect through starvation or septicemia.

f)      Subsequent Bt spore germination and septicemia may enhance mortality.

 

Isolation of Bacillus thuringiensis

• Bacillus thuringiensis are isolated from the soil sample by Serial Dilution Method (Spread Plate Method or Pour Plate Method).
• The common culture medium used for the isolation of Bacillus thuringiensis is Nutrient Agar.

Microscopic characteristics of Bacillus thuringiensis

• Gram staining – Bacillus thuringiensis was observed as Violet coloured, rod shaped Gram positive bacteria arranged as single, pair or chains.
• Motility test (Hanging Drop Method) – Actively motile rods due to Peritrichous flagella.

Cultural characteristics of Bacillus thuringiensis

• In Nutrient Agar, Bacillus thuringiensis colonies are round, white, slimy shape, smooth edges and raised elevation. At the edge of this bacteria is slightly shrunken or bumpy. The bacteria have an embossed elavance and a rough surface.

Figure – 1: Colony morphology of Bacillus thuringiensis in Nutrient Agar

Biochemical characteristics of Bacillus thuringiensis

a)     Catalase test – Positive

b)     Oxidase test – Negative

c)     Urease test – Negative

d)     Indole test – Positive

e)     Methyl Red (MR) test – Variable

f)      Voges Proskauer (VP) test – Negative

g)     Citrate utilization test – Negative

h)    Starch hydrolysis test – Positive

i)      Gelatin hydrolysis test – Positive

j)      Carboxy methyl cellulose (CMC) hydrolysis - Positive

k)     Growth at pH 9 – Positive

l)      Growth in 0.2 % Chitin - Positive

m)   Esculin hydrolysis – Negative

n)    Lecithinase production test – Positive

o)     Gas production in Glucose fermentation – Positive

p)     Mannose fermentation test – Positive

q)     Sucrose fermentation test - Positive

r)      Salicin fermentation test - Positive

Formulation of Bacillus thuringiensis

• Commercial preparations of Bt comes in various forms such as solids, liquids, powders, tablets, granules, ball agents, liquid suspensions, and oil emulsions

Advantages of Bacillus thuringiensis

• Unlike most insecticides, which target a Broad spectrum of species, including both pests and beneficial insects, Bt is toxic to a Narrow range of insects.
• Research suggests that Bt does not harm the natural enemies of insects, nor does it impair honeybees and other pollinators critical to agroecological systems.
• Bt is used for Integrated Pest Management (IPM) by many organic farmers.
• Reduce the use of chemical insecticide sprays, which are extremely toxic and expensive. 

Disadvantages of Bacillus thuringiensis

• Short self-life and remain effective for short periods because the toxic protein is broken down by sunlight or UV light and other climatic factor.
• Much higher cost.