Bio-control agents:Insecticidal toxins of Bacillus thuringiensis

Presented by:Manisha.GM.Sc. BiochemistryFinal yearDept. of biochemistry &bioinformaticsGIS, GITAM university

Contents

History Definition of biological control Pros and cons of bio control agents Introduction to Bacillus thuringiensis Properties Examples Its mode of action Administration

History Insects have largest number of described

species- >7,50,000. Cause massive crop damage and act as vectors

for both human and animal diseases. During 1940s, a number of chemical insecticides

were developed as a means of controlling the proliferation of insect populations.

DDT- chlorinated hydrocarbon, nervous system and muscle tissue

• Dieldrin, aldrin, chlordane, lindane, toxophene were applied against crop pests and insects that carry infectious agents.

• Malathion, parathion, diazinon- organophosphates, inhibit enzyme acetylcholinesterase in insects, which hydrolyses the nerve transmitter acetylcholine.

• These were found to persist in the environment for >20years.• During 1950s higher concentrations were applied to control pests.• They lack specificity and beneficial insects were killed along with pests.• Given all the drawbacks associated with the use of chemical insecticides,

alternative means of controlling harmful insects have been sought.

Definition of biological control:

In entomology, it has been defined as the use of live predatory insects, entomopathogenic nematodes, or microbial pathogens to suppress populations of different pest insects.

In plant pathology, the term applies to the use of microbial antagonists to suppress diseases as well as the use of host specific pathogens to control weed populations.

In Toto, the organism that suppresses the pest or pathogen is referred to as the Biological Control Agent (BCA).

The most commonly used biopesticides are living organisms, which are pathogenic for the pest of interest.

These include: biofungicides (Trichoderma), bioherbicides (Phytopthora) and bioinsecticides (Bacillus thuringiensis). One bacterial species like Bacillus thuringiensis may be effective on Aedes aegypti while another B.

sphaericus strain can be effective on a different types of mosquito like Culex quinquefasciatus.

Pros and cons of bio control agents

On the positive side,

1) These compounds are highly specific for a target insect species

2) Biodegradable

3) Slow to select for resistance

On the negative side,

4) Their low potency

5) High cost of production limit their use for a variety of applications.

Introduction

Bt is a naturally occurring soil bacterium. It was first detected in 1902 in the dying larvae of Bombyx

mori by Ishiwata, who reported his finding in the book:"Pathology of the Silkworm".

• It was first isolated from the larvae of Ephestia kuehniella by Berliner in 1911 after he noted that it had the capacity to kill certain insects in their larva stage.

• Natural Bt is highly specific, with toxicity limited to only some species of one of the major groups of insects—typically Lepidoptera (butterflies/moths), Coleoptera (beetles), or

Diptera (flies/mosquitos).

Bacillus thuringiensis is a gram-positive, spore-forming bacterium which, during sporulation, produces protein crystals (CRY). It is characterized as a widespread insect pathogen, and its insecticidal activity is attributed to the parasporal crystals.

A variety of strains have been isolated from different habitats and, to date, more than 100 crystal protein genes have been sequenced.

There are more than 150 different subspecies of B.thuringiensis.

Each of the subspecies produces a different toxin that can kill specific insect.

The toxicity of these crystal proteins against certain insects and their high specificity led to the development of bio-insecticides for the control of pest insect species among the orders Lepidoptera, diptera, and coleoptera.

PROPERTIES OF INSECTICIDAL TOXINS:

Examples:

B.thuringiensis subsp kurstaki -lepidopteran larvae (moths, butterflies, skippers, cabbage worms and spruce bud worms).

B.thuringiensis subsp tenebrionis – coleopteran (beetles) such as potato beetle and boll weevil.

Some species of B.thuringiensis produce insecticidal toxins that are directed against hymenoptera (sawflies, wasps, bees and ants), orthoptera (grasshoppers, crickets) and mallophaga (lice).

Different domains involved in the toxicity of B.thuringiensis toxin in the mid-gut of targeted insect. Source:Sharma et al., 2000. Bt bacteria are used by farmers, foresters and gardeners to destroy butterfly larvae, mosquito larvae and beetles. The Danish field study, which was undertaken in 1993 and 1994, is one of the first in the world where plants have been systematically sprayed with Bacillus thuringiensis bacteria, and where the research has been ecologically oriented.

Mode of action

1. Bacillus thuringiensis is only effective when eaten by specific family of insects with a specific (usually alkaline) gut pH and the specific gut membrane structures required to bind the toxin. (typically butterflies, moths, beetles, flies and mosquitoes).

2. Not only must the insect have the correct and be at a susceptible stage of development, but the bacterium must be eaten in sufficient quantity.

3. When ingested by a susceptible insect, the spores feed on natural intestinal flora then it burst releasing the protein toxin (Crystalline protein) damaging the gut lining (the intestinal walls), leading to a kind of leaky gut condition.

4. Affected insects stop feeding and die from the combined effects of starvation, tissue damage and gastrointestinal infections by other pathogens like bacteria and fungus.

5. The natural Bt spores do not usually spread to other insects or cause disease outbreaks on their own as occurs with many pathogens.

BT crystalline Toxin 200px.

Normal gut bacteria

BT SPORES

Formation of ion channel:

• In its active form, the toxic protein inserts itself into the membranes of the gut epithelial cells of the insect.

• Creates an ion channel.

• Leads to an excessive loss of cellular ATP.

DEATH

Summary:

• In 1979, approximately 1% of the forest area in Canada that was treated with an insecticide to combat the spruce budworm (about 2 million hectares, or 8,000 square miles) was sprayed with B. thuringiensis subsp. kurstaki. The remainder of the treated forests were sprayed with chemical insecticides.

• By 1986, the use of B. thuringiensis subsp. kurstaki had increased dramatically. It was used to treat approximately 74% of the forests sprayed in that year for spruce budworm.

• In other countries, B. thuringiensis subsp. kurstaki has been used against tent caterpillars, gypsy moths, cabbage worms, cabbage loopers, and tobacco hornworms.

Usage:

• For the biological control (bio control) of insect pests, B. thuringiensis subsp. kurstaki is typically applied by spraying approximately 1.3 × 108 to 2.6 × 108 spores per square foot (1 square foot is equivalent to 0.093 m2) of the target area.

• Administration of the spores is timed to coincide with the peak of the larval population of the target organism, because the parasporal crystals, being sensitive to sunlight, are short-lived in the environment.

• Under simulated conditions, sunlight degrades over 60% of the tryptophan residues of the parasporal crystal within a 24-hour period, thereby rendering the protein inactive.

• Depending on the amount of sunlight present, parasporal crystals may persist in the environment for as little as a day or as long as a month. The lack of persistence of the insecticidal protoxin in the natural environment means that natural selection of resistant insects is highly unlikely.

Administration:

Reference:

Aronson, A. (1993) In Bacillus subtilis and Other Grampostive Bacteria. Sonenshein, A., Hoch, J.A., and Losick, R. (eds). Washington D.C.: Am Soc Microbiol, pp. 953- 963.

Bacillus thuringiensis: an environmental bio pesticide. Theory and practical. Donovan, W.P., Rupar, M.J., Slaney, A.C., Malvar, T., Gawron-Burke, M.C., and Johnson,

T. (1992) Appl Env Microbio158: 3921-3927. Li., J., Carroll, J., and Ellar, D.J. (1991) Nature353: 815-821. Molecular biotechnology : principles and applications of recombinant DNA (4th

edition) by Bernard R. Glick, Jack J Pasternak, Cheryl L Patten.