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Among the plant pests, insects are most significant. Insects either act as carriers (vectors) of many pathogenic organisms or themselves act as pathogenic organisms. Insect pests cause a bulk of the damage to crop plants.

It is, therefore, imperative to control the pests to increase crop yield. Over the years, farmers have been using pesticides to control the pests and pathogenic organisms. Two common groups of pesticides are in use by farmers:

(i) Chlorinated hydrocarbons like DDT, aldrin, and dialdrin

(ii) Organophosphates like malathion and parathion.

The use of these chemical pesticides has dramatic and immediate effects. However, a long term use has detrimental side effects. All these chemical pesticides are non- bio-degradable. They have a tendency to persist in the environment as such for around 15-20 years and accumulate in increasing concentrations in the food chains.

This phenomenon, known as bioaccumulation, has significant biological impacts on many organisms. Secondly, the targeted pest population becomes increasingly resistant to treatments. Consequently, higher doses of the pesticides have to be applied. Moreover, these pesticides do not have specificities. These kill beneficial insects along with the harmful pests.

Given all these drawbacks, associated with the use of chemical pesticides, alternative methods of controlling the pests have been investigated over the past few years. Pesticides that are the products of microorganisms and plants have been discovered. These natural pest killing agents or bio-pesticides have become an obvious choice due to the following two reasons. These are: (i) highly pest-specific; (ii) bio­degradable.

Bacillus thuringiensis, a Gram-positive soil bacterium and a virus, baculovirus are noteworthy in this context. They encode for products, having insecticidal properties.

The encoding genes have successfully been cloned and introduced into crop plant cells in culture by tissue culture technique and transgenic plants generated.

These transgenic plants possess the endogenous ability of producing the specific bio-pesticide and protect from the harmful effects of pests. These pesticidal molecules are known as biopesticides or microbial pesticides.

Insecticidal toxin of Bacillus thuringiensis

A microbial pesticide is a microorganism, which produces a toxic substance that kills an insect pest species. The most often studied and utilized microbial pesticide contains toxins synthesized by the bacterial species, Bacillus thuringiensis (BT).

It is a Gram-positive soil bacterium, which forms endospores within a sporangium during adverse environmental conditions. Sporulating cells produce parasporal crystalline inclusions composed of proteins. These crystalline proteins are referred to as 8 -endotoxins / insecticidal crystal proteins (ICP) / BT protein.

This protein is hydrolyzed by an alkali’ into 250 kD units, Known as protoxins. Each protoxin sub-unit consists of two 130 kD polypeptides. This protein is lethal to some insect larvae, when ingested. The 130kD polypeptide is digested by an intestinal protease into a 68 kD toxin polypeptide, at an alkaline p^H. When caterpillars eat the leaves, on which bacterial spores have been deposited, the spores germinate. The parasporal crystalline protein insecticidal crystalline protein (ICP) /BT protein] of Bacillus thuritigiensis. (a) Parasporal crystalline protein; (b) 250 kD protoxin sub-unit; (c) 130 kD polypeptide; and (d) 68 kD toxin polypeptide. And the bacteria grow in the alimentary canal and produce the BT protein. This protein is digested into 68 kD toxin polypeptides in the intestine of the caterpillar larva.

This polypeptide changes the osmotic potential so that no absoiption takes place. This eventually kills the caterpillar. The alimentary canal of mammals and human produces an acid that degrades the BT protein, when ingested. Thus, it is apparently harmless to human and other mammals.

This bacterium comprises of a number of different strains (subspecies), each of which produces a different toxin that can kill specific insects. For example, B. thuringiensis kurstaki is toxic to lepidopteran larvae including moths, butterflies, skippers and cabbage worms. B. thuringiensis israelensis kills diptera such as mosquitoes and black flies. B. thuringiensis tenebrionis is effective against coleoptera (beetles) such as potato beetle and boll weevil.

Strategy for protection by BT protein

There are two strategies for developing insect resistance in crop plants by employing the BT protein. In the first strategy, B. thuringiensis spores containing BT protein are mixed in water and the resultant water and spore mixture is sprayed over the insect infested area.

However, the insecticidal effects are transient due to limited survival of the spores. Therefore, repeated spraying is necessary for a long term effect. This problem has been circumvented by the application of genetic engineering. The BT protein is encoded on a 75 kb plasmid of B. thuringiensis.

There are two lines of treatment in the genetic engineering approach. In the first approach, the BT protein gene is isolated and inserted into other organisms like Escherichia coli or Pseudomonasfluorescens, which are better suited for survival in the field. BT protein gene transformed P. fluorescens, when sprayed on corn plant, colonize the root area, thus endowing the corn plant with endogenous-fit- protein producing property.

A second genetic engineering strategy employs a constitutive expression of the Zfrprotein gene in the transformed cells of the host plant. In this process, the BT protein gene is first inserted into a Ti plasmid of Agrobacterium tumefaciens containing a constitutive promoter.

In the next step, host plant cells in culture are transformed by the recombinant 77 plasmids. The transformed cells are grown in culture and plants generated. These transformed plants have acquired the property of synthesizing the BT protein endogenously.

Potential risks of BT technology

One major problem of an accelerated use of BT technology is an increasing resistance of pests to the BT protein and rapid appearance of resistant varieties of pests. BT cotton marketed by Monsanto under the trade name of Bollgard and grown by US farmers is a case in point.

The cotton crop in US is prone to lepidopteran larvae, particularly, cotton bollworm (Helicoverpa zea), pine bollworm (Pectinophora gossypiella) and tobacco budworm (Heliothis virescens). Insect resistant variety of cotton was raised by using the BT technology. The variety was marketed by the US Biotechnological firm, Monsanto under the trade name, Bollgard. However, in 1996, following a commercial scale planting, there was an unexpected mid-season outbreak of cotton bollworm.

This required a supplementary spraying of insecticides. This incident raised concerns about the effectiveness of the BT protein. The rise and fall of the StarLink corn is another case in point. All products manufactured from the StarLink, marketed by the firm, Aventis (formerly AgrEvo) had to be recalled, following a finding that the BT protein was stable in the hydrochloric acid of the stomach of human.

It caused an extensive change in the mucosal epithelium. Finally, it was declared unsuitable for human consumption. In the event of all this, an extensive study in all aspects of the case-is required before a full-scale commercialization of the crop. There may be two possible solutions for overcoming the pest resistance problem.

Firstly, the BT crop and non- BT crop should be alternately cultivated. Secondly, it is suitable to use more than one BT transgene by crossbreeding between two BT varieties of the same crop. This phenomenon has been termed as pyramiding.

Insecticidal property of baculovirus

Baculoviruses are rod shaped double stranded DNA viruses that can infect and kill a large number of invertebrate organisms. They infect mostly the larval stages of various insect orders such as Lepidoptera, Diptera, Hymenoptera, Coleoptera and Homoptera.

Larvae of insects cause most of the damage to crop plants. Therefore, baculovirus is used as a potential bio-pesticicide for controlling the insect pests. An added advantage is that this virus does not harm non-target organisms, since it has a specific host range. Insect larvae are infected, when they ingest the plant materia] contaminated with baculoviruses. In the alimentary canal, the protein coat of the virus is digested by the digestive enzymes and eventually, the DNA is released.

This DNA enters into the nuclei of the intestinal epithelial cells, where it replicates and forms a large number of viral particles. The cell consequently breaks down releasing the viral particles. Then the second phase of infection commences.

This progressive cycle of infection and lyses causes a mass destruction of tissues of the larva. This causes the death of the insect and releases a large number of viral particles, ready to be consumed by other larvae. However, this process is very slow such that by the time the larva dies, it has consumed a lot of the plant material. The entire purpose is defeated.

The strategy is to shorten the larval life so that it will consume a small amount of the plant material minimizing the damage.

Strategy for protection by baculovirus

Two strategies are employed by genetic engineering to impart insecticidal properties to baculoviruses. In the first strategy, the larval growth is shortened such that the damage caused by it is minimized.

Juvenile hormone is necessary for larval growth and maintenance of the larval stage. The enzyme, juvenile hormone esterase inactivates the juvenile hormone synthesis so that the larva metamorphoses into the pupa and ceases feeding.

The gene encoding this enzyme is transferred to baculoviruses and expressed in the host insect upon infection. This will shorten the growth period and reduce feeding.

This effect was observed in the tobacco budworm, Heliothis virescens. However, one drawback is that the larvae are sensitive to the juvenile hormone only during a specific stage of development.

In experiments, this effect was observed, only when the recombinant virus infected the first instars larvae. The other stages were insensitive to the juvenile hormone level.

In an alternative strategy, insect toxin genes may be transferred to baculoviruses, which may prove more effective. The neurotoxin gene from the North African scorpion, Androctomus australis Hector is transferred to baculovirus and this has exhibited extended specificity and good result.

In another experiment, a scorpion toxin gene has been inserted into theAutographa califomica multicapsi’d nucfear polyhedrosis virus (AcMNPV), a baculovirus that kills larvae of butterflies and moths by paralyzing them. Field trials are underway, in which the virus is sprayed • on the infested crop plants.

Insect resistance by plant protease inhibitors

Plant protease inhibitors have proved to be effective insecticides. When the insect larvae ingest these inhibitors, the digestive enzymes are inhibited.

This results in starvation and consequently death. The cowpea trypsin inhibitor has successfully been transferred into the tobacco plant by Agrobacterium tumefaciens mediated transformation.

A more effective pesticide is generated by fusing the BT protein with a protease inhibitor. Storage pests destroy stored cereal grains and cause a great deal of loss. Recently, a garden pea is genetically engineered, which resists the destructive effects by two species of weevils.

A protein, a -amylase inhibitor blocks the enzyme, a-amylase, which digests the starch in the seeds. Interestingly, the inhibitor protein is expressed only in the seeds. The weevils, which fed the transformed pea seeds either died or suffered inhibited development.