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Chapter: Environmental Biotechnology: Genetic Manipulation

Microbial pesticides - Biotechnology Integrated Agricultural Applications

Chemical pesticides are problematic for many reasons.

Microbial pesticides

Chemical pesticides are problematic for many reasons. Firstly, although some degradation occurs, pesticides are notoriouslyrecalcitrant and consequently their use may lead to a build-up of chemicals damaging to the environment. This is an increasing problem with the intensive drive towards more and more cost-effective crop production. Secondly, insects are known to develop resistance to pesticides and so new and in some ways, more poisonous chemicals might be introduced to maintain the same level of effectiveness. Thirdly, chemical pesticides are rarely targeted to specific problematic species and may kill other organisms of no harm or even of some benefit to the crop plants. Balanced natural environments have an equilibrium between assailant and victim, however, it may take a commercially unacceptable time for this balance to establish, sometimes incurring quite extreme swings in either direction. For example, one season may see a flourishing of citrus trees due to an outbreak of disease leading to a dearth of butterflies, the caterpillars of which feed voraciously on citrus. The lack of insect host reduces the level of infection, leading to a recovery of the butterfly population the following year and, consequently, seriously damaged citrus trees. One means by which insect numbers are controlled in nature is by bacteria which produce toxins killing the insect which consumes it. Although this may serve to create the balance described above, it may not be sufficient to satisfy commercial crop production. Sufficient time may elapse between ingestion of the toxin by the larva and its ensuing death, for it to have caused considerable damage by feeding on the crop. Perhaps the best studied pesticidal bacterial toxin is the δ-endotoxin from Bacillus thuringiensis. This protein, frequently abbreviated to ‘Bt toxin’, is activeagainst some members of the Lepidoptera (butterflies and moths), Diptera (flies, midges and mosquitoes) and Coleoptera (beetles) families and has been used in its native, unmodified form as a pesticide for many years. There are several strains of the bacterium each one producing a toxin active against a limited number of insect species; a relationship which is continuously evolving. Already successful, it would appear to be the current leading candidate for development into a more generally useful and effective biopesticide, thus hopefully reducing the dependence on chemical pesticides.

 There are limitations associated with its use, all of which are being addressed in active research. These include a limited range of insects susceptible to each toxin, requiring dosing with multiple toxins, insufficient ingestion by the insect to prove lethal in a usefully short time, stability of the toxin when sprayed on crops and the development of resistance by the insect. The last stumbling block has attracted particular interest (Roush 1994, Gould 1994, Bohorova et al. 2001). The genes coding for the toxins have been isolated opening the way to their alteration and introduction into suitable ‘delivery systems’, either bacterial or into the plant itself, offering the plant inbuilt protection, thus attempting to overcome the various limitations introduced above.

 However, even without genetic engineering, Bacillus thuringiensis in its native form, remains a widely used and successful product for commercial crop pro-tection especially in ‘organic’ farming. In practice, the only major developmentsfound to be necessary, are improvements to the physical formulation of the crop spray; dry, flowable formulations being an advance on the original wettable pow-ders. Increasingly, nematodes especially Steinernema sp., are demonstrating great potential as biological control agents applied as sprays. It is anticipated that they will complement Bacillus thuringiensis in extending the spectrum of pests controlled by ‘environmentally friendly’ means (Knight, R., Koppert Biological Systems, personal communication).

 There are other Bacillus species which have also been used effectively as microbial insecticides. These are Bacillus sphaericus which produces a toxin more potent but more specific than Bt, and Bacillus popillae which although it does not produce a toxin, kills its host by weight of bacterial numbers. The latter is active against Japanese beetle, while the former is quite specific against mosquito larvae. Both the mosquito larvae and Bacillus sphaericus abound in heavily polluted water such as cesspits where the bacterium may exert control on the proliferation of mosquitoes. A different approach to microbial pesticides has been to examine the exploitation of Baculoviruses. The drive to use Baculoviruses as a means of biological pest control has dominated its research in the past, but currently these viruses are being recognised more as vectors to express proteins of various origins at a very high level indeed and so have become enormously useful tools in the major branches of biotechnology. Several Baculoviruses are registered for use in the USA as insecticides against maize bollworm (Heliothiszea SNPV), gypsy moth (Lymantira dispar MNPV) Douglas fir tussock moth(Orgyia pseudotsugata MNPV) and in the UK against pine sawfly (Neodiprionsertifer MNPV) and pine beauty moth (Panolis flammea MNPV). Members ofthe Baculovirus family, also called Nuclear Polyhedrosis Virus for reasons which become clear with a knowledge of their replication cycle, have been isolated from Lepidoptera, Homoptera (aphids and their relatives) and Diptera. The infectious cycle passes through a stage where several virus particles are bound together in a large crystal of protein. This protects the virus particles until the crystal is ingested by an insect where enzymes in the gut digest this polyhedrin protein releasing the viruses. These enter the insect’s cells where they are uncoated, make their way into the cell nucleus, and the viral replication cycle commences. Some 12 hours after initial infection, virus particles are released which spread the infection to neighbouring cells. By 24 hours post infection, the protective polyhedrin protein coded for in the viral DNA, is being produced in sufficient quantities to start assembling the crystal structures. By this time all the insect tissues are suffering severe damage such that at the time of death, the insect is effectively a mass of virus particles surrounded by the insect’s cuticle. This cadaver is eaten by birds, and consequently may be spread some distance. The virus is carried intact in the bird’s gut protected in the polyhedra, which is resistant to digestion by the enzymes found in avian gut. Polyhedrin protein is made in enormous quantities in the infected cell, but since its only known function is to protect the virus in vivo, in the wild, then it is redundant when culturing thevirus in vitro, in the laboratory. This being the case, the coding sequence for the polyhedrin protein may be replaced by a ‘foreign’ gene where under the control of the polyhedrin promoter it may, depending on the gene being replaced, have a good chance of being expressed at a very high level. This methodology used to overexpress proteins, has been used with great success but increasingly in biotechnology fields other than environmental, notably pharmaceuticals.

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