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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