Contents - Faperta

268 Biotechnological Approaches for Pest Management and Ecological Sustainability
transconjugant and naturally occurring strains producing Cry proteins distinct from those
of B. thuringiensis subsp. kurstaki, including strains of B. thuringiensis subsp. aizawai and
B. thuringiensis subsp. morrisoni, have been developed for use in pest control. It has been
reported that the encapsulated products persist on crops twice as long as conventional
products (Feitelson, Payne, and Kim, 1992). However, some fi eld data have shown that
persistence is still not long enough to increase the risk to nontarget organisms. Two encapsulated
products called MVP R (lepidopteran active) and M-Trak R (coleopteran-active) have
also been developed (Gelemter and Schwab, 1993).
Genetic Engineering of Bacteria
The immediate challenge for genetic engineering of bacteria is to: (1) increase the potency
of the toxin(s), (2) broaden the activity spectrum, (3) improve the persistence under fi eld
conditions, and (4) reduce the production costs. The problems associated with specifi city,
shelf life, potency, and presence of viable spores have been overcome by using modern
tools in microbiology and genetic engineering. Attempts have also been made to develop
high-temperature- and ultraviolet-resistant strains of Bt (Salama, Ali, and Sharaby, 1991).
The ultraviolet-resistant strains show high toxicity, although it is known that UV causes
plasmid curing. However, the heat-resistant strains have shown considerable reduction in
toxicity, which may be attributed to either plasmid curing during incubation at 48°C or to
the introduction of foreign DNA. The genetically engineered bacteria can be killed by heat
or chemical treatment and processed into a form that can be sprayed on crops. Due to its
rigidity, the dead bacterial cell walls form a protective microcapsule that prevents the toxin
from degrading. However, there are too many unknowns to determine whether the new
molecular approach for developing transgenic Bt toxin-based pesticides will be benefi cial
in IPM. It is possible that the approach could be used to tailor-make Bt-based pesticides
that affect only a single insect species. This could be quite benefi cial since there will be no
disruption of ecological dynamics of the system. The products could also be tailored in a
way that they do not cause selection for resistance in other insects that do not damage the
treated crop. It may be diffi cult to develop an on-farm IPM program with so many specifi c
pesticides. Therefore, the use of engineered strains might be limited to farming systems
that have only a few major pest problems.
Electroporation technology has been widely used to transform vegetative cells with
plasmid DNA (Bone and Ellar, 1989; Lereclus et al., 1989a, 1989b; Mahillon et al., 1989;
Masson, Prefontaine, and Brousseau, 1989; Schurter, Geiser, and Mathe, 1989). Macaluso
and Mettus (1991) reported that some B. thuringiensis strains restrict methylated DNA.
Plasmid DNA isolated from Bacillus megaterium De Bary or E. coli transformed B. thuringiensis
with much higher frequencies than did DNA isolated from B. subtilis or E. coli.
A number of shuttle vectors have been used to introduce cloned cry genes into B. thuringiensis
(Gawron-Burke and Baum, 1991). Integrational vectors have also been used to insert
cry genes by homologous recombination into plasmids (Lereclus et al., 1992; Adams et al.,
1994) or chromosomes (Kalman et al., 1995). Plasmid vector systems employing B. thuringiensis
site-specifi c recombination have been used to construct recombinant B. thuringiensis
strains for new bioinsecticide products (Baum, Kakefuda, and Gawron-Burke, 1996; Sanchis
et al., 1996, 1997). Applications of this technique have included disruptions of cry and cyt
genes to assess their contribution to biological activity (Delécluse et al., 1993; Poncet et al.,
1993), and inactivation of protease production genes to increase crystal production and
stability (Tan and Donovan, 1995; Donovan, Tan, and Slaney, 1997). Progress in understanding
cry gene expression has allowed the construction of asporogenous B. thuringiensis