Contents - Faperta

Genetic Engineering of Entomopathogenic Microbes for Pest Management 273
fungi (Srinivasan, 1997). For commercial-scale production, however, one would require a
solid-state fermentation system. The advantages associated with the use of fungi for insect
control is the fact that they can infect an insect through the cuticle, thus removing the need
for ingestion. They are generally not hazardous to mammals, have no toxic residues, and
give long-term control. Recent breakthroughs in formulation, strain selection, and production
have provided new impetus for the inclusion of fungal-based pesticides in IPM programs.
A number of facilities for mass production of M. anisopliae and B. bassiana have now
been established in several countries. Several commercial fungal products are currently
available. Bio 1020 has been registered in Germany for the control of black vine weevil,
Otiorhynchus sulcatus (F.). This product is composed of dried mycelial granules, and has a
shelf life of up to six months at low temperatures.
The major drawbacks associated with fungal pesticides include relative instability,
requirement for moist conditions for spore germination, invasion, and growth, and slow
rates of mortality. The problems associated with moisture requirement may be overcome
by selecting isolates that are less humidity dependent. In order to improve the insect
mortality, it is also possible to select naturally occurring potent fungi and use them to
improve other strains through protoplast fusion or anastomosis. The prospects for genetic
improvement of entomopathogenic fungi are quite good (Ferron, Fargues, and Riba, 1991;
St. Leger and Roberts, 1997). Developments in molecular biology of entomopathogenic
fungi will provide the basic understanding of mechanisms of pathogenesis and produce
recombinant fungi with increased virulence (Charnley, Cobb, and Clarkson, 1997). There
is considerable genetic variability in natural strains of B. bassiana and their virulence to the
spotted stem borer, Chilo partellus (Swinhoe) (Uma Devi et al., 2001). Genetic manipulation
of fungi still remains a diffi cult task considering the large size of the fungal genome and
the fact that most fungal toxins are complex molecules encoded by several genes. Davila,
Zambrano, and Castillo (2001) used 40 primers to gain an understanding of the mechanisms
for producing recombinant fungi with increased virulence. Molecular markers
can also be used for detecting gene sequences that confer on fungi the capacity to cause
disease in insects (Zambrano, Davila, and Castillo, 2002).
Genetic manipulation can be used to improve tolerance of entomopathogenic fungi
to fungicides (Figure 8.2), thereby promoting their utility in IPM programs (Table 8.6).
A benlate-resistance gene from Aspergillus has been used to transform M. anisopliae (Goettel
et al., 1989). The transformants grew at benomyl concentrations up to 10 times that inhibit
wild type, and were mitotically stable on either selective or nonselective medium or insect
tissue. The transformants were pathogenic to the sphingid, M. sexta, producing both
appressoria and the cuticle-degrading enzyme chymoelastase in the presence of 50 μg mL 1
of benomyl. The gene encoding the cuticle-degrading protease (Pr1) has been inserted
into the genome of the same fungus. Bernier et al. (1989) introduced benomyl resistance
(beta-tubulin) gene from Neurospora crassa (Draft) (encoding resistance to benomyl) into
M. anisopliae. The transformants were mitotically stable when subcultured on nonselective
agar and retained the ability to infect and kill larvae of M. sexta. The benomyl-resistant
phenotype persisted in re-isolates from insect cadavers. St. Leger et al. (1995) cotransformed
M. anisopliae with two plasmids (pNOM102 and pBENA3) containing the betaglucuronidase
and benomyl resistance genes, using electroporation and biolistic delivery
systems. The cotransformants showed pathogenicity to B. mori. The bar gene from
Streptomyces hygroscopicus (Jensen) Waksman and Henrici under the control of Aspergillus
nidulans (Eidam) Winter trpC promoter and terminator sequences has been inserted into
Paecilomyces fumosoroseus (Wise) Brawn & Smith (Cantone and Van denberg, 1999).
Evaluation of selected transformants revealed two mutant strains with altered sporulation