Insect Control: Biological and Synthetic Agents - Index of

11: Entomopathogenic Fungi and their Role in Regulation of Insect Populations 415
when compared to other groups of organisms, but
some interesting research has been done. The most
advanced research has been on the hyphomycete
fungi M. anisopliae and B. bassiana.
Transformation systems have been developed for
some entomopathogenic fungi. The first transformations
of an entomopathogenic fungus were by
Bernier et al. (1989) and Goettel et al. (1990b),
who expressed a benomyl resistance gene in M.
anisopliae. Electroporation and biolistics have also
been used to transform M. anisopliae (St. Leger et al.,
1995). Polyethylene glycol (PEG) mediated transformation
of protoplasts is another method for transformation
of entomopathogenic fungi, as done with
P. fumosoroseus and P. lilacinus (Inglis et al., 1999b)
using benomyl as the selective agent. Sandhu et al.
(2001) developed a heterologous transformation
system for B. bassiana and M. anisopliae based on
the use of the Aspergillus nidulans nitrate reductase
gene (niaD), after EMS treatment of protoplasts
and regeneration on chlorate medium.
Genetic modification of entomopathogenic fungi
by direct manipulation has been led by St. Leger,
and the area was recently reviewed by St. Leger and
Screen (2001), covering both insect and plant pathogenic
fungi. In the numerous publications, these
researchers described the identification and cloning
of protease genes, particularly the pr1 cuticle degrading
protease gene from M. anisopliae (St. Leger
et al., 1992a). Several other entomopathogenic
Hyphomycetes (A. flavus, B. bassiana, Paecilomyces
farinosus, Tolypocladium niveum, and L. lecanii)
also have pr1-type enzymes (St. Leger et al.,
1991, 1992a). Efforts to produce more effective
strains of fungi have included the overexpression
of pr1 by insertion of multiple copies, resulting in
increased speed of kill of a host insect, although
transformants had very poor sporulation ability
(St. Leger, 2001). Modification of pr1 gene expression
in M. anisopliae resulted in melanization and
cessation of feeding 25–30 h earlier than the wildtype
disease in caterpillars (St. Leger et al., 1996).
The group has gone on to place the pr1 gene under
control of a constitutive promoter and included a
green fluorescent protein encoding gene for tracking
the fungi after field release (Hu and St. Leger, 2002).
Genes involved in regulation of PR1 expression have
also been identified from M. anisopliae (Screen et al.,
1997, 1998).
Chitinase, produced by many insect pathogenic
fungi and implicated in pathogenesis, has also been
the target of molecular studies. Several chitinase
gene sequences from Beauveria and Metarhizium
have been submitted to GenBank and described in
publications (e.g., Bogo et al., 1998). Overexpres-
sion of extracellular chitinase has also been demonstrated
for M. anisopliae var. acridum, but did not
alter virulence to the caterpillar, M. sexta, compared
to the wild-type fungus (Screen et al., 2001).
11.6.3.3. Formulation and application strategies
There are a number of limitations that still
affect the use of fungi. Formulation and application
of fungal propagules has the potential to overcome
some of these limitations. Jones and Burges (1998)
defined four basic functions of formulation: (1) stabilization
of the agent during production, distribution,
and storage; (2) aiding the handling and
application of the product to the target in the most
appropriate manner and form; (3) protection of
the agent from harmful environmental factors at
the target site, thereby increasing persistence; and
(4) enhancing efficacy of the agent at the target site
by increasing activity, reproduction, contact, and
interaction with the target pest.
Progress has been made in formulation science,
and formulations for entomopathogens have been
comprehensively reviewed by Burges (1998). Early
formulations of entomopathogenic fungi often
used just a weak detergent to get the hydrophobic
spores into solution, without any efforts to use the
formulations to improve efficacy. More recently, the
use of oil as a formulation ingredient has been successfully
used to overcome several problems. In the
LUBILOSA program developing M. anisopliae
var. acridum for control of locusts in Africa, the
combination of oil formulations with ultra low
volume (ULV) spraying has led to successful
development of a biopesticide (Lomer et al., 2001).
Although it would seem impossible to use an entomopathogenic
fungus requiring high humidity
against locusts in the hot, dry conditions prevalent
in much of their environment, studies have shown
that ambient humidity may not be as critical as once
thought (Fargues et al., 1997; see also Section
11.3.1). Formulating Metarhizium conidia in nonevaporative
diluents such as oils allowed the conidia
to attach, spread (Inglis et al., 1996a), and germinate
on susceptible locusts, even at low ambient
humidities (Bateman, 1997).
Formulation can also assist in extending shelf
stability of fungal propagules generally desired by
commercial producers. Spore survival after mass
production varies greatly, depending on the species,
but few entomopathogenic species of fungi can
achieve the 1–2 years of shelf stability at formulation.
Formulation in clay material is known to improve
the shelf life of many conidial preparations
(e.g., Shi, 1998). This appears to be due to the ability
of clay to reduce the moisture content of the spores,