Insect Control: Biological and Synthetic Agents - Index of

408 11: Entomopathogenic Fungi and their Role in Regulation of Insect Populations
and interest in the release and monitoring of genetically
modified organisms in the environment, including
entomopathogenic fungi. Bidochka (2001)
identified three main benefits from monitoring the
fate of fungi: (1) identification of the introduced
agent in a biological impact assessment, (2) economic
cost benefit assessment, and (3) valuable information
on fungal epidemiology.
Despite many years of research into entomopathogenic
fungi, there remains an inadequate understanding
of epizootiology, due in part to lack of
specific methods for tracking individual isolates in
the environment. The fate of released fungi, the
genetic structure of fungal populations, and how
to measure these components has not been well
explored.
There are several possible ways in which to study
persistence of artificially augmented fungal inoculum
levels, including leaf and soil washes, leaf
impressions, differential staining, bioassay of field
collected substrates, and monitoring of sentinel
insects (Goettel et al., 2000). Comparisons can be
made to untreated areas. When there are no longer
differences in population levels between treated and
untreated areas, it can be concluded that the augmented
fungus has returned to background levels.
The number of fungal propagules in the environment
has often been used as a measure of the potential
success of biocontrol applications. Propagule loadings
have been measured by colony counts on semiselective
media, where the fungus can be cultured, or
through spore traps for fungi that discharge their
spores into the air, such as the Entomophthorales.
However, these methods do not distinguish between
applied and background strains.
Methods that have been used to study the fate
of entomopathogenic fungi at the specific strain
level include phenotypic markers, vegetative compatibility,
mating types and, more recently, a plethora
of molecular approaches, which allow specific
tracking of individual strains in most environments.
When strains can be cultured, studies have used
markers such as colony appearance or antibiotic
resistance, but these techniques suffer from genetic
instability and alterations due to environmental factors.
Other nonmolecular based markers have been
investigated, such as commercially available carbohydrate
utilization or enzyme production strips system
of API (i.e., API50CH; APIZyms), but with
limited success (St. Leger et al., 1986; Todorova
et al., 1994; Rath et al., 1995). These systems can
be used to distinguish groups, but are unlikely to
distinguish individual isolates in most cases. Therefore
their usefulness in tracking released strains is
usually minimal.
Vegetative compatibility groups (VCGs) are subspecific
groups within the same fungal species
from which isolates can fuse hyphae to form heterokaryons
and, thus, exchange genetic information
(the parasexual cycle). Couteaudier and Viaud
(1997) demonstrated that compatibility groups,
within Beauveria spp. in Europe, could be used
to delimit genetically incompatible groups and aid
monitoring.
Many entomopathogenic fungi cannot be
cultured and the lack of simple methods for isolation
and specific strain characterization has made it
difficult to conduct ecological studies on fungal persistence.
The most important advances in monitoring
the fate of entomopathogenic fungi in the
environment have come from the application of
molecular biology techniques. Precursors to DNA
techniques were the use of allozymes to identify
strains, and use of enzyme-linked immunosorbent
assay (ELISA) for tracking in the environment.
Application of allozyme analysis to strain detection
in the environment has been attempted, although
single isolate identification amongst strains from a
single host has not proved possible. For example,
the allozymes of Beauveria and Metarhizium spp.
have a high degree of variability, indicative that the
species had maintained a large effective population
size for a long time (St. Leger et al., 1992b). In some
cases, isoenzymes have not been useful in separating
applied and background strains (e.g., Trzebitzky
and Lochelt, 1994) or showed little correlation
with virulence (Reineke and Zebitz, 1996), but Lin
and Lin (1998) found esterase patterns of Beauveria
isolates correlated with geographical distribution
and host. Allozymes have also been used with the
Entomophthorales. Silvie et al. (1990) used allozymes
to study the fate of P. neoaphidis released for greenhouse
aphid control. Milner and Mahon (1985) used
similar techniques to distinguish between an introduced
Israeli strain of Z. radicans for aphid control
and endemic strains pathogenic to flies and caterpillars.
Hajek et al. (1990) used allozymes to confirm
that the gypsy moth pathogen causing epizootics
in North America was E. maimaiga. In further studies
using allozymes and RFLPs, they demonstrated that
E. maimaiga and E. aulicae, two very similar species,
were attacking different caterpillars in the same
area (Hajek et al., 1991b). ELISA was also developed
for E. maimaiga based on allozymes, but was
cross-reactive for E. aulicae (Hajek et al., 1991a).
Application of DNA techniques is making major
advances in monitoring the fate of entomopathogenic
fungi in the environment. There are numerous
studies that have demonstrated the power of molecular
biology in strain, species, and genera separation