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