Insect Control: Biological and Synthetic Agents - Index of

394 11: Entomopathogenic Fungi and their Role in Regulation of Insect Populations
Figure 15 Earwig (Forficula auricolaria) infected with Zoophthora
forficulae. The insect is completely covered with the fungus and
is firmly attached to the vegetation by sticky rhizoids produced
by the fungus. (Photo courtesy of J. Eilenberg.)
the subspecies level. Identification of specific isolates
is necessary in biocontrol, both as a regulatory
requirement and to advance understanding of
ecology and epizootiology (see Section 11.4).
There have been many different approaches to
classifying insect pathogenic fungi. In some cases,
fungi have been defined functionally or ecologically,
rather than phylogenetically, especially when techniques
to directly determine phylogeny did not
exist. The entomogenous fungi provide an example
where the entomogenous habitat was seen as a
defining criterion in many cases, whereas morphology
and physiology formed the basis of many of
the earlier efforts with other fungi. Early descriptions
of M. anisopliae refer to Entomophthora
anisopliae (Zimmermann et al., 1995), recognizing
the relationship between two common entomogenous
groups over dissimilarities in morphology
and physiology. The mode and morphology of
conidiation has been a main character used for
classification.
In recent times, classification systems have
attempted to take a more phylogenetic approach to
systematics (e.g., Humber, 1984), but a true phylogenetic
classification had not emerged for most
organisms until the advent of molecular biology.
Other approaches are still being explored. Chemotaxonomy
has been attempted with some entomopathogenic
groups such as Beauveria, utilizing the
commercially available carbohydrate utilization
strips system of API (i.e., API50CH) (Todorova
et al., 1994; Rath et al., 1995). Isoenzymes have
been investigated for their ability to discriminate
between fungal strains (e.g., May et al., 1979; St.
Leger et al., 1992b), but their power in phylogenetic
classification is limited.
Molecular techniques that target RNA and DNA
have been applied at every level of the taxonomic
hierarchy. Many techniques have been applied,
some of which directly sequence genes, and some
of which represent differences in DNA sequence,
such as random amplified polymorphic DNA
(RAPD) and restriction fragment length polymorphism
(RFLP) approaches. Pulse field gel electrophoresis
(PFGE) allows the separation of DNA
fragments as large as chromosomes and can be
used for whole genome restriction digest comparisons.
Chromosome length polymorphism was
used by Viaud et al. (1996) to study nine isolates
of B. bassiana. Mitochondrial DNA has been used
to estimate intraspecies variation in Lecanicillium
(¼ Verticillium) lecanii and M. anisopliae isolates
(Typas et al., 1998). There are a number of reviews
on the use of molecular characterization for entomogenous
fungi (e.g., Clarkson, 1992; Driver and
Milner, 1998).
With the development of molecular biology as a
mainstream tool in classification, it became possible
to test assumptions formed by studies based on
morphology and other characteristics reliant on
phylogenetic relationships. In macroevolution, the
seven kingdoms are based largely on sequencing of
small subunit ribosomal genes. While classifying
fungi solely on the sequence of any gene is not
wise, a general consensus is being reached over the
evolutionary position of the fungi in respect to other
groups based on rRNA gene sequences (e.g., Berbee
and Taylor, 2001). Molecular analysis has shown
that a number of species and genera classified as
fungi, or in the kingdom Eumycota, are actually
grouped with the Chromista. Many species have
been placed in the fungi simply on the basis of
hyphal growth, but are now being reconsidered.
The genera of entomopathogenic fungi are being
reconsidered on the basis of sequencing of conserved
genes and introns. Recent efforts to clarify classification
of entomopathogenic fungi demonstrate the
utility of molecular characterization at several levels.
Zare et al. (2000), Gams and Zare (2001),
Zare and Gams (2001) have redefined the genus
Verticillium using rDNA sequencing, placing all
insect pathogens in the genus Lecanicillium. Similarly,
sequencing of large subunit rRNA gene
revealed Paecilomyces may not be monophyletic
(Obornik et al., 2001). Sequencing of the small
subunit rDNA has also been used to examine
phylogenetic relationships among the order Entomophthorales
( Jensen et al., 1998). These molecular
studies supported the use of spore discharge
characteristics as an identifying characteristic for
Entomophthorales.