Molecular Identification And Tracking

In addition to improving our understanding of the genetics of disease caused by entomopathogenic fungi, molecular techniques have also been used to aid in identification, classification, and environmental monitoring of fungi. It is now almost a standard practice to perform some form of genetic characterization of fungi to specifically identify strains used for biocontrol purposes. The utility of molecular techniques, however, has been demonstrated beyond simple strain identification. There are a number of reviews on the use of molecular characterization for entomogenous fungi in the literature [e.g., Driver et al. (1998) and Glare (2002)]. An example of the importance of molecular characterization was the clarification of the taxonomic position of M. anisopliae strain IMI 330189 used in the LUBILOSA program for locust control. Often described as a M. flavoviride strain because of the morphology of the conidia and some other features, the correct classification was debated. Recently, Driver et al. (2000) published a revision of the subspecific relationships between M. anisopliae and M. flavoviride strains, based largely on the sequence of the ITS-5.8s regions of rRNA. They demonstrated that IMI 330189 and related strains formed a discrete clade on the M. anisopliae branch of the Metarhizium trees, a finding that appears to have been well received by those working on locust control. Driver et al. (2000) named the subspecies M. anisopliae var. acridium, as well as describing several other subspecies, some of which can only presently be distinguished by ITS sequencing. This demonstrates a problem with molecular characterization, as fungal species cannot be solely erected on sequence data and requires supporting morphological or biological descriptions.

Molecular markers have been used to characterize the genotypes of individual fungal strains by examining gene products, but new techniques allow direct examination of variability at the DNA level. Pulsed field gel electrophoresis has been used to study karyotype variation in other fungi, and could be used with entomopathogens. Several studies have examined the number of chromosomes and mapped genes on those chromosomes. Viaud et al. (1996) studied the level of chromosome length polymorphism among nine isolates of B. bassiana to obtain a more extensive knowledge of the genomic organization. While extensive use of molecular characterization has proved useful, there are currently no standard techniques or agreement on even how many regions of the genome should be sampled to provide taxonomic data.

While this is not a problem for strain identification or comparison, it reduces the ability to compare between studies. Many of the molecular studies on entomogenous fungi have used the nuclear ribosomal DNA, but there are a number of other DNA regions used, such as mitochondrial DNA (mtDNA) restriction fragment length polymorphisms. The MtDNA has been used to estimate intraspecies variation in V. lecanii and M. anisopliae isolates.

The contribution of molecular techniques to the development of entomopathogenic fungi has been enormous. The techniques have been used to clarify evolutionary relationships [e.g., Driver et al. (2000) and Jensen et al. (1998)]. Molecular techniques have also allowed development of theories of evolution around these often obligate pathogens. Generally, studies on the entomogenous fungi using conserved mitochondrial or nuclear regions have failed to find a link between fungal species and host species. For example, Bidochka et al. (2001) found that habitat rather than host selection drives population structure of M. anisopliae. There have been exceptions, such as B. bassiana strains from Sitona weevils (Maurer et al. 1997) and some Entomoph-thorales [e.g., Jensen and Eilenberg (2001)]. In the order Entomophthorales, sequencing of the small subunit rDNA has been used to examine phylogenetic relationships (Jensen et al. 1998). The molecular studies supported the use of spore discharge characteristics as an identifying characteristic for Entomophthorales. The role of horizontal gene transfer in microbial evolution has been the topic and studies by St Leger et al. (2001) have found some evidence for the involvement of horizontal gene transfer in evolution of fungal parasitism, finding similarity between genes in M. anisopliae and Streptomyces bacteria. Monitoring of specific strains of entomopathogenic fungi in the field after release is crucial for advancing and understanding of biopesticide ecology. It has often been difficult to conduct ecological studies on fungal persistence and spread after application, because there has been a lack of simple methods for isolation and specific strain characterization of these fungi. The molecular characterization of strains of entomogenous fungi has improved the ability to track specific fungi in the field. Specific identification of the B. brongniartii strain used for control of the scarab pest, Hoplochelus marginalis, in the ReUnion Islands was based on introns (insertions) in the 28s gene of the rDNA (Neuveglise et al. 1997). Genetic modification is also a method to allow tracking following release of a strain into the environment or in a host. For example, a ^-glucuronidase gene has been inserted in M. anisopliae to allow detection of hyphae in infected hosts (St Leger et al. 1995) and the expression of a green fluorescent protein-encoding gene for tracking purposes (St Leger 2001).

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