Chapter VIII-3 - The application of molecular techniques to insect pathology with emphasis on entomopathogenic fungi

Abstract

This chapter discusses the application of molecular techniques to insect pathology with emphasis on entomopathogenic fungi. Entomopathogenic fungi represent an unconsidered, and therefore untapped, reservoir of pesticidal genes for the production of advanced engineered pesticides. The application of molecular techniques to pathogen genomes often depends on the ability to purify high molecular weight DNA. Two major factors influencing satisfactory purification of high-molecular-weight DNA are shearing and the presence of nucleases. Identifying the presence of a particular pathogen genotype using specific DNA probes is greatly facilitated by the ability to extract fungal DNA from infected insects or soil. Protoplasts are prepared from young mycelia growing in liquid shake cultures. The mycelia are centrifuged and washed with osmotic stabilizer containing varying amounts of sorbitol/ NaC1/MgSO4. Techniques developed to transform filamentous fungi have followed the same three conceptual steps applied to all organisms. It is suggested that high-velocity ballistic transformation of M. anisopliae germlings can be performed with the DuPont particle delivery system PDS 1000/HE by modifying methods used with other filamentous fungi.