Molecular and biochemical aspects of the mechanisms of pathogenesis for the entomopathogenic fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana can infect and decimate insect populations, including the migratory grasshopper, Melanoplus sanguinipes. The objective of this work was to use genetic, molecular biological and biochemical means to analyze aspects of B. bassiana pathology in grasshoppers. This included the development of temperature-sensitive mutants to delineate critical stages in the infection as well as the creation of specific DNA-based systems for detection and classification.;Heat-sensitive (HS) and cold-sensitive (CS) mutants were isolated and characterized. Mutants were found that were affected in cell wall and macromolecular synthesis, septum formation, or non-specific functions. All of the HS mutants examined were infective at the permissive temperature (PT) of 20;There was a correlation between mutant physiology at the NPT and the outcome of the infection. Evidence was also found to support the postulate that B. bassiana can circumvent the host's defense responses through avoidance of phagocytosis. Newly formed hyphal elements are refractory to phagocytosis and perpetuate the infection beyond cuticle penetration and initial interaction with circulating hemocytes.;A series of genomic DNA probes which specifically recognize B. bassiana were developed. The probes were used to generate strain-specific restriction fragment length polymorphisms (RFLPs) that allowed for inter-strain discriminations. The sequence of one of the probes, pBb22, was used to select B. bassiana-specific primers for the polymerase chain reaction (PCR). Analysis of PCR products with restriction enzymes, DNA sequencing and single-stand conformation polymorphisms was used to differentiate Beauveria spp. and B. bassiana strains. Protocols were developed for the isolation of PCR-amplifiable DNA that allowed for the detection of the fungus within infected insects. Cloned mitochondrial (mt) DNA sequences from B. bassiana were used to generate strain and species-specific RFLPs. The mt DNA of B. bassiana was grouped into two categories, type A and type B, based on RFLP similarities.;The systems described here have proven useful for the study of the mechanisms of pathogenesis as well as epizootiological considerations for B. bassiana.