CONSTRUCTION OF CLONED DNA PROBES FOR THE SPECIFIC DETECTION OF THE ENTOMOPATHOGENIC FUNGUS BEAUVERIA-BASSIANA IN GRASSHOPPERS

A series of four DNA probes which specifically hybridize to the entomopathogenic fungus Beauveria bassiana have been developed. The B. bassiana-specific DNA fragments ranged in size from 1180 to 2700 bp. The probes did not hybridize to DNA from other Beauveria spp. (brongniartii, caledonica, and densa), Tolypocladium nivea, Tolypocladium cylindrospora, Metarhizium anisopliae strains, Verticillium lecanii, Paecilomyces farinosus, other nonentomopathogenic fungi (Aspergillus niger and Neurospora crassa), or grasshoppers. The probes are derived from moderately repetitive DNA and produce distinct hybridization patterns with all strains of B. bassiana tested. B. bassiana strain DAOM 71453 produced hybridization patterns that differed extensively from other strains when probed with the B. bassiana-specific probe pBb12 or the mitochondrial small ribosomal (sr) RNA-derived probe pBbmtE3. As well, several probes were identified which hybridized to Beauveria densa DAOM 57904 as well as to B. bassiana GK 2010 under conditions of reduced stringency. Two of these probes produced identical hybridization bands for the two strains, thus supporting previous biochemical evidence that B. densa is an aberrant strain of B. bassiana. The most sensitive of the probes detected 152 ng of total fungal DNA on a routine basis. This level of sensitivity proved to be suitable for the identification of the fungus within field-collected grasshoppers of the following species: Melanoplus sangainipes, Melanoplus bivittatus, Melanoplus packardii, Melanoplus infantalis, and Camnula pellucida that were infected with B. bassiana. These probes should be useful in the study of mechanisms of pathogenesis as well as epizootiological considerations for B. bassiana, both in the susceptible hosts and nontarget organisms. © 1993 Academic Press, Inc.