Genetic variation among isolates of the entomopathogenic fungus, Beauveria bassiana (Bals.) Vuill. (Ascomycota: Hypocreales): use of rRNA internal transcribed spacers and group I introns, and a novel polymorphic minisatellite locus for the identification of strains
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The filamentous ascomycete fungus Beauveria bassiana is a pathogen of certain crop insects and is used in insect biological control regimes, yet little investigation has been devoted to the description of reliable genetic fingerprinting techniques or the analysis of population genetic diversity. An allele-specific molecular genetic approach was devised for the differentiation of 120 isolates of the entomopathogenic fungus B. bassiana using nuclear ribosomal RNA (rRNA) mutation and allelic variation at a single minisatellite locus. High resolution PCR-RFLP and direct DNA sequence analysis was used to describe the level of variation in two nuclear rRNA internal transcribed spacer (ITS) regions and four rRNA large subunit (LSU) group I introns. Differential cleavage at seven restriction sites within the ITS region identified 31 unique genotypes among 111 B. bassiana isolates. Inter- and intraspecific PCR-RFLP variation in four group I introns inserted into the B. bassiana LSU rRNA identified thirty-seven different fungal strains. A second locus was isolated from a partial B. bassiana genomic library and shown to contain a novel polymorphic minisatellite repeat, BbMin1. Size variation in PCR amplified fragments identified eight allelic types within the sample set that varied in the number of complete repeat units. In conjunction with rRNA loci and allelic variation at the BbMin1 locus, 89 genetic groups were delineated. Fixation indices (Fst) were calculated separately for each genetic marker data set, and neither indicated statistical correlation between genotype and geographical origin or pathogenic phenotype. All genetic markers were suggested to be selectively neutral since similar genotypes were randomly distributed between arbitrary subpopulations of Beauveria.