Random amplified polymorphic DNA (RAPD) analysis of Bacillus sphaericus

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Virginia Tech

Mosquito pathogenic strains of Bacillus sphaericus are indistinguishable from nonpathogenic strains based on simple phenotypic tests. DNA-DNA hybridizations performed in 1980 placed the 7 pathogens included in that study in a distinct homology group separate from 5 groups of nonpathogens. The overall homology of the pathogenic strains to the species type strain was only 19% indicating that these pathogens should be a separate species.

Since the DNA homology study was published in 1980, many more pathogenic strains have been isolated worldwide. Pathogenic strains have been differentiated from other strains of B. sphaericus by rRNA sequencing, fatty acid analysis, and isozyme analysis. The pathogens have been further classified by type of toxin produced, serotyping, and phage typing.

I have used random amplified polymorphic DNA (RAPD) fingerprinting to determine the phenetic relationships among 31 pathogenic and 14 nonpathogenic strains of B. sphaericus. DNA Bands in agarose gel migrating the same distance were verified as being homologous using PCR-generated probes made from the RAPD bands. Band patterns resulting from 8 10-mer primers were examined by three coefficients, Jaccard, Dice, and simple matching. Each coefficient was able to distinguish DNA homology groups, although the relative similarity values differed. In agreement with DNA homology studies, pathogenic strains showed less than 10% similarity to nonpathogens using Jaccard and Dice coefficients. This value was 68% based on the simple matching coefficient.

Individual serotypes were clearly indicated among the pathogenic strains by each coefficient. This suggests an overall genetic homogeneity among strains within serotypes. It also parallels the uniform toxicity pattern found within each serotype (unlike the toxin diversity found within B. thuringiensis serotypes). These results together with DNA homology data support the establishment of a new species for the pathogenic strains.