Summary auto-generated
This study applied random amplified polymorphic DNA (RAPD) fingerprinting to examine genetic relationships among 31 mosquito-pathogenic and 14 nonpathogenic strains of Bacillus sphaericus. The researchers used eight decamer primers to generate DNA fragments via PCR and verified that bands migrating the same distance in agarose gels were homologous using digoxigenin-labeled probes. Clustering analysis identified six groups corresponding to known DNA homology groups, with pathogenic strains of homology group IIA showing only 5.2-6.3% similarity to nonpathogenic strains. Within serotypes of pathogenic strains, genetic homogeneity was high (minimum 84% similarity), and each serotype produced distinctive RAPD patterns. Individual serotypes could be clearly identified by serotype-specific bands. The results support previous DNA homology, fatty acid analysis, and rRNA sequence data indicating that B. sphaericus comprises genetically distinct groups. Importantly, toxin production was stable and not plasmid-encoded, unlike Bacillus thuringiensis. The authors conclude that sufficient genetic and phenotypic evidence supports creating a new species designation for mosquito-pathogenic B. sphaericus strains.
Key findings
- RAPD fingerprinting confirmed five DNA homology groups in B. sphaericus, with pathogenic group IIA strains showing 5.2-6.3% similarity to nonpathogenic groups, supporting distinct species classification
- Individual serotypes within pathogenic strains showed high genetic homogeneity (≥84% similarity) with distinctive RAPD patterns and serotype-specific DNA bands
- DNA hybridization experiments verified that comigrating RAPD bands represent homologous DNA sequences, validating the fingerprinting approach
- Toxin genes in mosquito-pathogenic B. sphaericus are chromosomal rather than plasmid-encoded, making pathogenicity a stable taxonomic trait unlike in Bacillus thuringiensis
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Abstract
Random amplified polymorphic DNA fingerprinting was used to examine 31 mosquito-pathogenic and 14 nonpathogenic strains of Bacillus sphaericus. We verified that DNA bands that migrated the same distance in an agarose gel were homologous by using PCR-generated probes made from the random amplified polymorphic DNA bands. The band patterns obtained with eight primers were analyzed by using the Jaccard coefficient and unweighted pair group with arithmetic average clustering. Pathogenic strains belonging to DNA homology group IIA were similar to strains belonging to nonpathogenic homology groups at an average level of similarity of 6.3%. Individual serotypes were clearly identified among the pathogenic strains. This suggests that there is overall genetic homogeneity among strains within serotypes. It is also consistent with the uniform toxicity pattern found for each serotype (unlike the toxin diversity found in Bacillus thuringiensis serotypes). These results, together with DNA homology data, support the proposal that a new species should be described for the pathogenic strains.