Characterization of two DNA clones specific for identification of Corynebacterium sepedonicum

Escherichia coli TB1 was transformed with pUC9 containing fragmented DNA (4–10 kilobases (kb)) from Corynebacterium sepedonicum. The resulting genomic bank was screened by a dot blot assay to identify clones specifically hydridizing to C. sepedonicum DNA and not to the DNA of several other Gram-positive and Gram-negative bacteria. Two clones (III24 and III31) were selected because of their ability to strongly hybridize to C. sepedonicum DNA and weakly hybridize to the DNA of C. michiganense, Erwinia carotovora, Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas solanacearum, Micrococcus luteus, and Arthrobacter globiformis. These two clones were also specific for C. sepedonicum DNA when tested against the DNA from 30 isolates of soil bacteria. Restriction enzyme analysis has shown that the two clones have an insert of 8 kb (III24) and 4 kb (III31). On the basis of restriction enzyme patterns, one clone (III24) does not correspond to plasmid pCL 50, a cryptic plasmid found in several C. sepedonicum isolates. Because purified III24 and III31 DNA can be used to detect approximately 1 ng of C. sepedonicum genomic DNA, the two clones can complement serological or biological detection methods. This could be useful, especially when a high degree of specificity is required for detection or identification of this plant pathogen.