Characterization of two DNA clones specific for identification of Corynebacterium sepedonicum

Abstract

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.

Detection of Bacterial and Phytoplasmal Pathogens

“…atroseptica (Eca) De Boer 1984, 1990) and C. michiganensis subsp. sepedonicus (Cms) (Verreault et al 1998) in potato tubers. Cms, the causative agent of potato ring rot disease, was detected by labeled probes obtained from unique three single-copy DNA fragments designated Cms 50, Cms 72 and Cms 85 isolated from CS3 strain of Cms by subtractive hybridization.…”

Detection and Identification of Postharvest Microbial Pathogens

“…Different probes have been employed for the detection of Cms in symptomless potato tissues and it is known to persist in potato tissues for long periods without being detected, if cultural and immunological methods are applied (Verreault et al, 1988;Johansen et al, 1989;Mirza et al, 1993;Drennan et al, 1993). A 1.1 kb multicopy repeated sequence (Rs) from Cms was used as a probe in direct blotting of potato plant tissue (Drennan et al, 1993).…”

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

“…The extraction method described by Verreault et al (1988) was modified as follows. The pronase was replaced by proteinase K. The lysis of the bacterial cells was followed by a phenol extraction and an ethanol precipitation at )20°C for 1 h (Maniatis et al 1989).…”

“…A simplified version of the RNA isolation method according to Verreault et al (1988). In this method, bacteria were suspended in 0AE3% (w ⁄ v) glycine and concentrated by centrifugation for 10 min at 10 000 g. The pellet was resuspended in 30 ll TSE (10 mmol l )1 Tris ⁄ HCl, 10 mmol l )1 NaCl and 0AE8 mmol l )1 EDTA, pH 7AE5) and 30 ll lysozyme (Sigma, 10 mg ml )1 ) and incubated for 1 h at 37°C.…”