Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/29/1996
Publication Date: N/A
Interpretive Summary: Plant pathogenic coryneform bacteria have been recently classified into two genera, Clavibacter and Rathayibacter. Some species and subspecies in these genera cause serious diseases in various plant species. Ring rot disease of potato, caused by C. michiganensis subsp. sepedonicus (C.m.s.), is one such serious disease. Because potato ring rot has been assigned a zero tolerance level for both import and export of seed potatoes in several countries, an ability to detect very low titers of C.m.s. in seed potatoes is indispensable for disease control. Polymerase chain reaction (PCR) assay has been shown to be more sensitive than other means available. The present study was undertaken to develop a nested-PCR that can be used for an ultrasensitive detection of potato ring rot bacterium. Two sets of primer pairs were developed and can be used to specifically detect C.m.s. or to detect all subspecies in C. Michiganensis. A classification system based on restriction fragment length polymorphism analysis of PCR-amplified 16S rDNA developed in this study can be used to rapidly classify and identify species and subspecies in the genus Clavibacter and the closely related coryneform bacteria. The technology will be of benefit to diagnosticians and APHIS and will facilitate the pathogen screening for potato ring rot in commercial seed potatoes.
Technical Abstract: Oligonucleotide primers derived from sequences of 16S ribosomal RNA (rRNA)gene (CMR16F1, CMR16R1, CMR16F2, CMR16R2) and the insertion element IS1121 of Clavibacter michiganensis subsp. sepedonicus (C.m.s.) CMSIF1, CMSIR1, CMSIF2, CMISR2) were used in nested-PCR (polymerase chain reaction to detect potato ring rot bacterium C.m.s. Nested-PCR, using primer pair CMSIF1/R1 followed by a second primer pair CMSIF2/R2, specifically detected C.m.s. In another nested-PCR, using CMR16F1/R1 followed by CMR16F2/R2, only C.m.s. and the other subspecies in C. Michiganensis were detected. In the latter case, C.m.s. can be differentiated from the other subspecies by restriction fragment length polymorphism (RFLP) analysis of nested-PCR products. The nested-PCR assays developed in this work allow for an ultrasensitive detection of very low tiers of C.m.s. which may be present in symptomless potato plants or tubers and which can not be readily detected by direct PCR (single PCR amplification). These separate nested-PCR assays, using primers derived from 16S rDNA and insertion element sequences, provide for an unambiguous confirmation of presence and identification of CMS A rapid classification system based on RFLP analysis of the PCR-amplified 16S rDNA sequences was developed for differentiating species and subspecies in the genus Clavibacter and the closely related genus Rathayibacter.