3.3.40
USE OF BIO-PCR AND AN AUTOMATED FLUORESCENCE, SENSITIVE DETECTION OF CLAVIBACTER MICHIGANENSIS SUBSP. SEPEDONICUS IN POTATO TUBERS

NW SCHAAD1, Y BERTHIER-SCHAAD1, A SECHLER1 and D KNORR2

1ARS/USDA Foreign Disease-Weed Science Research Unit, Frederick, MD 21702, USA; 2 PE Applied Biosystems, Foster City, CA 94404, USA

Background and objectives
Ring rot of potato is one of the most regulated diseases of potatoes world wide. The only control for the disease is clean seed. All potato producing countries maintain a zero tolerance quarantine on the pathogen, Clavibacter michiganensis subsp. sepedonicus (CMS). The bacterium has been difficult to detect in soil or infected tubers because CMS grows very slowly, is present in low numbers, and exists in different morphological forms. Present methods for detecting the pathogen include immunofluorescence and ELISA. These serological methods generally require >1 0,000 cfu/ml for a positive result and often have specificity problems. In contrast, BIO-PCR requires only 1 cfu/l00ml of test sample. The objective of this work was to determine the reliability of using BIO-PCR together with the ABI PRISM 7700 Sequence Detection System (TaqMan) for detecting CMS. A major advantage of the TaqMan system is that results are based on PCR amplification coupled with oligonucleotide hybridization.

Materials and methods
A fluorescent oligonucleofide probe and PCR primers were designed using sequences from the unique DNA fragment designated Cms 50 (2). Thirty tubers suspected of being infected with CMS, collected from three widely separated locations were sampled by the core tissue extraction shaker incubation procedure(1). Tuber extracts were assayed by 1) plating aliquots onto agar media, 2) classical PCR, and 3) BIO-PCR and TaqMan.

Results and conclusions
Results of screening the primers against a large number of CMS strains and other bacteria showed the primers to be specific for CMS. Four tubers tested positive by agar plating and pathogenicity tests, eight by classical PCR, and 26 by BIO-PCR and TaqMan. We conclude that BIO-PCR combined with the TaqMan automated detection system is a rapid, reliable, and inexpensive method of assaying large numbers of potato tubers for CMS.

References
1. Dinsen, IG, De Boer SH, 1995. Amer. Potato J. 72:133-142.
2. Mills D, Russell BW, Williams Hanus J, 1997. Phytopathology 87:853-861.