PCR-BASED DETECTION OF BACTERIAL RING ROT INFECTIONS IN POTATO
PCR-BASED DETECTION OF BACTERIAL RING ROT INFECTIONS IN POTATO R KARJALAINENl2, A KANGASNIEMIl, M HEITH l, J TEGEL3 AND T KERVINENl lUniversity of Helsinki, Box 28, Fin-00014, Finland; 2 Agricultural Research Centre, Plant Protection, Fin-31600, Jokioinen, Finland; 3Plant Protection Inspection Centre, Box 42, Fin-00501 Helsinki, Finland Background and objectives Clavibacter niichiganensis subsp. sepedonicus is the causal agent of bacterial ring rot of potato. This is a seed-bome pathogen and restrictions have been set up on seed imports from countries were infections have occurred (e.g. Europe and Canada). This bacterial pathogen is a very difficult to detect reliably by visual inspections of symptoms because this disease can occur in latent infections. In Europe and Canada, aerological methods based on immunofluorescence are widely used. However, detection of bacterial infections by immunofluorescence may have problems with cross-reacting saprophytic bacteria. Therefore, following the positive result found in aerological tests, further analysis based on bacterial isolation and inoculation into eggplant is subsequently carried out. In practice, eggplant test is time consuming and rather difficult to perform reliably. Consequently, improved diagnostic methods with high sensitivity and specificity is urgently needed in the detection of latent infections from potato seeds. We show here that the PCR is a suitable tool for also routine analysis of ring rot infections from potato seeds. Results and discussion In order to develop a PCR procedure suitable for analysing the latent infections in potato tubers from routine samples of large composite material, specific primers were successfully designed from sequence information of genomic fragments of C.m.sepenodicus. DNA from several related Coryneform and other bacteria were amplified by specific primers but only positive signal was produced by DNA from ring rot bacteria. We optimized a PCR procedure so that the annealing temperature was raised up to 55 C which eliminated the nonspecific PCR products and the dilution of original tissue sap 1: 10 improved further the resolution. As a consequence of these improvements, a single clear PCR product of 0,3 kb was detectable when tuber tissue was contaminated by C.m.sepenodicus. PCR procedure described here allowed a detection limit about 1 pg of C.m.sepenodicus DNA using of 40 cycle of amplification. For the testing of our PCR procedure for analysing practical samples of potato for ring rot detecton, two types of material were analyzed by both PCR and immunofluoreseence methods. Composite samples consisting of 200-400 tubers from 20 000-25 000 kg of potatoes constituted a single sample in a latent infection test. Our results indicate that generally the same positive and negative results could be revealed in parallel tests with PCR and immunofluorescence, but in some cases were the results of aerological test was difficult to analyze, PCR gave clear negative results. Thus, our experience suggest that PCR is a reliable test system for practical diagnosis of difficult samples.