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- Title
FAST TEMPLATE PREPARATION FOR MOLECULAR DETECTION OF RALSTONIA SOLANACEARUM AND CLAVIBACTER MICHIGANENSIS subsp. SEPEDONICUS.
- Authors
Lucchese, C.; Galeone, A.; Mazzucchi, U.; Bertaccini, A.
- Abstract
Member states of European Union (EU) conduct systematic official surveys to confirm in the potato production system the absence of Clavibacter michiganensis subsp. sepedonicus, the causal agent of potato ring rot, and of Ralstonia solanacearum, the causal agent of potato brown rot. PCR on DNA extracted from potatoes and processing water samples is the most frequently first screening test used among those regulated by EU Directives. The considerable amount of sample lots and the necessity of quick diagnostic responses opens to the need for a more rapid detection of these two bacteria with a sensitivity comparable to that of the official methods. Different types of samples, artificially contaminated by 107 to 10• CFU/ml bacterial suspensions, were submitted to a preliminary additional centrifugation to obtain crude extracts. Three methods were also tested: (i) lysis with 10% 0.5 N NaOH; (ii) lysis plus purification on FTA ™Card (Whatman) and (iii) digestion with amylase. The sensitivity of PCR, additioned with 2% blotto, on crude extracts and on those treated with the above three methods was compared to PCR on DNA extracted using a commercial kit. PCR detection limit on DNA extracted from potato and potato processing water by a commercial kit was 10³ CFU/ml and 104 CFU/ml, respectively. PCR assays performed on crude extract of potato processing water showed a sensitivity increase to 10² CFU/ml, whereas the assays on potato samples lysated and purified on FTA ™ card showed the same sensitivity of official DNA extraction, allowing also the detection of latently infected tubers.
- Subjects
POTATO diseases &; pests; RALSTONIA solanacearum; CLAVIBACTER michiganensis; BACTERIAL wilt of potato; BROWN rot; EUROPEAN Union
- Publication
Journal of Plant Pathology, 2012, Vol 94, Issue 4, pS4.49
- ISSN
1125-4653
- Publication type
Article