Detection and Early Diagnosis of Peronospora tabacina Adam in Tobacco Plant with Systemic Infection

A disease characterized by the absence of sporulation on leaves and evident necrosis underneath the bark tissues affected adult tobacco plants during 2005 in south Italy. In the field, diagnosis was based mainly on recognition of symptoms, often non-specific, resulting in the application of the wrong control strategy. Two current diagnostic methods based on microscopic observations were compared with polymerase chain reaction (PCR) technique. DNA extraction protocol was modified by replacing sonication with a further lysis step at 60 C and PCR was performed using primers that amplified a specific DNA fragment (230 bp). Microscopy methods were able to detect the pathogen in 50% and 6% of cases, respectively, while PCR was able to detect Peronospora tabacina in all diseased plants. Elimination of sonication in DNA extraction rendered the method easier to perform and avoided the possibility of DNA fragmentation. The results demonstrated that PCR makes the diagnosis fast and reliable while microscopy gives discontinuous results, is time-consuming and not applicable for monitoring