Detecting Pathogenic Fungus And Bacteria Of Main Potato Diseases By Multiplex Real-Time PCR

China is the biggest potato producer in the world and is ranked #1 in both total yield and acerage. Potato production in China is threatened by a number of diseases. Major potato diseases in China include late blight caused by oomycete pathogen Phytophthora infestans, dry rot caused by fungal pathogens Fusarium spp., and soft rot and bacterial wilt caused by bacterial pothgens Fusarium spp. and Ralstonia solanacearum, respectively. Currently, the primary approach for controlling these diseases in China is by application of disease-free seed potatoes. Thus, development of efficient and accurate methods for detecting various potato disease pathogens is essential for production and application of quality seed potatoes. Various techniques have been developed to detect the pathogens that cause different potato diseases. However, the recently developed real-time polymerase chain reaction (PCR) methodology is widely used in detecting different potato pathogens. The real-time PCR methodology is simple, fast, highly sensitive, and more reproducible than most of the traditional techniques.To establish real-time PCR procedures for detecting pathogens associated with several potato diseases, DNA primers were designed from conserved DNA sequences associated with pathogens that are responsibe for potato late blight (Pi), potato black scurf (Rhs), potato soft rot (Ec), potato dry rot (Fz), and potato bacterial wilt (Rs). The potato gene Cox was used as a internal control to eliminate false negatives in the detection procedure. In our study, we establish Real time PCR using DNA binding dye SYRB GREEN I and labelled fluorescence Taqman probe. In addition, we developed a double Real time PCR to detect the diseases according to their different fluorescence:Rhs and Cox, Fz and Ec, Pi and Rs. But Pi and Fz can not be combined together. Sensitivity test showed to us Real time PCR is 10～100 times more than conventional PCR. The new detection procedure is simple, rapid and reliable. It can be used for control the seed production areas strictly so that ensure the quality of seed.