Detection Of Tomato Bacterial Canker Disease (Clavibacter Michiganensis Subsp. Michiganensis) With Real-Time Fluorescent PCR

Clavibacter michiganensis subsp. michiganensis is the most destructive bacterial pathogen of tomato. The infected seeds are the major way to spread the pathogen distantly. EPPO has listed Cmm as an A2 quarantine pest. The traditional methods such as: isolation and culture, physiological and biochemical identification, phage specificity, serological test and host reaction-were applied to detect and identify the bacterium. These methods are time-consuming, low efficiency,low accuracy and sensitivity, which can not meet the custom-quarantine expectations. PCR was applied to detect the phytopathogen widely in recently years, which is more rapid and efficient. However, the gel electrophoresis is needed to determine the sizes of amplified product after normal PCR and the contamination, safety and time-consuming problems have been noted as significant issues. So a safer, more accurate, sensitive and rapid PCR method are required for phytopathogen detection. The series of primers and Taqman probe were designed based on the region of ITS to detect Cmm with normal PCR and Real-Time fluorescent PCR (RTF PCR) in this experiment. The specific primers and probes were found that they could detect all the Cmm isolates collected from different sources. The protocol was approximately 100 times more sensitive than normal PCR and the amplified products were confirmed by specific fluorescent probe used during amplification. Strong fluorescent signal could be collected in the reaction when the DNA were directly extracted from the inoculated seed and seedling which had no symptom. Furthermore, the pathogen isolation is not required and contamination is controlled because the whole detection process is finished in the contained tubes. This method can provide a specific, sensitive, rapid detection of Cmm for quarantine department and the seed industry.