LNA probe based real-time PCR for detection and identification of tomato bacterial canker (Clavibacter michiganensis subsp. michiganensis) and tomato bacterial leaf spot (Xanthomonas axonopodis pv. vesicatoria)

In this study, a sensitive and selective Real-Time PCR method was developed for identification and detection of Clavibacter michiganensis subsp. michiganensis (Cmm) causal agent of Tomato Bacterial Canker and Wilting Disease and Xanthomonas axonopodis pv. vesicatoria (Xav) causal agent of Bacterial Leaf Spot Disease of tomato and peppers.The sensitivity of primer and prob sets developed in this study were tested against different Cmm and Xav strains, plant pathogenic bacteria from different genus and species, tomato and pepper total genomic DNA. Although 73 bp and 69 bp DNA fragments were amplified from all Cmm and Xav strains, respectively, no amplified product was obtained from bacterial genus and species except Cmm and Xav, and tomato genomic DNA. The detection limits of Real-Time PCR were found to be 1 cfu/ml and 2 cfu/ml from seeds, 1 cfu/ml and 2 cfu/ml from direct bacterial cell and 10 pg DNA from purified genomic DNA, for Cmm and Xav, respectively. This Real-Time method developed by using LNA probe providing internal control of the sequence is selective, sensitive and repeatable.This method can be used for rapid and sensitive identification of Cmm and Xav strains from direct bacterial cell and detection of Cmm and Xav strains from seed and diseased plant tissues.KEY WORDS : Tomato, Clavibacter michiganensis subsp. michiganensis (Cmm),Xanthomonas axonopodis pv. vesicatoria (Xav), Real-Time PCR (RT-PCR),Real-Time Bio-PCR,Diagnosis, Detection