Abstract:

In order to establish a rapid and specific detection method for tomato diseases，a quadruple PCR assay was designed for identifing the pathogens including Pseudomonas syringae pv. tomato（Pst），Clavibacter michiganensis subsp. michiganensis（Cmm），Ralstonia solanacearum（Rs）and Xanthomonas campestris pv. vesicatoria（Xcv）. The specific primers（BW-F/BW-R）of Pst were designed based on the gap1 gene sequence，which can amplify 375 bp specific fragment by optimizing the PCR conditions. Besides，three pairs of specific primers of Cmm（Fan1/Fan2），Rs（RS-1-F/RS-3-R），and Xcv（XCVF/XCVR）used in this study were referenced previous studies. The primer concentration，the annealing temperature，amplification cycles，and the extension time were optimized to obtain the best ratio of primers and amplification conditions. Then，the rapid quadruple PCR detection of pathogens in tomato was established. The annealing temperature was 57.1 ℃，the extension time was 45 s and the number of cycles was 35. The final concentrations of BW-F/BW-R primers，Fan1/Fan2 primers，RS-1-F/RS-3-R primers and XCVF/XCVR primers were 0.24，0.16，0.16 and 0.08 μmol ? L-1，respectively. The expected sizes of amplification bands were 375，146，716 and 517 bp for BW-F/BW-R，Fan1/Fan2，RS-1-F/RS-3-R and XCVF/XCVR，respectively. The quadruple PCR can simultaneously detect Pst，Cmm，Rs and Xcv in infected plants tissue，with the detection limits of 0.1 ng ? μL-1 gDNA. This study indicated that quadruple PCR might be a useful tool for rapid and sensitive diagnosis and provided an effective technical support for pathogenic identification.

Key words: tomato, Pseudomonas syringae pv. tomato, Clavibacter michiganensis subsp. michiganensis, Ralstonia solanacearum, Xanthomonas campestris pv. vesicatoria, quadruple PCR