E-Book 2nd Congress

  • Isothermal amplification of syrD gene as a tool for detection of pseudomonas syringae pv. Syringae
  • Keihan Kookli,1 Pouyan Asadi,2 Elham Talebi,3 Rabee Movagharnia,4 Danial Jafari,5,*
    1. International Campus, Iran University of Medical Sciences, Tehran, Iran
    2. Medical Cellular & Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran
    3. Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
    4. Department of Genetics and Biotechnology, School of Biological Sciences, Varamin-Pishva Branch, Islamic Azad University, Varamin, Iran
    5. MD, Medicine faculty of Islamic Azad University of Shahrood, Iran


  • Introduction: Pseudomonas syringae pv. syringae is one of the most important bacterial pathogens infecting stone fruits throughout the world. It can lead to diseases in more than 180 plant species such as fruit trees, annual, and perennial plants. P. syringae damages are determined by the growing region of stone fruits and host plants. Biochemical assays such as LOPAT (Levan production, oxidase production, pectinolytic activity, arginine dihydrolase production, tobacco hypersensibility) and GATTa (Gelatin liquefaction, aesculin hydrolysis, tyrosinase activity, tartrate utilization) are common tools for detecting this pathogen. Serological tests and culturing on King B (KB) medium were also used to detect/isolate this bacterium. Nevertheless, mentioned methods are not enough accurate to differentiate the strains. On the other hand, PCR-based techniques are sensitive and efficient in detecting plant diseases. However, these techniques are not practicable for those researchers who do not have access to a thermal cycler. In the current study, we have used Loop-mediated isothermal amplification (LAMP) as a fast, highly specific tool to couple with a target.
  • Methods: 65 bacterial canker samples taken from stems, buds, twigs, and shoots were collected from gardens of apricot (Prunus armeniaca) fruits. A total of 65 bacteria were isolated from 65 infected parts of apricot trees. The 65 samples were divided into two groups; one cultured on a King B medium for detection of the strains. For this purpose, 65 samples were kept in nutrient broth containing 20% glycerol at −85◦C and cultured on KB at 25◦C for 48 h before usage. After 24-48 h of incubation, fluorescence on KB was observed under UV light. Another group was used for genomic DNA extraction. Genomic DNA was extracted and PCR was carried out for syrD (Syringomycin D). PCR amplification reactions were performed in a C1000 Touch™ Thermal Cycler. The amplified products were stained by SYBR Gold after running on the agarose gel. Equal dilutions were prepared for both LAMP and PCR products and run on the electrophoresis gel to perform the sensitivity assay of PCR and LAMP products. To remove the electrophoresis step optionally and direct visualization of PCR products by SYBR Gold, it was directly added to the PCR products in the microtube to be visualized by a UV transilluminator. To eliminate the electrophoresis step optionally and direct visualization of LAMP products by SYBR Gold, it was directly added to the product’s microtubes to be visualized by a UV transilluminator. The specificity of the LAMP with 60°C and PCR methods performed with an annealing temperature of 55°C. Similar dilutions were prepared for both LAMP and PCR products to compare the PCR and LAMP products’ sensitivity. To confirm whether Pseudomonas syringae pv. syringae was identified correctly, the suspected samples were cultured on KB medium at 28°C. After 48-72 h of incubation, Fluorescence on King’s medium B was observed under Ultra Violet light.
  • Results: SyrD gene amplification using LAMP and PCR primers resulted in molecular detection. The results indicated that the direct add-on of SYBR Gold in microtubes was 100 times more sensitive than electrophoresis in direct visualization. The results revealed that the sensitivity of gel-free staining is 10 times greater than that of gel-based staining in direct visualization. The findings indicated that the bacterium has been identified correctly. 20 Pseudomonas syringae strains from 65 ones were fluorescent on the KB medium.
  • Conclusion: The results confirmed that molecular detection (LAMP, PCR, and electrophoresis) assays have more efficiency in comparison to direct culture (King B medium).
  • Keywords: Pseudomonas syringae pv. Syringae, syrD gene, King B medium, PCR, LAMP