Loop-mediated isothermal amplification: a rapid molecular technique for early diagnosis of Pseudomonas syringae pv. syringae of stone fruits

Abstract

Background: Pathogenic bacteria cause significant economic damages in agriculture. The detection of such bacteria is considered as a continual interest for plant pathologists to prevent disease dissemination. Pseudomonas syringae pv. syringae is one of the most important bacterial pathogens infecting yield and quality of stone fruits throughout the world. Biochemical assays such as a LOPAT and GATTa are common methods to detect this pathogen. Serological tests and culturing on King’s B selective medium also used to isolate this bacterium. Selective media is composed of specific and effective ingredients to inhibit the growth of certain species of microbes in a mixed culture while allowing others to grow. These are used for the growth of only selected microorganisms. King’s B medium can be used as a general medium for the non-selective isolation cultivation and pigment production of Pseudomonas species from foods, cosmetic samples, plants, etc. Nevertheless, the 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. We have used loop-mediated isothermal amplification to couple with a target. The amplification of syrD gene using loop and bumper primers can be used to prevent disease dissemination. Results: The outcome of this investigation indicated more sensitivity of LAMP in comparison to PCR. The direct addition of SYBR Gold in microtube is more sensitive than gel in both LAMP and PCR byproducts so we can eliminate gel electrophoresis, while the LAMP showed high sensitivity and high specificity in comparison to results obtained by cultivation. The described molecular test could detect Pseudomonas syringae pv. syringae type in nearly 1 h, and this is the first time that Lamp molecular detection of Pseudomonas syringae pv. syringae particularly on stone fruits is described and introduced. Conclusions: The obtained data confirmed that LAMP is a fast, cheap, and high specific method for the rapid detection of Pseudomonas syringae pv. syringae to the comparison of PCR and culture.