Quantitative real-time PCR assay for rapid detection of Pseudomonas amygdali pv. lachrymans in cucumber leaf rinse

Abstract

Pseudomonas amygdali pv. lachrymans (Pal) is a bacterium that causes angular leaf spot disease of cucumbers. There is no direct protection against this pathogen, only prevention. Previously published detection systems are labour-intensive and time-consuming. The aim of this study was to create a rapid and reliable diagnostic assay for the detection of Pal in cucumber plants without the need to destroy or damage the plant. The created method is based on washing the bacteria off the leaf surface with saline solution and using the sample directly in a quantitative real-time PCR assay. This detection system is based on the specific amplification of a small coding region of the bacteriophage N4 adsorption protein B gene. The assay was tested on 120 plant samples inoculated with seven different Pal strains. The detection limit of the assay was 2.60\u2009×\u2009102\xa0CFU\xa0ml−1, and the concentration of Pal in the leaf rinse ranged from 2.71\u2009×\u2009102 to 6.94\u2009×\u2009107\xa0CFU\xa0ml−1 depending on the particular strain. This assay is a reliable and rapid diagnostic method for the detection of Pal from cucumber leaves and is valuable for contributing to angular leaf spot management.