Detection of Pseudomonas syringae pv. tomato by PCR

Abstract

Pseudomonas syringae pv. tomato is the causal agent of bacterial speck of tomato. This bacterium may cause spots on leaves, with or without a chlorotic halo, and on fruits, with consequent economic damage. The identification of the P. s. pv. tomato is mainly based on isolation of bacteria on semiselective media (e.g. VBTar) coupled to DNA hybridisation using coronatine synthesis genes and on serological techniques such as indirect fluorescent antibody staining (IFAS) and enzyme-linked immunosorbent assay (ELISA). A method based on PCR amplification of coronatine gene sequences was reported as well. Unluckily not only are cor genes conserved among several P. syringae pathovars, but also Cot P. s. pv. tomato strains have been described, leading to potential false negative or positive misdiagnosis. In this study we present a molecular detection method based on PCR amplification of stable chromosomal sequences that are located in the Hrp_PST pathogenicity island. Pathovar-specific oligonucleotide primers were designed on nonconserved regions of the hrpZ gene. A specific amplification product of 532 bp was obtained from all of 35 isolates of P. s. pv. tomato but not from 98 pathogenic and symbiotic bacterial isolates belonging to the genera Pseudomonas, Xanthomonas, Erwinia, and Rhizobium. Moreover, no amplicons were obtained from a plethora of unidentified bacterial saprophytes isolated from tomato plants in the field. On experimentally and naturally infected tomato plant materials, the expected amplicon was always obtained using crud extracts from leaves and fruit spots.