APS Homepage

Poster: Diseases of Plants: Disease Detection & Diagnosis


Real-time PCR detection and discrimination of the parsley pathogens Pseudomonas syringae pv. apii and Pseudomonas syringae pv. coriandricola
J. REINTKE (1), G. Hiddink (2), D. Sanchez Mendez (2) (1) Enza Zaden BV, Netherlands; (2) Enza Zaden BV., Netherlands

Bacterial leaf spot in parsley is caused by two different Pseudomonas syringae pathovars, Pseudomonas syringae pv. apii and Pseudomonas syringae pv coriandricola (Koike, Bull , 2011 Bull et al, 2011). Both were identified as causal agents in outbreaks of bacterial leaf spot in parsley. Seed was claimed to be the initial source of infection. Current seed test methods include a pathogenicity tests but lack confirmation methods. Therefor a realtime PCR assay was developed to detect and discriminate the two pathovars causing leaf spot disease in parsley. The primers and probes were developed on sequence variation in the gap1-gene of 35 closely related Pseudomonas syringae pathovars. Both P.s. apii and P.s.coriandricola were amplified with the forward primer 5’-ACCCAGTCGATGATCCCGA-3’, reverse primer 5’-AGCGAAACGTTGATCACCGG-3’, while probes differentiated P.s. apii (5’-6FAM-CAGGCAAACTGACCGGCATGGCCGTT-BHQ1-3’) from P.s. coriandricola (5’- HEX-CGGGCAAGCTCACGGGGATGGCTGTT-BHQ1-3’). Primers and probe showed sufficient discriminative power at the genomospecies level when tested on 52 closely or non-related strains. Primers were used in an improved seed health tests for the detection of P.s. apii and P.s. coriandricola on parsley seeds. The seed health test was validated and test results were confirmed for biological relevance using grow-outs of all used seed lots.