title: Simultaneous detection of major blackleg and soft rot bacterial pathogens in potato by multiplex polymerase chain reaction.
abstract: A multiplex polymerase chain reaction (PCR) assay for simultaneous, fast and reliable detection of the main soft rot and blackleg potato pathogens in Europe has been developed. It utilises three pairs of primers and enables detection of three groups of pectinolytic bacteria frequently found in potato, namely:Pectobacterium atrosepticum,Pectobacterium carotovorumsubsp.carotovorumtogether withPectobacterium wasabiaeandDickeyaspp. in a multiplex PCR assay. In studies with axenic cultures of bacteria, the multiplex assay was specific as it gave positive results only with strains of the target species and negative results with 18 non-target species of bacteria that can possibly coexist with pectinolytic bacteria in a potato ecosystem. The developed assay could detect as little as 0.01 ng µL-1ofDickeyasp. genomic DNA, and down to 0.1 ng µL-1ofP. atrosepticumandP. carotovorumsubsp.carotovorumgenomic DNAin vitro. In the presence of competitor genomic DNA, isolated fromPseudomonas fluorescenscells, the sensitivity of the multiplex PCR decreased tenfold forP. atrosepticumandDickeyasp., while no change was observed forP. carotovorumsubsp.carotovorumandP. wasabiae. In spiked potato haulm and tuber samples, the threshold level for target bacteria was 101cfu mL-1plant extract (102cfu g-1plant tissue), 102cfu mL-1plant extract (103cfu g-1plant tissue), 103cfu mL-1plant extract (104cfu g-1plant tissue), forDickeyaspp.,P. atrosepticumandP. carotovorumsubsp.carotovorum/P. wasabiae, respectively. Most of all, this assay allowed reliable detection and identification of soft rot and blackleg pathogens in naturally infected symptomatic and asymptomatic potato stem and progeny tuber samples collected from potato fields all over Poland.
authors.affiliation: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk Gdansk, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Potrykus
authors.affiliation: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk Gdansk, Poland.
authors.forename: W
authors.initials: W
authors.lastname: Sledz
authors.affiliation: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk Gdansk, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Golanowska
authors.affiliation: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk Gdansk, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Slawiak
authors.affiliation: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk Gdansk, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Binek
authors.affiliation: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk Gdansk, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Motyka
authors.affiliation: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk Gdansk, Poland.
authors.forename: S
authors.initials: S
authors.lastname: Zoledowska
authors.affiliation: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk Gdansk, Poland.
authors.forename: R
authors.initials: R
authors.lastname: Czajkowski
authors.affiliation: Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk Gdansk, Poland.
authors.forename: E
authors.initials: E
authors.lastname: Lojkowska
keywords: Dickeya; Pectobacterium; differentiation; identification; pectinolytic Erwinia; sampling; specific primers