Simultaneous detection of major blackleg and soft rot bacterial pathogens in potato by multiplex polymerase chain reaction.

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: , subsp. together with and spp. 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 of sp. genomic DNA, and down to 0.1 ng µL of and subsp. genomic DNA . In the presence of competitor genomic DNA, isolated from cells, the sensitivity of the multiplex PCR decreased tenfold for and sp., while no change was observed for subsp. and . In spiked potato haulm and tuber samples, the threshold level for target bacteria was 10 cfu mL plant extract (10 cfu g plant tissue), 10 cfu mL plant extract (10 cfu g plant tissue), 10 cfu mL plant extract (10 cfu g plant tissue), for spp., and subsp. /, 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.