Specific identification and detection of Pantoea stewartii subsp. stewartii using a membrane-based multi-gene oligonucleotide array

The Stewarts wilt pathogen (Pantoea stewartii subsp. stewartii; Pss) of sweet corn is classified as a quarantine bacterium, requiring certification of grain shipments made to over 60 countries. This study reports the development and validation of the first multi-gene array for accurate and specific differentiation of Pss from Pantoea stewartii subsp. indologenes (Psi) and other Pantoea species using DNA, or RNA after generation of cDNA. The technique consisted of multiplex (16S rRNA, leuS, gyrB, rpoB, cpsD) PCR amplifications using universal and specific primers in a single reaction, followed by the hybridization of the digoxigenin-labelled amplicons to 22 specific oligonucleotide probes (19- to 24-mers) immobilized on a nylon membrane. The sensitivity of the array (10 fg of DNA) compared favourably with TaqMan real-time PCR. The specificity and reliability of the array was tested on 65 bacterial strains consisting of Pantoea species, closely and distantly related bacterial genera. Specific detection of Pss in infected corn leaves and seed homogenates was also validated using growth chamber-inoculated plants. The reported multi-gene DNA array could be a reliable tool for routine and specific detection of Pss.