Identification of Pantoea stewartii subsp. stewartii by PCR and Strain Differentiation by PFGE.

Abstract

Stewart's bacterial wilt and leaf blight of sweet corn and maize is caused by Pantoea stewartii subsp. stewartii. This bacterium can be seed transmitted at a low frequency, so it is subject to quarantine restrictions by many countries. To develop a polymerase chain reaction assay for the identification of this pathogen from field samples and for use in seed health tests, four primer pairs were tested. These were selected from the sequences of hrpS, cpsDE, and the 16S rRNA intergenic transcribed spacer (ITS) region. Under optimal reaction conditions, about 20 and 200 cells of P. stewartii could be detected in pure cultures and leaf lesions, respectively. Other plant-associated enteric bacteria (e.g., P. agglomerans pv. herbicola, P. ananas, Erwinia amylovora, and E. carotovora) either did not produce amplicons or they were not the correct size for P. stewartii. To test further for possible false positives, 29 yellow-pigmented bacteria, mainly other Pantoea spp., were isolated from lesions on old corn leaves and assayed with the ITS primer sets. Except for weak, variable reactions with three P. ananas strains, the bacteria did not test positive. Pulsed field gel electrophoresis (PFGE) was evaluated as an additional test to confirm the identity of P. stewartii. After digestion with SpeI and XbaI, P. stewartii strains could be easily distinguished from related Erwinia and Pantoea spp. and each other.