AimsAssays to detect Pantoea stewartii from maize seeds should include differentiation of P.stewartii subsp. stewartii and P.stewartii subsp. indologenes. Methods and ResultsPreviously published PCR primers for the identification of P.stewartii subsp. stewartii amplified signals from both subspecies using both conventional and quantitative PCR. In MALDI-TOF mass spectroscopy analysis with the Biotyper software (Bruker), subspecies stewartii and indologenes produced identical score values. Analysis against the Biotyper database produced similar score values for both subspecies. From the subtyping methods provided by the Biotyper software, only composite correlation indexing (CCI) separated both groups. By alignment of 16S rRNA sequences, no subspecies distinction was possible. To develop new techniques for the separation of these subspecies, the partial sequences of several housekeeping genes were compared. The type strains of the two subspecies showed characteristic single-nucleotide polymorphisms (SNPs) in the genes galE, glmS and recA. Other reference strains of P.stewartii subsp. stewartii followed the same nucleotide pattern, whereas known P.stewartii subsp. indologenes strains were different. Based on single-nucleotide polymorphisms in galE and recA, PCR primers were created to separate the subspecies by stepdown PCR analysis. Two putative P.stewartii strains were isolated from imported maize seeds. They were not virulent on maize seedlings, were positive in the indole assay with Kovacs reagent and identified as P.stewartii subsp. indologenes. The subspecies-specific PCR primers confirmed they were subspecies indologenes. ConclusionsBy stepdown PCR, P.stewartii subsp. indologenes can be differentiated from P.stewartii subsp. stewartii. Significance and Impact of the StudyA possible detection of P.stewartii subsp. stewartii, the causative agent of Stewart's wilt of maize, in plant material by immunological or molecular assays must exclude contamination with P.stewartii subsp. indologenes, which would create false positives in seed tests and affect quarantine measurements.