Molecular differentiation of Pantoea stewartii subsp. indologenes from subspecies stewartii and identification of new isolates from maize seeds.
AIMS
Assays to detect Pantoea stewartii from maize seeds should include differentiation of P. stewartii subsp. stewartii and P. stewartii subsp. indologenes.


METHODS AND RESULTS
Previously 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.


CONCLUSIONS
By stepdown PCR, P. stewartii subsp. indologenes can be differentiated from P. stewartii subsp. stewartii.


SIGNIFICANCE AND IMPACT OF THE STUDY
A 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.
['Gehring|I|I|', 'Wensing|A|A|', 'Gernold|M|M|', 'Wiedemann|W|W|', 'Coplin|D L|DL|', 'Geider|K|K|']
D015373:Bacterial Typing Techniques D001483:Base Sequence D017931:DNA Primers D005798:Genes, Bacterial D008969:Molecular Sequence Data D020636:Pantoea D010935:Plant Diseases D016133:Polymerase Chain Reaction D020641:Polymorphism, Single Nucleotide D012336:RNA, Ribosomal, 16S D012639:Seeds D016415:Sequence Alignment D013045:Species Specificity D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization D003313:Zea mays
stepdown PCR Stewart's wilt MALDI-TOF MS maize disease