Molecular differentiation of Pantoea stewartii subsp. indologenes from subspecies stewartii and identification of new isolates from maize seeds

Abstract

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.

Validation of a specific PCR screening test for Pantoea stewartii subsp. stewartii in maize (Zea mays) samples

“…indologenes-specific 3614gal primers of Gehring et al (2014) both routine maize seed samples mentioned above were confirmed to be infested with P. stewartii subsp. indologenes-specific 3614gal primers of Gehring et al (2014) both routine maize seed samples mentioned above were confirmed to be infested with P. stewartii subsp.…”

“…indologenes which can be found on maize is of particular importance, as the latter subspecies is neither virulent on maize nor a regulated quarantine organism in any country (Gehring et al, 2014). stewartii in maize samples within the framework of the implementation of quarantine regulations it is essential for diagnostic laboratories to have a specific and sensitive first screening test available that reliably selects for potential positives, which will then have to undergo further tests according to an official testing scheme.…”

“…stewartii the primer pair DC283galE/ DC283galEc of Gehring et al (2014) was eventually selected, designed to amplify a 268-bp fragment of the UDP-glucose 4-epimerase gene galE. stewartii the primer pair DC283galE/ DC283galEc of Gehring et al (2014) was eventually selected, designed to amplify a 268-bp fragment of the UDP-glucose 4-epimerase gene galE.…”

“…indologenes-specific primers 3614-galE/3614galEc of Gehring et al (2014) at 0.2 lM were used as an additional confirmation that particular samples from routine testing were infested with P. stewartii subsp. indologenes Pantoea stewartii subsp.…”

“…stewartii PCR test with primer pair DC283galE/DC283-galEc (Gehring et al, 2014) were analysed according to PM 7/98 (EPPO, 2014). stewartii PCR test with primer pair DC283galE/DC283-galEc (Gehring et al, 2014) were analysed according to PM 7/98 (EPPO, 2014).…”