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Molecular differentiation of P antoea stewartii subsp. indologenes from subspecies stewartii and identification of new isolates from maize seeds
Author(s) -
Gehring I.,
Wensing A.,
Gernold M.,
Wiedemann W.,
Coplin D.L.,
Geider K.
Publication year - 2014
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12467
Subject(s) - subspecies , pantoea , biology , polymerase chain reaction , microbiology and biotechnology , genetics , 16s ribosomal rna , bacteria , gene , ecology
Aims Assays to detect P antoea 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 B iotyper software ( B ruker), subspecies stewartii and indologenes produced identical score values. Analysis against the B iotyper database produced similar score values for both subspecies. From the subtyping methods provided by the B iotyper software, only composite correlation indexing ( CCI ) separated both groups. By alignment of 16 S r RNA 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 ( SNP s) in the genes gal E , glm S and rec A . 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 gal E and rec A , 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 K ovacs 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.