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GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter
Authors:Zaluga Joanna  Heylen Kim  Van Hoorde Koenraad  Hoste Bart  Van Vaerenbergh Johan  Maes Martine  De Vos Paul
Institution:aLaboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium;bPlant-Crop Protection, Institute for Agricultural and Fisheries Research – ILVO, Burg. Van Gansberghelaan 96, B-9820 Merelbeke, Belgium;cFaculty of Applied Engineering Sciences, University College Ghent, Schoonmeersstraat 52, B-9000 Ghent, Belgium
Abstract:The bacterial genus Clavibacter has only one species, Clavibacter michiganensis, containing five subspecies. All five are plant pathogens, among which three are recognized as quarantine pests (mentioned on the EPPO A2 list). Prevention of their introduction and epidemic outbreaks requires a reliable and accurate identification. Currently, identification of these bacteria is time consuming and often problematic, mainly because of cross-reactions with other plant-associated bacteria in immunological tests and false-negative results in PCR detection methods. Furthermore, distinguishing closely related subspecies is not straightforward. This study aimed at evaluating the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fragment of the gyrB sequence for the reliable and fast identification of the Clavibacter subspecies. Amplification and sequencing of gyrB using a single primer set had sufficient resolution and specificity to identify each subspecies based on both sequence similarities in cluster analyses and specific signatures within the sequences. All five subspecies also generated distinct and reproducible MALDI-TOF MS profiles, with unique and specific ion peaks for each subspecies, which could be used as biomarkers for identification. Results from both methods were in agreement and were able to distinguish the five Clavibacter subspecies from each other and from representatives of closely related Rathayibacter, Leifsonia or Curtobacterium species. Our study suggests that proteomic analysis using MALDI-TOF MS and gyrB sequence are powerful diagnostic tools for the accurate identification of Clavibacter plant pathogens.
Keywords:Clavibacter  Bacterial identification  Mass spectrometry  gyrB  MALDI-TOF MS
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