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PMID: 21802235 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter.

Systematic and applied microbiology ·Vol. 34 ·No. 6 ·2011-09-00 ·Pages 400-7

Zaluga J, Heylen K, Van Hoorde K, Hoste B, Van Vaerenbergh J, Maes M, De Vos P

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.

MeSH Terms

Base Sequence DNA Gyrase/chemistry,genetics DNA, Bacterial/chemistry,genetics Gene Amplification Genes, Bacterial Genetic Variation Micrococcaceae/enzymology,genetics Molecular Sequence Data Plant Diseases/microbiology Sequence Analysis, DNA Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods

Chemicals

DNA, Bacterial DNA Gyrase

Authors & Affiliations

7 authors, click to expand affiliations / ORCID

Zaluga Joanna

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium. [email protected]

Heylen Kim

Van Hoorde Koenraad

Hoste Bart

Van Vaerenbergh Johan

Maes Martine

De Vos Paul

Article Info

Journal

Systematic and applied microbiology

Abbr.

Syst Appl Microbiol

ISSN

1618-0984

DOI

10.1016/j.syapm.2011.05.001

[email protected]

Published

2011-09-00

Epub

2011-00-28

Pages

400-7

Language

English

Region

Germany

NLM ID

8306133

Subset

Databases

GENBANK

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PubMed source DOI: 10.1016/j.syapm.2011.05.001 View journal details

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GyrB sequence analysis and MALDI-TOF MS as identification tools for plant pathogenic Clavibacter.

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