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

Area of Science:

• Plant Pathology
• Bacteriology
• Microbial Diagnostics

Background:

• The bacterial genus Clavibacter comprises one species, Clavibacter michiganensis, with five subspecies, all of which are plant pathogens.
• Three Clavibacter subspecies are designated as quarantine pests by the European and Mediterranean Plant Protection Organization (EPPO), necessitating stringent identification protocols.
• Current identification methods, including immunological tests and PCR, are time-consuming, prone to cross-reactions, and can yield false negatives, hindering accurate subspecies differentiation.

Purpose of the Study:

• To evaluate matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and gyrB gene sequencing as rapid and reliable diagnostic tools for Clavibacter subspecies identification.
• To assess the ability of these methods to differentiate between the five Clavibacter subspecies and closely related bacterial species.

Main Methods:

• Amplification and sequencing of a fragment of the gyrB gene using a single primer set.
• Proteomic analysis of Clavibacter subspecies using MALDI-TOF MS to generate distinct spectral profiles.

Main Results:

• The gyrB sequencing provided sufficient resolution and specificity to identify each subspecies through cluster analysis and sequence signatures.
• MALDI-TOF MS generated unique and reproducible proteomic profiles for each of the five subspecies, with specific ion peaks serving as potential biomarkers.
• Both MALDI-TOF MS and gyrB sequencing successfully distinguished between the five Clavibacter subspecies and closely related genera such as Rathayibacter, Leifsonia, and Curtobacterium.

Conclusions:

• MALDI-TOF MS and gyrB sequencing are powerful and accurate diagnostic tools for identifying Clavibacter plant pathogens.
• These methods offer a significant improvement over existing techniques for the rapid and reliable differentiation of Clavibacter subspecies.
• The findings support the implementation of these advanced techniques for regulatory purposes and disease management strategies.