Rapid identification of plant-health related bacteria by MALDI-TOF mass spectrometry (MALD-ID)

The Matrix Assisted Laser Desorption Ionization - Time of Flight (MALDI-TOF) mass spectrometry is an analytical method that can be used as a diagnostic tool to identify microorganisms. The method is rapid (less than 1h from sample preparation to results) and accurate and it is widely used in other fields, such as human health and animal health. Mass spectrometry allows to obtain a unique molecular (mainly proteins) fingerprint of the sample analyzed, the mass spectrum, that can be used to characterize and eventually identify the sample through comparison with reference spectra of known samples. The quality and completeness of the reference databases is crucial for an accurate and reliable identification. The commercial reference databases available to support MALDI-TOF mass spectrometry identification are not specialized for plant-pathogenic bacteria. This precludes the use of MALDI-TOF mass spectrometry for the identification of plant-pathogenic bacteria. In this framework, the aim of the Euphresco project 2018-A-271 ‘Rapid identification of plant-health related bacteria by MALDI-TOF mass spectrometry (MALD-ID)’ was i) to build a reference database that can support the identification of selected plant-pathogenic bacterial genera and ii) to evaluate the possibility to use the MALDI-TOF mass spectrometry method as a diagnostic tool. The project gathered three partners from JKI (Germany), NIVIP (The Netherlands), and INRAE (France). The project allowed to produce 117 reference spectra for the identification of bacterial strains from eight genera: Clavibacter, Curtobacterium, Erwinia, Pantoea, Pectobacterium, Pseudomonas, Ralstonia, and Xylophilus. These strains were chosen to represent the known diversity of each genus or to better distinguish between regulated and not regulated in a same genus. These reference spectra, compatible with the Bruker System, are freely available from a public repository on the following url: https://doi.org/10.57745/7OJNOO.The project also allowed to validate the use of MALDI-TOF mass spectrometry for the analysis of more strains from these genera and to determine the accuracy and reliability of the tests. The project showed that MALDI-TOF mass spectrometry allows to correctly identify plant-pathogenic bacterial strains at the genus level in all cases. For Erwinia, Pantoea, Ralstonia, and Xylophilus MALDI-TOF mass spectrometry is even able to correctly identify strains at the species level. However, the method is unable to discriminate accurately at the intra-specific level in all cases. In plant-pathology, strain identification should often be performed at intra-specific level. Consequently, MALDI-TOF mass spectrometry alone is not sufficient for a complete diagnosis. However, MALDI-TOF mass spectrometry can reliably identify the genus of the isolates and can be used as a rapid first screening that can help to further choose the most relevant test for a precise identification.Report of the Euphresco project 2018-A-271 'Rapid identification of plant-health related bacteria by MALDI-TOF mass spectrometry (MALD-ID)