title: In situ identification of plant-invasive bacteria with MALDI-TOF mass spectrometry.
abstract: Rhizobia form a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes. The study of rhizobial populations in nature involves the collection of large numbers of nodules found on roots or stems of legumes, and the subsequent typing of nodule bacteria. To avoid the time-consuming steps of isolating and cultivating nodule bacteria prior to genotyping, a protocol of strain identification based on the comparison of MALDI-TOF MS spectra was established. In this procedure, plant nodules were considered as natural bioreactors that amplify clonal populations of nitrogen-fixing bacteroids. Following a simple isolation procedure, bacteroids were fingerprinted by analysing biomarker cellular proteins of 3 to 13 kDa using Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) mass spectrometry. In total, bacteroids of more than 1,200 nodules collected from roots of three legumes of the Phaseoleae tribe (cowpea, soybean or siratro) were examined. Plants were inoculated with pure cultures of a slow-growing Bradyrhizobium japonicum strain G49, or either of two closely related and fast-growing Sinorhizobium fredii strains NGR234 and USDA257, or with mixed inoculants. In the fully automatic mode, correct identification of bacteroids was obtained for >97% of the nodules, and reached 100% with a minimal manual input in processing of spectra. These results showed that MALDI-TOF MS is a powerful tool for the identification of intracellular bacteria taken directly from plant tissues.
authors.affiliation: Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland.
authors.forename: Dominik
authors.initials: D
authors.lastname: Ziegler
authors.forename: Anna
authors.initials: A
authors.lastname: Mariotti
authors.forename: Valentin
authors.initials: V
authors.lastname: Pflüger
authors.forename: Maged
authors.initials: M
authors.lastname: Saad
authors.forename: Guido
authors.initials: G
authors.lastname: Vogel
authors.forename: Mauro
authors.initials: M
authors.lastname: Tonolla
authors.forename: Xavier
authors.initials: X
authors.lastname: Perret
mesh_terms: D001419:Bacteria / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods; D007887:Fabaceae / Q000382:microbiology; D027343:Rhizome / Q000382:microbiology; D053204:Root Nodules, Plant / Q000382:microbiology*; D042181:Sinorhizobium fredii / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*