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The rapid and sensitive identification of invasive plant pathogens has important applications in biotechnology, plant quarantine, and food security. Current methods are far too time-consuming and need a pre-enrichment period ranging from hours to days. Here, a micro-Raman spectroscopy-based bioassay for culture-free pathogen quarantine inspection at the single cell level within 40 min is presented. The application of this approach can readily and specifically detect plant pathogens Burkholderia gladioli pv. alliicola and Erwinia chrysanthemi that are closely related pathogenically. Furthermore, the single-bacterium detection was able to discriminate them from a reference Raman spectral library including multiple quarantine-relevant pathogens with broad host ranges and an array of pathogenic variants. To show the usefulness of this assay, Burkholderia gladioli pv. alliicola and Erwinia chrysanthemi are detected at single-bacterium level in plant tissue lesions without pre-enrichment. The results are confirmed by the plate-counting method and a genetic molecular approach, which display comparable recognition ratios to the Raman spectroscopy-based bioassay. The results represent a critical step toward the use of micro-Raman spectroscopy in rapid and culture-free discrimination of quarantine relevant plant pathogens.