MinION Nanopore-Enabled Identification and Genomic Characterization of Pseudomonas syringae Complex Infecting Blueberry Using Symptomatic Plant Samples and Pathogen Pure Culture.

Pseudomonas syringae complex (Psc) causes bacterial blight and canker on blueberry and other fruit crops. Reliable and cost-effective pathogen detection is essential for managing crop diseases. The Oxford Nanopore MinION has emerged as a powerful tool in plant disease diagnostics and genetic characterizations of plant pathogens due to streamlined library preparation protocols, rapid sequencing turnaround, the ability to generate long reads, and enhanced taxonomic resolution. However, standardized and reproducible methods for whole-genome sequencing of the plant pathogenic Psc remains limited, especially for pathogens infecting blueberry tissues. Therefore, we describe several protocols including DNA extraction and quality control from both pure bacterial cultures and infected blueberry tissues (e.g., infected leaves and stem tissues), library preparation, and whole-genome sequencing using the MinION platform. The challenges of working with low bacterial pathogen biomass in plant tissue, contamination from host DNA, and sample variability are addressed through optimized workflows and troubleshooting strategies. Furthermore, we provide a modular bioinformatics software pipeline ranging from user-friendly EPI2ME tools to command-line analyses with Kraken2 and Krona for genome assembly and annotation and taxonomic identification, and comparative and evolutionary studies of Psc. These methods provide a reproducible framework for the genomic characterization and diagnostics of the Psc in the context of agricultural disease diagnostics, with the potential flexibility for adaptation to other bacterial taxa and sample types. © 2026 His Majesty the King in Right of Canada. Current Protocols published by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Agriculture and Agri-Food. Basic Protocol 1: Isolation of pseudomonad cultures from infected plant tissues Basic Protocol 2: DNA extraction from pure cultures of pseudomonads Basic Protocol 3: DNA isolation from infected host tissues Basic Protocol 4: Quality control, library preparation, and whole-genome sequencing Basic Protocol 5: Taxonomic identification using EPI2ME Desktop application Support Protocol: Taxonomic identification using Kraken2 and Krona in Linux Basic Protocol 6: Bioinformatics analyses for genome assembly and annotation.