MinION Nanopore-Enabled Identification and Genomic Characterization of Pseudomonas syringae Complex Infecting...

Area of Science:

• Plant Pathology
• Genomics
• Microbial Diagnostics

Background:

• The Pseudomonas syringae complex (Psc) causes significant damage to blueberry and other fruit crops.
• Accurate and affordable detection of Psc is crucial for effective disease management.
• Existing whole-genome sequencing methods for Psc, particularly from blueberry tissues, lack standardization and reproducibility.

Purpose of the Study:

• To establish standardized and reproducible protocols for whole-genome sequencing of Psc from both pure cultures and infected blueberry tissues.
• To address challenges associated with low pathogen biomass, host DNA contamination, and sample variability.
• To provide a comprehensive bioinformatics pipeline for Psc genome assembly, annotation, and taxonomic identification.

Main Methods:

• DNA extraction and quality control from pure bacterial cultures and infected blueberry tissues (leaves, stems).
• Library preparation and whole-genome sequencing utilizing the Oxford Nanopore MinION platform.
• Development of a modular bioinformatics pipeline including EPI2ME, Kraken2, and Krona for data analysis.

Main Results:

• Optimized workflows and troubleshooting strategies were developed to overcome challenges in sequencing Psc from plant tissues.
• A reproducible framework for genomic characterization and diagnostics of Psc was established.
• The protocols demonstrated flexibility for adaptation to other bacterial taxa and sample types.

Conclusions:

• The described protocols offer a reliable and cost-effective approach for Psc whole-genome sequencing and diagnostics.
• These methods enhance agricultural disease management by providing detailed genomic insights into plant pathogens.
• The study contributes to advancing plant disease diagnostics and pathogen characterization using long-read sequencing technologies.