Microneedle-Assisted CRISPR Biosensing Platform for Presymptomatic Detection of Plant Diseases

Presymptomatic diagnosis of tomato bacterial speck caused by Pseudomonas syringae requires rapid sample preparation and specific molecular readout. Although existing methods offer high reliability, their requirement for destructive sampling and centralized laboratory infrastructure severely limits their suitability for monitoring of plant diseases. We address this gap with a microneedle-assisted CRISPR platform that integrates on-leaf sample extraction with pathogen-specific detection. A hydrophilic and swelling polyvinyl alcohol microneedle patch mechanically bypasses the cuticle to extract pathogen-associated nucleic acids from the plant apoplast within minutes, and a CRISPR-based fluorescence assay reports the presence of plant pathogens with high detection specificity. The platform can detect unamplified pure target DNA at 0.16 nM, and as few as 103 copies following PCR preamplification. For bacterial suspensions, this platform can achieve a limit of detection at a concentration of 104 CFU/mL without amplification and 103 CFU/mL with PCR within 2 h. In inoculated plants, the platform reliably achieved presymptomatic detection of P. syringaeon day 3 postinoculation, preceding macroscopic necrotic specks by 24 h. These results establish a rapid and minimally invasive sampling-to-molecular biosensing pipeline that significantly accelerates conventional diagnostic turnaround and provides a versatile foundation for presymptomatic plant disease surveillance.