High-Throughput Sequencing Methods for the Detection of Two Strawberry Viruses in Post-Entry Quarantine

Area of Science:

• Plant pathology
• Molecular biology
• Genomics

Background:

• Imported plant germplasm requires robust pathogen detection methods.
• High-throughput sequencing (HTS) offers potential for broad-spectrum pathogen identification.
• Existing PCR-based assays often lack strain-generic detection capabilities.

Purpose of the Study:

• To evaluate Metatranscriptomics (RNAseq) and tiled amplicon PCR (TA-PCR) as HTS techniques for detecting low-titre plant viruses.
• To compare the sensitivity and efficacy of RNAseq and TA-PCR against quantitative PCR (qPCR).
• To establish HTS protocols for flexible and comprehensive plant virus detection.

Main Methods:

• RNAseq was performed on ribosomal RNA-depleted samples from plants infected with Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV).
• Custom TA-PCR primer panels were designed for SMoV and SVBV detection.
• Sensitivity was assessed by comparing HTS results with qPCR assays at low viral titres.

Main Results:

• RNAseq achieved PCR-like sensitivity, detecting as few as 10 viral copies/µL with 60 million paired-end reads.
• TA-PCR successfully recovered significant portions of viral genomes, detecting viruses at approximately 10 viral copies/µL with a 500 bp minimum sequence recovery.
• The detection limits of both RNAseq and TA-PCR were comparable to those of qPCR.

Conclusions:

• HTS methods, specifically RNAseq and TA-PCR, are effective for detecting plant viruses at low concentrations.
• These HTS techniques provide strain-generic detection, enhancing capabilities beyond traditional PCR.
• The study provides a foundation for developing more flexible and sensitive HTS-based plant virus diagnostics.