Sclerotinia sclerotiorum is a global fungal pathogen affecting numerous plant species, leading to significant crop yield reductions. This pathogen, responsible for Sclerotinia Stem Rot, severely damages crops, rendering vegetables unmarketable and decreasing seed weight, quantity, and quality in grain and oilseed crops, causing substantial economic losses. Early detection is vital for effective disease management. This study aimed to detect S. sclerotiorum in oilseed rape leaves from naturally infected fields using quantitative polymerase chain reaction (qPCR) and nanopore sequencing. qPCR is valued for its speed, accuracy, reproducibility, and reliability in quantifying DNA during amplification, while nanopore sequencing provides comprehensive genomic analysis with rapid, accurate pathogen identification through long reads.
DNA was extracted from ten oilseed rape plants across five naturally infected fields. After optimizing the annealing temperature (Ta) for polymerase chain reaction (PCR), the internal transcribed spacer (ITS) region of fungal DNA was amplified on extracted DNA using specific primers. The amplicons were then analyzed with qPCR and Oxford Nanopore sequencing. The qPCR results did not detect S. sclerotiorum in any samples. However, Oxford Nanopore sequencing identified Sclerotinia species in three fields and specifically detected S. sclerotiorum in the remaining three fields (five fields and 1 spiked) along with other pathogens. These findings indicate that nanopore sequencing offers a more comprehensive detection capability compared to qPCR, which may have been hindered by contamination, inhibitors, or incorrect sample dilution, suggesting areas for improvement in the qPCR assay development process.