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Multiplex TaqMan qPCR Assay for Detection, Identification, and Quantification of Three Sclerotinia Species
Dong Jae Lee1, Jin A Lee1, Dae-Han Chae2
1Department of Biological Science, Kunsan National University, Gunsan, Korea.
Abstract:
White mold (or Sclerotinia stem rot), caused by Sclerotinia species, is a major air, soil, or seed-transmitted disease affecting numerous crops and wild plants. Microscopic or culture-based methods currently available for their detection and identification are time-consuming, laborious, and often erroneous. Therefore, we developed a multiplex quantitative PCR (qPCR) assay for the discrimination, detection, and quantification of DNA collected from each of the three economically relevant Sclerotinia species, namely, S. sclerotiorum, S. minor, and S. nivalis. TaqMan primer/probe combinations specific for each Sclerotinia species were designed based on the gene sequences encoding aspartyl protease. High specificity and sensitivity of each probe were confirmed for sclerotium and soil samples, as well as pure cultures, using simplex and multiplex qPCRs. This multiplex assay could be helpful in detecting and quantifying specific species of Sclerotinia, and therefore, may be valuable for disease diagnosis, forecasting, and management.
Insights
A new multiplex quantitative PCR (qPCR) assay accurately detects and quantifies three key Sclerotinia species. This rapid molecular tool aids in diagnosing and managing white mold disease in crops.
Area of Science:
- Plant Pathology
- Molecular Biology
- Agricultural Science
Background:
- White mold, caused by Sclerotinia species, is a significant plant disease transmitted through air, soil, or seeds.
- Current detection methods (microscopy, culture-based) are slow, labor-intensive, and prone to errors.
- Accurate identification of Sclerotinia species is crucial for effective disease management.
Purpose of the Study:
- To develop a multiplex quantitative PCR (qPCR) assay for discriminating, detecting, and quantifying three economically important Sclerotinia species.
- To enable rapid and accurate identification of Sclerotinia sclerotiorum, Sclerotinia minor, and Sclerotinia nivalis.
Main Methods:
- Designed TaqMan primer/probe combinations targeting aspartyl protease gene sequences for species-specific detection.
- Validated the assay using simplex and multiplex qPCR on sclerotium, soil, and pure culture samples.
- Assessed specificity and sensitivity of the developed molecular assay.
Main Results:
- The multiplex qPCR assay demonstrated high specificity and sensitivity for all three target Sclerotinia species.
- Successful discrimination and quantification of S. sclerotiorum, S. minor, and S. nivalis were achieved.
- The assay performed effectively on various sample types, including soil and sclerotia.
Conclusions:
- The developed multiplex qPCR assay provides a reliable and efficient tool for identifying and quantifying Sclerotinia species.
- This molecular diagnostic method can significantly improve disease diagnosis, forecasting, and management strategies for white mold.
- The assay holds potential value for researchers and agricultural professionals involved in plant disease management.
