Multiplex TaqMan qPCR Assay for Detection, Identification, and Quantification of Three Sclerotinia Species

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.

Keywords: TaqMan probe; asp gene; phylogeny; sclerotia; white mold.

Figure 1.

Design of TaqMan assay primers and probes for detection of Sclerotinia species. The sequences shown in the alignment are fragments of the DNA sequences encoding the asp gene. In the reference sequence, the forward primer located at 1,675,730 bp and reverse primer located 1,675,897 bp were indicated in blight green. PSm3 = S. minor-specific TaqMan^® probe at 1,675,843–1,675,862 bp in green; PSs3 = S. sclerotiorum-specific TaqMan^® probe at the 1,675,845 (with 2 gaps)–1,675,867 bp in red; PSn2 = S. nivalis-specific TaqMan^® probe at the 1,675,845 (with 1 gap)–1,675,867 bp in yellow; Sasp1-1F = nonspecific forward primer (marked with a mint bar); Sasp1-1R = nonspecific reverse primer (marked with a mint bar). SNPs were highlighted in red within the probe sequence.

Figure 2.

Concomitant amplification (A) and standard curves (B) of three DNA targets by multiplex qPCR: Sclerotinia sclerotiorum (red), S. minor (blue), and S. nivalis (green). The log rDNA copy number is plotted against the quantification cycle (Cq) values. All data points are from an average of four technical replicates.

Figure 3.

Multiplex qPCR assays of three target species for soil (A-H) and sclerotia (I-L) samples. Autoclaved soil was inoculated with a mycelium plug of Sclerotinia minor (A), S. sclerotiorum (B), S. nivalis (C), and three Sclerotinia species (D) or a sclerotium of S. minor (E), S. sclerotiorum (F), S. nivalis (G), and three Sclerotinia species (H). Each sclerotium was harvested the PDA media of S. minor (I), S. sclerotiorum (J), S. nivalis (K), and three Sclerotinia species (L). Colored lines mean the qPCR amplification curves specific for S. sclerotiorum (red), S. minor (blue), and S. nivalis (green).