The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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A Duplex PCR Assay for Rapid Detection of Phytophthora nicotianae and Thielaviopsis basicola

  • Liu, Na (College of Life Sciences, Henan Agricultural University) ;
  • Jiang, Shijun (College of Plant Protection, Henan Agricultural University) ;
  • Feng, Songli (College of Life Sciences, Henan Agricultural University) ;
  • Shang, Wenyan (College of Life Sciences, Henan Agricultural University) ;
  • Xing, Guozhen (College of Life Sciences, Henan Agricultural University) ;
  • Qiu, Rui (Key Laboratory for green preservation & control of tobacco diseases and pests in Huanghuai Area, Tobacco Research Institute of Henan Academy of Agricultural Science) ;
  • Li, Chengjun (Key Laboratory for green preservation & control of tobacco diseases and pests in Huanghuai Area, Tobacco Research Institute of Henan Academy of Agricultural Science) ;
  • Li, Shujun (Key Laboratory for green preservation & control of tobacco diseases and pests in Huanghuai Area, Tobacco Research Institute of Henan Academy of Agricultural Science) ;
  • Zheng, Wenming (College of Life Sciences, Henan Agricultural University)
  • Received : 2018.09.04
  • Accepted : 2018.12.28
  • Published : 2019.04.01
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Abstract

A duplex PCR method was developed for simultaneous detection and identification of tobacco root rot pathogens Phytophthora nicotianae and Thielaviopsis basicola. The specific primers for P. nicotianae were developed based on its internal transcribed spacer (ITS) regions of ribosomal gene, ras gene and hgd gene, while the specific primers for T. basicola were designed based on its ITS regions and ${\beta}$-tubulin gene. The specificity of the primers was determined using isolates of P. nicotianae, T. basicola and control samples. The results showed that the target pathogens could be detected from diseased tobacco plants by a combination of the specific primers. The sensitivity limitation was $100fg/{\mu}l$ of pure genomic DNA of the pathogens. This new assay can be applied to screen out target pathogens rapidly and reliably in one PCR and will be an important tool for the identification and precise early prediction of these two destructive diseases of tobacco.

Keywords

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Fig. 1. Duplex PCR results for Phytophthora nicotianae and Thielaviopsis basicola. (A, B) Duplex PCR for primer combination TBITS1419-F/TBITS1419-R of T. basicola with YYI-F/ YYI-R and YYH-F/YYH-R of P. nicotianae. 1: DNA mixture of T. basicola and P. nicotianae; 2: DNA mixture of P. nicotianae and T. basicola; 3: DNA of T. basicola; 4: DNA of P. nicotianae; M: DM2000 DNA Marker; CK: control (distilled water). (C, D) Duplex PCR for primer TBβ1419-F/TBβ1419-R of T. basicola combined with YYI-F/YYI-R and YYH-F/YYH-R of P. nicotianae. 1: DNA mixture of T. basicola and P. nicotianae; 2: DNA mixture of P. nicotianae and T. basicola; 3: DNA of P. nicotianae; 4: DNA of T. basicola; M: DM2000 DNA Marker; CK: control (distilled water).

Table 1. Primers for the detection of Phytophthora nicotianae and Thielaviopsis basicola

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