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Rapid and quantitative detection of Pythium inflatum by real-time fluorescence loop-mediated isothermal amplification assay

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Abstract

Pythium inflatum is the causal agent of Pythium maize stalk rot, which is one of the most devastating diseases of maize (Zea mays L.). P. inflatum is currently a major concern in global maize production. To the best of our knowledge, no effective resistance to P. inflatum is known in maize, and no effective measures have been reported for the control of this pathogen once maize plants have been infected. Early and accurate detection of P. inflatum is essential to guide maize planting and to protect maize production. A real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid quantitative detection of P. inflatum in soil. The detection limit of the RealAmp assay was approximately 0.1 pg/μl plasmid DNA when mixed with extracted soil DNA or 103 spores/g of artificially infested soil. No cross-reactions with other related pathogens were observed. Results of the RealAmp assay for quantifying the genomic DNA of P. inflatum were confirmed by testing with artificially and naturally infested samples. The quantification of the soil-borne pathogen DNA of P. inflatum in naturally infested samples was not significantly different compared with classic real-time PCR (P < 0.05). Additionally, the RealAmp assay could be detected via an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification. Consequently, the inhibitory effects of the stain on DNA amplification were avoided. Therefore, the assay could be used more conveniently in the field as a simple, rapid, and effective technique and has the potential to become an alternative tool for detecting and monitoring P. inflatum in the field.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (#U1204320) and the HNAAS (Henan Academy of Agricultural Sciences) Foundation for Excellent Young Scholars (2013YQ03). We thank Professor Xiaoming Wang (Chinese Academy of Agricultural Sciences, Beijing, China) for providing the P. inflatum strain; Professor Jiguang Wei (Guangxi University, Nanning, China) for providing the P. graminicola, P. aphanidermatum, P. arrhenomanes, P. guangxiense, and P. periilum; and Dr. Junjie Hao (Henan Academy of Agricultural Sciences, Zhengzhou, China) for providing the F. graminearum.

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Authors and Affiliations

  1. Institute of Cereal Crops, Henan Academy of Agricultural Sciences, Zhengzhou, 450002, China

    Yanyong Cao, Jingjing Li, Lifeng Wang, Zeqiang Cheng, Hao Wang & Huiyong Li

  2. Plant Science and Technology College, Beijing University of Agriculture, Beijing, 102206, China

    Yongqiang Li

  3. State Key Laboratory for Agro-Biotechnology, Ministry of Agriculture Key Laboratory of Plant Pathology and Department of Plant Pathology, China Agricultural University, Beijing, 100094, China

    Zaifeng Fan

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  1. Yanyong Cao
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Fig. S1

Specific identification of Pythium inflatum among Pythium spp. via the real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay. a. Agarose gel electrophoresis analysis of RealAmp assay amplicons showing the specific test for P. inflatum. Lane 1 to lane 2, genomic DNAs of positive and negative controls, respectively; Lane 3 to lane 8, the DNAs of P. inflatum (HNAAS-19), P. inflatum (GuoLD001447), P. graminicola (CY-A156), P. acanthicum (CY-B424), P. arrhenomanes (CY-B386), and P. debaryanum, respectively. Lane M, D2000 DNA marker (Tiangen Biotech). The same samples were used for lane 1 to lane 8 of Fig. S1a to Fig. S1d. b. The RealAmp assay was validated by specific PCR amplification using the specific Pinf1/ITS2 primer set; Lane D, 50 bp DNA ladder (Tiangen Biotech). c. Visual inspection of the RealAmp amplification products. The original orange color of SYBR Green I turned green in the positive reaction mixture. d. The fluorescence vs. time graph was plotted automatically by the ESE-Quant Tube Scanner. The graph reports the fluorescence in mV on the y-axis and time in min on the x-axis. (GIF 35 kb)

High Resolution Image (TIFF 596 kb)

Fig. S2

The sensitivity of Pythium inflatum real-time fluorescence loop-mediated isothermal amplification (RealAmp) test. a. Agarose gel electrophoresis analysis of RealAmp amplication products correspond to serial diluted plasmid DNA template. Lane 2 to lane 7 are 10-fold dilutions of the pPiITS plasmid DNA ranging from 10 ng/μl to 1.0 × 10-5 ng/μl. Lane 8, negative control. b. Visual detection of the RealAmp amplification products. The original orange color of SYBR Green I turned green in the positive reaction mixture. (GIF 19 kb)

High Resolution Image (TIFF 320 kb)

Fig. S3

Screening of different sets of primers used for Pythium inflatum RealAmp reactions. The real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) products were amplified from P. inflatum DNA (P1 and P2) and the DNA of other pathogens (N1 and N2). The fluorescence vs. time graph was plotted automatically by the ESE-Quant Tube Scanner. The graph reports the fluorescence in mV on the y-axis and time in min on the x-axis. (GIF 47 kb)

High Resolution Image (TIFF 885 kb)

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Cao, Y., Li, Y., Li, J. et al. Rapid and quantitative detection of Pythium inflatum by real-time fluorescence loop-mediated isothermal amplification assay. Eur J Plant Pathol 144, 83–95 (2016). https://doi.org/10.1007/s10658-015-0752-2

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