Development of Multiplex PCR to Detect Five Pythium Species Related to Turfgrass Diseases

Journal of Phytopathology (Impact Factor: 0.82). 08/2010; 158(9):609 - 615. DOI: 10.1111/j.1439-0434.2009.01660.x


The objective of this study was to develop multiplex PCR detection method for five Pythium species associated with turfgrass diseases, Pythium aphanidermatum, Pythium arrhenomanes, Pythium graminicola, Pythium torulosum and Pythium vanterpoolii. Species-specific primers and two common primers were designed based on the sequences of the internal transcribed spacer region of ribosomal DNA. Another primer set by which all organisms would be amplified in 18S rDNA was used as a positive control. When these total nine primers were applied to the multiplex PCR, all species were individually discriminated in the mixture of five species culture DNA. Furthermore, all five Pythium species were detected in naturally infected plants using the multiplex PCR.

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Available from: Koji Kageyama, Jun 17, 2014
    • "In addition, some Pythium species are morphologically similar (Asano et al. 2010). In particular, P. inflatum and closely related species such as P. graminicola, are remarkably similar in appearance and ecology, thereby causing confusion when these Pythium species are identified using traditional methods (Chen and Hoy 1993; Kageyama et al. 2005; Asano et al. 2010). "
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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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    • "In Pythium and Phytophthora research, these techniques have been applied for the qualitative and quantitative detection of pathogens and for the analysis of population structures. We developed 10 species-specific primer sets for conventional PCR detection and five sets for quantitative real-time PCR detection of Pythium and three for conventional PCR and two for real-time PCR detection of Phytophthora (Asano et al. 2010; Ishiguro et al. 2013, 2014b; Kageyama et al. 1997; Li et al. 2010, 2011, 2013, 2014). PCR detection was used in a survey of serious pathogens such as Ph. "

    Full-text · Article · Sep 2015 · Journal of General Plant Pathology
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    • "Recently, molecular methods based on the analysis of DNA sequences, such as ITS regions of the nuclear ribosomal RNA gene, have been widely applied to the identification and detection of plant pathogenic fungi. A species-specific PCR primers designed within the ITS1 and ITS2 regions have been developed to detect plant pathogenic fungi (Asano et al., 2010; Chen et al., 2006; Grote et al., 2002; Ma and Michailides, 2002; Ni et al., 2012). Further, PCR-based random amplified polymorphic DNA (RAPD) is a frequently applied molecular approach for the identification of inter-and intra-species specific molecular markers (Williams et al., 1990). "
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    ABSTRACT: The fungus Venturia nashicola is the causal agent of scab on Asian pears. For the rapid and reliable identification as well as sensitive detection of V. nashicola, a PCR-based technique was developed. DNA fingerprints of three closely related species, V. nashicola, V. pirina, and V. inaequalis, were obtained by random amplified polymorphic DNA (RAPD) analysis. Two RAPD markers specific to V. nashicola were identified by PCR, after which two pairs of sequence characterized amplified region (SCAR) primers were designed from the nucleotide sequences of the markers. The SCAR primer pairs, designated as D12F/D12R and E11F/E11R, amplified 535-bp and 525-bp DNA fragments, respectively, only from genomic DNA of V. nashicola. The specificity of the primer sets was tested on strains representing three species of Venturia and 20 fungal plant pathogens. The nested PCR primer pair specific to V. nashicola was developed based on the sequence of the species-specific 525-bp DNA fragment amplified by primer set E11F/E11R. The internal primer pair Na11F/Na11R amplified a 235-bp fragment from V. nashicola, but not from any other fungal species tested. The nested PCR assay was sensitive enough to detect the specific fragment in 50 fg of V. nashicola DNA.
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