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
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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. "

    Journal of General Plant Pathology 09/2015; 81(3). DOI:10.1007/s10327-015-0617-8 · 0.97 Impact Factor
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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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    • "AsPyF/APH2B was described by Asano et al. (2010) d Specific primer kkMYRR was designed based on the internal transcribed spacer region for P. myriotylum e Specific primers kkhel F1mod2/kkhel R2 were designed based on the internal transcribed spacer region for P. helicoides f Lèvesque and de Cock (2004) g Blair et al. (2008) other culturable isolates listed in Table 2 by the method of Meherun et al. (2011). The microorganisms were incubated for 7–10 days in V8 agar at 25 °C. "
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    ABSTRACT: The objective of this study was to develop a multiplex PCR detection method for the high-temperature-growing pathogens Pythium aphanidermatum, P. helicoides and P. myriotylum. Species-specific primer pairs were designed that targeted the rDNA ITS regions. The multiplex PCR was constructed with a universal primer pair for eukaryotes directed at the 18S rDNA as a positive control, in addition to the three species-specific primer pairs. When the multiplex PCR was applied to naturally infested soils, the expected species were reliably identified, suggesting that the method is suitable for the detection of the three Pythium pathogens in environmental samples.
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