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TaqMan real-time PCR assay for the detection and quantification of Sclerospora graminicola, the causal agent of pearl millet downy mildew

European Journal of Plant Pathology volume 142, pages 149–158 (2015)Cite this article

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Abstract

Early detection and quantification of Sclerospora graminicola, causal agent of pearl millet downy mildew, from seed, plant and infested soil could help in initiating preventive measures to control the spread of the disease. Two sensitive assays, SgK PCR (conventional PCR) and SgTqK q-PCR (real-time PCR), were developed for the detection of S. graminicola. Both assays were targeted on the 28S region of the nuclear large subunit (nuLSU) of the rDNA cluster. In conventional PCR, the species-specific primers (SgK F/R) amplified a 436 bp product in S. graminicola. TaqMan real-time PCR specifically amplified an 86 bp fragment in diverse DNA samples of S. graminicola obtained from infected seed, root, leaves and infested soil. Both assays did not amplify or produce any fluorescent signal from DNA obtained from other test microbes or from healthy pearl millet leaves. The absolute quantity of target molecules (28S) in the DNA sample obtained from S. graminicola sporangia (6 × 108/ml) was estimated at 25 pg/μl and the copy number was calculated as 2.69 × 108 molecules/μl. The copy number of target molecules in a diploid nucleus (2n) of S. graminicola was estimated to be ~27 molecules/nucleus. This assay can be used as a rapid and efficient detection tool for the identification and diagnosis of S. graminicola, and will be helpful in ensuring safe exchange of S. graminicola-free pearl millet germplasm across borders.

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Acknowledgments

We thank Dr Harikishan Sudini, Groundnut Pathology, ICRISAT; Dr S Gopalakrishnan, Biocontrol Lab, ICRISAT and Dr R D Prasad, Directorate of Oilseeds Research, India for providing microbial cultures. We are also thankful to Dr Rajeev Varshney and Dr Mahender Thudi, Applied Genomic Lab, ICRISAT for providing constructive suggestions during this study.

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  1. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, 502324, Hyderabad, Telangana, India

    Bandamaravuri Kishore Babu & Rajan Sharma

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  1. Bandamaravuri Kishore Babu
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  2. Rajan Sharma
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Correspondence to Rajan Sharma.

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Babu, B.K., Sharma, R. TaqMan real-time PCR assay for the detection and quantification of Sclerospora graminicola, the causal agent of pearl millet downy mildew. Eur J Plant Pathol 142, 149–158 (2015). https://doi.org/10.1007/s10658-015-0599-6

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  • DOI: https://doi.org/10.1007/s10658-015-0599-6

Keywords

  • q-PCR
  • Species-specific primers
  • Downy mildew
  • Obligate pathogen
  • Sclerospora graminicola

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