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Quantification of downy mildew (Peronospora sparsa) in Rubus species using real-time PCR

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Abstract

Downy mildew disease caused by Peronospora sparsa, also known as ‘dryberry’ disease, is a serious threat to the cultivation of arctic bramble (Rubus arcticus) and boysenberry (Rubus spp. hybrid). A quantitative and sensitive screening method is necessary for the breeding of downy mildew resistant cultivars and for determining efficient disease control methods. A quantitative real-time PCR method using SYBR® Green I fluorescent dye was developed for the analysis of P. sparsa in arctic bramble, other Rubus species and roses. Primers were designed to amplify a P. sparsa specific 94-bp fragment from the internal transcribed spacer region (ITS1) and a 140-bp fragment from a conserved region of plant 5.8S ribosomal DNA, which served as an internal control in the samples. Linear amplification from genomic DNA and control plasmids was achieved with both primers, and even 37 fg of P. sparsa conidial DNA was detected. In the samples collected from the field, quantities as low as 0.2 ppm of P. sparsa DNA in plant DNA were detected, thus enabling the diagnosis of weak and latent infections. Arctic bramble cvs Pima, Mespi and Mesma, all showing distinct foliar symptoms, were tested to assess the relative amount of downy mildew DNA present. The symptoms and the amount of P. sparsa DNA detected correlated only in cv. Pima, indicating that visual inspection of symptoms is not a reliable method for assessing the extent of tissue infection. The number of conidiophores of P. sparsa on in vitro inoculated leaves of two arctic bramble cultivars correlated with the results obtained by real-time PCR.

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Abbreviations

bp:

base pair

Ct:

threshold cycle

ITS:

internal transcribed spacer

rDNA:

ribosomal DNA

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Acknowledgements

This research was funded by the Northern Periphery Programme (INTERREG IIIB, Grant no. 102-12874-02), Tiura Foundation, and Kuopio Naturalists’ Society. We also thank Dr A. Stensvand (Norwegian Crop Research Institute), Dr A. Buchala (University of Fribourg), and Prof. J. Hantula (Finnish Forest Research Institute) for providing us with the fungal samples.

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Authors and Affiliations

  1. Institute of Applied Biotechnology, University of Kuopio, Yliopistonranta 1E, P.O. Box 1627, FIN-70211, Kuopio, Finland

    Anne Hukkanen, Liisa Pietikäinen, Sirpa Kärenlampi & Harri Kokko

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  1. Anne Hukkanen
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  2. Liisa Pietikäinen
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  3. Sirpa Kärenlampi
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  4. Harri Kokko
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Correspondence to Harri Kokko.

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Hukkanen, A., Pietikäinen, L., Kärenlampi, S. et al. Quantification of downy mildew (Peronospora sparsa) in Rubus species using real-time PCR. Eur J Plant Pathol 116, 225–235 (2006). https://doi.org/10.1007/s10658-006-9054-z

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