caa a22 4500 467928851 CHVBK 20180406152937.0 cr unu---uuuuu 170328e20061101xx s 000 0 eng 10.1007/s10658-006-9054-z doi (NATIONALLICENCE)springer-10.1007/s10658-006-9054-z Quantification of downy mildew ( Peronospora sparsa ) in Rubus species using real-time PCR [Elektronische Daten] [Anne Hukkanen, Liisa Pietikäinen, Sirpa Kärenlampi, Harri Kokko] 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 37fg of P. sparsa conidial DNA was detected. In the samples collected from the field, quantities as low as 0.2ppm 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. Springer Science + Business Media B.V., 2006 arctic bramble nationallicence blackberry nationallicence ITS nationallicence ribosomal DNA nationallicence rose nationallicence bp : base pair nationallicence Ct : threshold cycle nationallicence ITS : internal transcribed spacer nationallicence rDNA : ribosomal DNA nationallicence Hukkanen Anne Institute of Applied Biotechnology, University of Kuopio, Yliopistonranta 1E, P.O. Box 1627, FIN-70211, Kuopio, Finland aut Pietikäinen Liisa Institute of Applied Biotechnology, University of Kuopio, Yliopistonranta 1E, P.O. Box 1627, FIN-70211, Kuopio, Finland aut Kärenlampi Sirpa Institute of Applied Biotechnology, University of Kuopio, Yliopistonranta 1E, P.O. Box 1627, FIN-70211, Kuopio, Finland aut Kokko Harri Institute of Applied Biotechnology, University of Kuopio, Yliopistonranta 1E, P.O. Box 1627, FIN-70211, Kuopio, Finland aut European Journal of Plant Pathology Kluwer Academic Publishers 116/3(2006-11-01), 225-235 0929-1873 116:3<225 2006 116 10658 https://doi.org/10.1007/s10658-006-9054-z text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10658-006-9054-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Hukkanen Anne Institute of Applied Biotechnology, University of Kuopio, Yliopistonranta 1E, P.O. Box 1627, FIN-70211, Kuopio, Finland aut NATIONALLICENCE 700 1- Pietikäinen Liisa Institute of Applied Biotechnology, University of Kuopio, Yliopistonranta 1E, P.O. Box 1627, FIN-70211, Kuopio, Finland aut NATIONALLICENCE 700 1- Kärenlampi Sirpa Institute of Applied Biotechnology, University of Kuopio, Yliopistonranta 1E, P.O. Box 1627, FIN-70211, Kuopio, Finland aut NATIONALLICENCE 700 1- Kokko Harri Institute of Applied Biotechnology, University of Kuopio, Yliopistonranta 1E, P.O. Box 1627, FIN-70211, Kuopio, Finland aut NATIONALLICENCE 773 0- European Journal of Plant Pathology Kluwer Academic Publishers 116/3(2006-11-01), 225-235 0929-1873 116:3<225 2006 116 10658 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer