H LINDQVIST1, H KOPONEN2, JPT VALKONEN 1 1Dept. Plant Biology, Genetic Centre, PO Box 7080, S-750 07 Uppsala, Sweden; 2Dept. Plant Biology, PO Box 28, FIN-00014 University of Helsinki, Finland Background and objectives
Arctic bramble (Rubus arcticus L.) is a wild berry plant growing in subarctic Eurasia, mainly between 62 and 66 degrees of latitude. It is commercially cultivated only in Finland and berries are mainly used in liqueur industry. Since 1994 farmers have reported serious yield losses due to berries drying in the middle of the growing season. The yield loss is commonly 50% but in some cases the yield is completely lost. Other symptoms in the diseased plants include interveinal, angular, purple-red lesions on leaves. These symptoms are typical for the downy mildew infection in other Rubus plants caused by Peronospora sparsa Berkeley (syn. P. rubi Rabenshorst), but it has not been previously reported in arctic bramble. The aim of this study was to isolate and identify fungi from the diseased plants of arctic bramble. Because the pathogenic fungus most often associated with the dry berry disease was P. sparsa, a quick and specific polymerase chain reaction (PCR) based method was developed for diagnosis of this fungus. Results and conclusions
Leaf samples of arctic bramble were collected in June and July in 1996 from plants that were affected by foliar and/or dry berry symptoms. Fungi were detected in leaves incubated on water agar at 17C for 14 days. P. sparsa was detected in 38 (88%) of the 43 samples from three different geographic regions, and in 14 samples it was the only fungus detected. Examination of the plants grown from rootstocks collected later in the fall of the same year showed that P. sparsa was maintained in the rootstocks and grew systemically in arctic bramble. DNA was isolated from spores of P. sparsa grown on detached leaves of arctic bramble in Petri dishes, from mycelia of four other fungi isolated from arctic bramble, and from leaves of healthy arctic bramble. The internal transcribed spacers (ITS1 and 1T52) between the 18S, 5.8S and 28S rRNA genes, respectively, were PCR-amplified using universal primers, cloned and sequenced. Based on sequence comparison, primers PRI and PR2 were designed that specifically amplified a product from P. sparsa. However, these primers also amplified a product of similar size from Phytophthora cactorum isolated from strawberry. Thus, the ITS regions of this latter mentioned fungus were also sequenced and new primers (PR3 and PR4) were designed that specifically amplified a product from P. sparsa only. The specificity of amplification using both pairs of primers was tested and amplification conditions optimized for two different types of thermal cyclers: a heat block cycler with heated lid (PTC-150, MiniCycler with Hotbonnet, MJ Research) using thin wall microcentrifuge tubes and an air thermo-cycler (Rapidcycler, Idaho Technology) using glass capillary tubes. With both types of cyclers and both primer pairs the specific amplification product was obtained using a mixture of 0.2 pg of fungal DNA and 3 ng of DNA from healthy arctic bramble as the template. The detection method developed in this study is a promising diagnostic tool to be employed in indexing arctic bramble for latent infections with P. sparsa.