dsRNA pattern and partial sequence data indicate plant virus infection.pdf

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dsRNA pattern and partial sequence data indicate plant virus infection.pdf

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dsRNA pattern and partial sequence data indicate plant virus infection

Arch Virol (2005) 150: 37–52 DOI 10.1007/s00705-004-0397-5 Double-stranded RNA pattern and partial sequence data indicate plant virus infection associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.) W. Benthack1, N. Mielke1, C. Bu?ttner2, and H.-P. Mu?hlbach1 1Institute of General Botany and Botanical Garden, University of Hamburg, Hamburg, Germany 2Department of Phytomedicine, Faculty of Agriculture and Horticulture, Humboldt University, Berlin, Germany Received March 16, 2004; accepted July 13, 2004 Published online September 24, 2004 c? Springer-Verlag 2004 Summary. Double-stranded RNA (dsRNA) has been extracted from tissue of European mountain ash trees (Sorbus aucuparia L.) showing typical ringspot and mottling symptoms on leaves and a gradual decay in general. A characteristic dsRNA pattern was found in leaf samples of symptomatic mountain ash trees from various stands in Germany. Bands of dsRNA molecules of approximately 7 kb, 2.3 kb, 1.5 kb, and 1.3 kb, respectively, were repeatedly detected. By random primed reverse transcription cDNA was synthesised from dsRNA and amplified by degenerate oligonucleotide primed PCR. After TA cloning, the cDNA clones obtained were screened with an enhanced-chemiluminescence-labelled dsRNA probe. Positive clones were further analysed by using them as hybridisation probes in Northern blots of total plant RNA and in Southern hybridisation with genomic DNA from Sorbus aucuparia leaves. From cDNA clones that were found to be specific for dsRNA in Northern analysis, primers were deduced for 5′-RACE analyses and further cloning. Finally, a cDNA fragment of 3,737 bp was obtained, which showed homology to viral proteins, particularly to the RNA- dependent RNA polymerase of members of the family Bunyaviridae, but without high similarity to a known genus. The dsRNA pattern and the sequence information strongly indicate a virus associated with the mountain ash ringspot disease. The putative virus remains sti