Ann. appl. Biol. (2002), 141:1-10 Printed in Great Britain *Corresponding Author E-mail: tjones@scri.sari.ac.uk © 2002 Association of Applied Biologists 1 Identification of Rubus yellow net virus as a distinct badnavirus and its detection by PCR in Rubus species and in aphids By A TEIFION JONES 1 *, WENDY J McGAVIN 1 , A D W GEERING 2 and B E L LOCKHART 3 1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK 2 Queensland Horticulture Institute, Department of Primary Industries, 80 Meiers Road, Indooroopilly, Queensland 4068, Australia 3 Department of Plant Pathology, University of Minnesota, 495 Borlaug Hall, 1991 Upper Buford Circle, St Paul, MN 55108 USA (Accepted 12 April 2002; Received 13 March 2002) Summary Rubus yellow net virus (RYNV) infects Rubus species and cultivars worldwide and is an essential component of raspberry veinbanding mosaic (RVBMD), a virus disease complex that causes serious decline in plant vigour and productivity. The virus is transmitted, probably in a semi-persistent manner, by the large raspberry aphid, Amphorophora idaei in Europe, and A. agathonica in North America. The particles of RYNV are bacilliform in shape and measure 80-150 ´ 25-30 nm, similar to those of badnaviruses. A 1.7 kb fragment of the viral DNA was amplified by PCR and then directly sequenced. Analysis of this sequence suggests that RYNV is possibly a distinct species in the genus Badnavirus and is most closely related to Gooseberry vein banding associated virus (GVBAV) and Spiraea yellow leaf spot virus, two other badnaviruses described recently. Using the sequence derived from the PCR- amplified viral DNA fragment, RYNV-specific primers were designed and used in PCR to assay for RYNV in a range of Rubus germplasm infected with RYNV, with other unrelated viruses and virus- like diseases found in Rubus, and in healthy plants. RYNV was detected in all glasshouse cultures of RYNV-infected plants, whether alone or in complex infections with other viruses, but not from healthy Rubus plants, nor from plants infected with other viruses. It was also detected in field-grown raspberry plants with and without symptoms of RVBMD and in raspberry plants infected with RYNV by viruliferous A. idaei. RYNV was also detected by PCR in A. idaei following access feeds on RYNV- infected plants of 1 h or more. PCR failed to amplify DNA from gooseberry infected with GVBAV confirming the specificity of the RYNV analysis. PCR detection of RYNV in dormant raspberry buds allows assays to be made outside the natural growing season, providing a useful application for plant introduction and quarantine programmes. Key words: DNA, raspberry veinbanding mosaic disease, Amphorophora idaei, aphid transmission Introduction Rubus yellow net virus (RYNV) is transmitted, probably in a semi-persistent manner, by the large raspberry aphid, Amphorophora idaei Börner in Europe, and A. agathonica Hottes in North America (Stace-Smith & Jones, 1987). RYNV is common in Rubus crops throughout Europe and is reported in North America (Stace-Smith & Jones, 1987). It infects all red raspberry (Rubus idaeus var. idaeus; R. idaeus var. strigosus) cultivars tested and most blackberry and hybrid berry cultivars; most are infected symptomlessly, or develop only very faint vein netting of leaves (Stace-Smith & Jones, 1987; Jones, 1991; Jones & McGavin, 1998). A very faint vein netting symptom also occurs in graft-inoculated black raspberry (R. occidentalis), a recommended indicator for RYNV, but symptoms are usually more intense when such plants are infected using viruliferous aphids (Stace-Smith & Jones, 1987). The virus is a major component in the virus complex inducing raspberry veinbanding mosaic (RVBMD), a disease that affects severely plant vigour and yield of infectible cultivars (Fig. 1A; Converse et al., 1987; Jones, 1991). In North America, this disease is reported to be induced by dual infection with RYNV and another Amphorophora-borne virus, identified as Black raspberry necrosis (Stace-Smith, 1956; Stace-Smith & Jones, 1987). However, in the UK, the disease has been shown to be caused only by dual infection with RYNV and a different Amphorophora-borne virus, Raspberry leaf mottle virus (RLMV), but disease symptoms are accentuated by the presence of additional Amphorophora-borne viruses (Jones, 1991). Because of the very faint symptoms induced in