ABSTRACT
Grapevine leafroll disease (GLD) has been recognized as one of the major constraints to the production of wine grapes in Washington State. At least nine distinct Grapevine leafroll-associated viruses (GLRaV-1 to -9) have been detected in grapevines showing GLD symptoms in grape-growing areas of several countries. Previous studies documented the presence of GLRaV-1, -2, and -3 in Washington State (3). We initiated a program to test grapevine cultivars with GLD symptoms for the occurrence of the other GLRaVs. Leaf samples were collected from individual grapevines of red-berried grapevine cultivars showing typical GLD symptoms and tested by single-tube reverse transcription (RT)-PCR. Of nearly 300 samples from 13 cultivars in 19 vineyards, 14 samples from 5 cultivars (Cabernet Sauvignon, Merlot, Pinot Noir, Mourvedre, and Lagrein) in different vineyards tested positive for GLRaV-9 using primers LR9 F/F (5'-CGG CAT AAG AAA AGA TGG CAC-3') and LR9 R/R (5'-TCA TTC ACC ACT GCT TGA AC-3'), specific for the HSP-70h gene of GLRaV-9 (1). To confirm the identity of the RT-PCR products, the 393-bp amplicons obtained from each of these five cultivars were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparisons of these sequences (GenBank Accession Nos. EF101737, EF101738, EF101739, EF101740, and EU252530) with corresponding sequences of other GLRaVs in GenBank showed 94 to 100 and 96 to 100% identity at the nucleotide and amino acid level, respectively, with the sequence of HSP-70h gene of GLRaV-9 (GenBank Accession No. AY297819). Antiserum specific to GLRaV-9 was not accessible, therefore, an additional 540-nucleotide fragment specific to the coat protein (CP) gene of GLRaV-9 was amplified from cv. Lagrein using primers LR9-CP-F (5' TAC CGT CGA CAC TTT CGA AGC ACT 3') and LR9-CP-R (5' TGA GGC GTC GTA ACC GAA CAA TCT 3'). PCR amplified fragments were cloned and sequenced. A comparison of this sequence (GenBank Accession No. EU251512) with corresponding nucleotide sequences of other GLRaVs in GenBank showed 96% identity with CP of GLRaV-9 (GenBank Accession No. AY297819), further confirming the presence of GLRaV-9. Previously, GLRaV-9 was reported in grapevines in California (1), Tunisia (2), and Western Australia (4). To our knowledge, our results are the first evidence for the occurrence of GLRaV-9 in Washington State vineyards. Results from our study and previous reports (1,2,4) indicate the wide distribution of GLRaV-9 in several Vitis vinifera cultivars. The economic impact of GLRaV-9 on wine grape cultivars, however, remains to be determined. References: (1) R. Alkowni et al. J. Plant Pathol. 86:123, 2004. (2) N. Mahfoudhi et al. Plant Dis. 91:1359, 2007. (3) R. R. Martin et al. Plant Dis. 89:763, 2005. (4) B. K. Peake et al. Aust. Plant Pathol. 33:445, 2004.
ABSTRACT
Perennial cultivars of Coreopsis, a genus native to the United States, are widely grown for aesthetics in home gardens and roadsides and are increasingly used in conservation projects and native-plant gardens. During the spring and summer of 2006 and 2007, Coreopsis auriculata 'Nana' plants with foliar symptoms showing chlorotic spots and rings were observed in wholesale and retail nurseries in Washington. Nicotiana benthamiana plants inoculated with crude sap extracts from symptomatic leaves of C. auriculata 'Nana' obtained from two different sources showed systemic mosaic mottling symptoms, indicating the presence of a virus. Symptomatic leaf samples from C. auriculata 'Nana' and N. benthamiana tested positive in antigen-coated plate-ELISA with potyvirus group-specific monoclonal antibodies (Agdia Inc., Elkhart, IN). Additional analysis by ELISA was positive for Lettuce mosaic virus (LMV; Agdia Inc.). To confirm these results, total RNA extracted from symptomatic N. benthamiana leaves was subjected to reverse transcription (RT)-PCR using potyvirus degenerate primers (PNIbF5: 5'-GCCAGCCCTCCACCGTNGTNGAYAA-3' and PCPR1: 5'-GGGGAGGTGCCGTTCTCDATRCACCA-3') covering the 3' end of the NIb gene and the 5' end of the CP gene (1). A single DNA band of approximately 1,000 bp amplified from symptomatic leaves of two independent plants was cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences with corresponding nucleotide sequences of potyviruses in GenBank showed 93 to 99% identity in the NIb/CP region with LMV sequences from France (GenBank Accession Nos. X97704, X65652, and X97705), China (GenBank Accession Nos. AJ306288 and AJ488153), and Brazil (GenBank Accession No. AJ278854). These results confirmed the presence of LMV in symptomatic leaves of N. benthamiana and C. auriculata 'Nana'. The occurrence of LMV has been reported in ornamental plants that included freeway daisy (Osteospermum fruticosum), lisianthus (Eustoma grandiflorum), and gazanias (Gazania spp.) (2-4). To our knowledge, this is the first documented evidence for the occurrence of LMV in Coreopsis, an economically important perennial ornamental widely grown in the United States. Although the origin of LMV in C. auriculata 'Nana' is not known, distribution of cuttings from LMV-infected C. auriculata 'Nana' plants to wholesale and retailers within Washington and across the country by movement of plant material could pose a risk to other ornamentals and crops like lettuce because of the broad host range of LMV and its potential transmission by several species of polyphagous aphids. Seed transmission as a potential means of dissemination of LMV in Coreopsis has not been examined, although the virus is seedborne in other plants such as lettuce. References: (1) Y.-C. Hsu et al. J. Virol. Methods 128:54, 2005. (2) V. Lisa et al. Inf. Fitopatol. 3:58, 1995. (3). D. C. Opgenorth et al Plant Dis. 75:751, 1991. (4) F. M. Zerbini et al. Plant Dis. 81:641, 1997.
