Detection and analysis of mycovirus-related RNA viruses from grape powdery mildew fungus Erysiphe necator.

The fungus, Erysiphe necator Schw., is an important plant pathogen causing powdery mildew disease in grapevines worldwide. In this study, high-throughput sequencing of double-stranded RNA extracted from the fungal tissue combined with bioinformatics was used to examine mycovirus-related sequences associated with E. necator. The results showed the presence of eight mycovirus-related sequences. Five of these sequences representing three new mycoviruses showed alignment with sequences of viruses classified in the genus Alphapartitivirus in the family Partitiviridae. Another three sequences representing three new mycoviruses showed similarity to classifiable members of the genus Mitovirus in the family Narnaviridae. These mycovirus isolates were named Erysiphe necator partitivirus 1, 2, and 3 (EnPV 1-3) and Erysiphe necator mitovirus 1, 2, and 3 (EnMV 1-3) reflecting their E. necator origin and their phylogenetic affiliation with other mycoviruses.

First Report of Tomato spotted wilt virus in Peppers and Tomato in the Dominican Republic.

In the Dominican Republic, green bell pepper (Capsicum annuum L.) and tomato (Solanum lycopersicum L.) are widely cultivated under protected greenhouse conditions as high value commercial crops for export. For the past 2 to 3 years, pepper and tomato have been observed in protected crop facilities in Jarabacoa and Constanza in the North Region with chlorotic and necrotic spots and rings on leaves, petioles, and stems, leaf bronzing, and tip necrosis. Fruits on symptomatic pepper and tomato plants showed concentric rings, irregular chlorotic blotches and deformation, and uneven maturation and development. Incidence on pepper and tomato was 20 to 100% and 5 to 20%, respectively. In initial tests, leaves and fruits from each of 20 symptomatic tomato and pepper plants from several greenhouse facilities were reactive in Tomato spotted wilt virus (TSWV; genus Tospovirus, family Bunyaviridae) immunostrip assays (Agdia, Inc., Elkhart, IN). Since these immunostrips are known to react with other tospoviruses, such as Tomato chlorotic spot virus (TCSV) and Groundnut ring spot virus, additional molecular diagnostic assays were conducted. Leaf and fruit samples from symptomatic plants were imprinted on nitrocellulose membrane (NCM) (2), air-dried, and sent to Washington State University for confirmatory tests. Viral nucleic acids were eluted from NCM discs (1) and subjected to reverse transcription (RT)-PCR using primers gL3637 (CCTTTAACAGTDGAAACAT) and gL4435 (CATDGCRCAAGARTGRTARACAGA) designed to amplify a portion of the L RNA segment of several tospoviruses (3). A single DNA product of ~800 bp was amplified from all samples. Amplicons from two tomato (leaf and fruit) and one pepper fruit samples were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced in both orientations. Sequence analyses of these clones (GenBank Accession Nos. KF 219673 to 75) showed 100% nucleotide sequence identity among themselves and 97% identity with corresponding L RNA sequences of pepper isolates of TSWV from Taiwan (HM180088) and South Korea (HM581940), 94 to 95% with tomato isolates of TSWV from South Korea (HM581934) and Hawaii (AY070218), and 89% with a tomato isolate from Indonesia (FJ177301). These results further confirm the presence of TSWV in symptomatic tomato and pepper plants. A comparison of TSWV sequences from the Dominican Republic with TSWV isolates from the United States and other countries in the Caribbean region could not be made due to the absence of corresponding sequences of the L-RNA of the virus from these countries in GenBank. TSWV-positive samples were negative for TCSV in RT-PCR, indicating the absence of this tospovirus that has been reported in the Caribbean region (data not shown). To our knowledge, this is the first confirmed report of TSWV in tomatoes and peppers in the Dominican Republic. The presence of vector thrips, Frankliniella occidentalis, on symptomatic plants was also confirmed, suggesting a role in the spread of TSWV under greenhouse conditions. Recent surveys identified some greenhouses with 100% symptomatic peppers. The presence of TSWV in tomato and pepper has important implications for the domestic and export vegetable industry in the Dominican Republic because of the broad host range of the virus (4). It is critical for commercial producers to monitor TSWV and deploy appropriate management strategies to limit virus spread. References: (1) O. J. Alabi et al. J. Virol. Methods 154:111, 2008. (2) P.-G. S. Chang et al. J. Virol. Methods 171:345, 2011. (3) F. H. Chu et al. Phytopathology 91:361, 2001. (4) G. Parrella et al. J. Plant Pathol. 85:227, 2003.

First Report of Potato virus Y in Potato in West Java, Indonesia.

Potato (Solanum tuberosum) is an important vegetable crop in Indonesia. A small survey was conducted for virus diseases in November 2011 in Lembang, West Java, as part of assessing the sanitary status of potatoes produced in farmers' fields. Among the six potato fields surveyed, one field had nearly 20% of plants displaying stunted growth with leaves showing mild chlorotic spots and reduced size of lamina. Tubers harvested from symptomatic plants showed no necrosis symptoms. Symptomatic leaves from three representative potato plants were positive for Potato virus Y (PVY) when tested with PVY-specific immunostrips (Agdia Inc., Elkhart, IN). Leaf samples from virus-positive plants were imprinted on FTA Classic Cards (Whatman International Ltd., Maidstone, UK), air dried, and shipped to Washington State University for confirmatory diagnostic tests. Total nucleic acids were eluted from FTA cards (1) and subjected to reverse transcription (RT)-PCR using primers (PVY/Y4A and PVY/Y3S) specific to the coat protein (CP) of PVY (3). Nucleic acid extracts from samples infected with PVY ordinary strain (PVYO), tuber necrosis strain (PVYNTN), tobacco veinal necrosis strains (PVYEU-N and PVYNA-N), and a recombinant strain (PVYN:O) were included as standards to validate RT-PCR assays. The approximately 480-bp DNA fragment, representing a portion of the CP, amplified in RT-PCR was cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA). DNA isolated from four independent recombinant clones was sequenced from both orientations. Pairwise comparison of these sequences (GenBank Accession Nos. KF261310 to 13) showed 100% identity among themselves and 93 to 100% identity with corresponding sequences of reference strains of PVY available in GenBank (JQ743609 to 21). To our knowledge, this study represents the first confirmed report of PVY in potato in West Java, Indonesia. Studies are in progress to assess the prevalence of PVY in other potato-growing regions of Indonesia and document the presence of different strains of the virus (2). Since the majority of farmers in Indonesia plant seed selected from their previous potato crop, there is an increased risk of primary and secondary spread of PVY through the informal seed supply system, leading to its increased significance to potato production in Indonesia. Therefore, strengthening foundation seed potato and supply chain programs will promote the production of virus-free potatoes in Indonesia. References: (1) O. J. Alabi et al. Plant Dis. 96:107, 2012. (2) A. Karasev and S. M. Gray. Am. J. Potato Res. 90:7, 2013. (3) R. P. Singh et al. J. Virol. Methods 59:189, 1996.

Pheromone-based monitoring of Pseudococcus maritimus (Hemiptera: Pseudococcidae) populations in concord grape vineyards.

The grape mealybug, Pseudococcus maritimus (Ehrhorn), is the dominant mealybug in Washington's Concord grape vineyards (Vitis labrusca L.). It is a direct pest of fruit clusters and a vector of grapevine leafroll-associated viruses. Using traps baited with the sex pheromone of Ps. maritimus, we determined the optimal trap density for monitoring Ps. maritimus, with the goal of providing a more rapid monitoring method for Ps. maritimus than visual surveys. Varying densities of pheromone-baited traps (one, four, and eight traps per 12.14 ha) were deployed in Concord vineyards to monitor Ps. maritimus seasonal phenology in 2010 and 2011. In both years, flights of adult males were detected in early May and captures peaked twice per season in mid-June and mid-August, indicating two generations each year. Trap data were analyzed using Taylor's Power Law, Iwao's patchiness regression, and the K parameter of the negative binomial model to determine optimal sample size. The formula using the K parameter provided the lowest required sample size, showing that four to eight traps per 12.14 ha were needed to provide 30% sampling precision efficiency throughout the entire season. Fewer traps were needed during flight peaks when trap capture numbers were great. Only one pheromone-baited trap per 12.14 ha was sufficient to provide Ps. maritimus flight phenology data to make informed management decisions. Species-specific pheromone-baited traps deployed for Planococcus ficus (Signoret), Pseudococcus longispinus (Targioni Tozzetti), and Pseudococcus viburni (Signoret) did not detect any of these species in the vineyards sampled.

First Report of Impatiens necrotic spot virus (INSV) Infecting Basil (Ocimum basilicum) in the United States.

Basil (Ocimum basilicum L.), a native of India belonging to the Lamiaceae family, is an aromatic herb with distinctive aroma, and several commercial varieties are used extensively for culinary and ornamental purposes. During the summer of 2011 and 2012, potted plants of basil in a commercial greenhouse in the Richland-Kennewick area of Washington State were observed showing foliar symptoms consisting of chlorotic spots, ring spots, leaf distortion, and stem necrosis. In initial tests, extracts of symptomatic leaves were positive for Impatiens necrotic spot virus (INSV; genus Tospovirus, family Bunyaviridae), when tested with INSV immnunostrips (Agdia, Inc., Elkhart, IN). These samples were negative with immunostrips specific to Tomato spotted wilt virus (genus Tospovirus) and group-specific potyviruses. The virus from symptomatic leaves of basil was transmitted by leaf rub inoculation to Nicotiana benthamiana and Emilia sonchifolia, where it produced necrosis on inoculated leaves followed by systemic necrosis in the former and chlorotic spots and mosaic mottling in newly developed leaves in the latter. Symptomatic leaves from both host plants tested positive with INSV, but not with TSWV, immunostrips. For additional confirmation of INSV, total RNA was extracted from symptomatic leaves of basil using RNeasy Plant Minikit (Qiagen, Inc., Valencia, CA) and used for reverse transcription (RT)-PCR amplification of the nucleocapsid (N) gene using forward (5'-AGCTTAAATCAATAGTAGCA-3') and reverse (5'-AGCTTCCTCAAGAATAGGCA-3') primers. RT was carried out at 52°C for 60 min followed by denaturation at 94°C for 3 min. Subsequently, 35 cycles of PCR was carried out with each cycle consisting of 94°C for 1 min, 58°C for 45 s, and 72°C for 1 min, followed by a final extension step at 72°C for 10 min. The amplicons of about 610 nt obtained from RT-PCR were cloned into pTOPO2.1 vector (Invitrogen Corporation, Carlsbad, CA) and three independent clones were sequenced in both directions. Sequence analyses of these clones (GenBank Accession No. KC218475) showed 100% nucleotide sequence identity among themselves and 99% nucleotide sequence identity with INSV isolates from the United States (DQ523598, JX138531, and D00914) and a basil isolate (JQ724132) from Austria. These results further confirm the presence of INSV in symptomatic leaves of basil. Previously, basil has been reported to be naturally infected with TSWV in the United States (3) and INSV in Austria (2). Therefore, this study represents the first confirmed report of the virus in basil in the United States. No species of thrips vector was observed on the affected basil plants. The discovery of INSV in basil has important implications for the nursery industry due to the broad host range of the virus (1); stock plants may serve as a source of inoculum in production areas and infected plants could be distributed to homeowners. It is important for commercial nurseries to monitor for INSV to identify infected mother plants to prevent virus spread. Since more than 31 viruses belonging to 13 different genera have been reported in basil ( http://pvo.bio-mirror.cn/famly073.htm#Ocimumbasilicum ), further studies are in progress to determine if the observed symptoms on basil are only due to single infection of INSV. References: (1) M. Daughtrey et al. Plant Dis. 81:1220, 1997. (2). S. Grausgruber-Gröger. New Dis. Rep. 26:12, 2012. (3) G. E. Holcomb et al. Plant Dis. 83:966.

Pseudococcus maritimus (Hemiptera: Pseudococcidae) and Parthenolecanium corni (Hemiptera: Coccidae) are capable of transmitting grapevine leafroll-associated virus 3 between Vitis x labruscana and Vitis vinifera.

The grape mealybug, Pseudococcus maritimus (Ehrhorn), and European fruit lecanium scale, Parthenolecanium corni (Bouché), are the predominant species of Coccoidea in Washington State vineyards. The grape mealybug has been established as a vector of Grapevine leafroll-associated virus 3 (GLRaV-3) between wine grape (Vitis vinifera L.) cultivars, elevating its pest status. The objective of this study was to determine if GLRaV-3 could be transmitted between Vitis x labruscana L. and V. vinifera by the grape mealybug and scale insects. Three transmission experiments were conducted with regard to direction; from V. vinifera to V. x labruscana L., from V. x labruscana L. to V. x labruscana L., and from V. x labruscana L. to V. vinifera. Each experiment was replicated 15 times for each vector species. Crawlers (first-instars) of each vector species were allowed 1-wk acquisition and inoculation access periods. The identities of viral and vector species were confirmed by reverse transcription-polymerase chain reaction, cloning, and sequencing of species-specific DNA fragments. GLRaV-3 was successfully transmitted by both species in all experiments, although Ps. maritimus was a more efficient vector under our experimental conditions. To the best of our knowledge, this study represents the first documented evidence of interspecific transmission of GLRaV-3 between two disparate Vitis species. It also highlights the potential role of V. x labruscana L. in the epidemiology of grapevine leafroll disease as a symptomless source of GLRaV-3 inoculum.

First Report of Potato virus Y in Potato in Tajikistan.

Potato (Solanum tuberosum L.) is widely grown as a staple food and cash crop in Tajikistan and is an important food security crop in the country. In June 2011, we conducted a survey of potatoes in farmers' fields in the Buston and Dushanbe regions (about 200 miles apart) of Tajikistan. Potato plants with stunted growth and leaves showing chlorotic spots, curling, and necrotic spots and rings were observed with the disease incidence monitored in 10 fields each in Buston and Dushanbe areas varying between 10 and 60%. Representative samples from symptomatic plants tested positive for Potato virus Y (PVY) using virus-specific immunostrips (Agdia Inc., Elkhart, IN). Leaf samples from symptomatic plants were collected from Buston and Dushanbe areas, imprinted on FTA Classic Cards (Whatman International Ltd., Maidstone, UK), air dried, and shipped to the lab at Washington State University for confirmatory diagnostic tests. Total nucleic acids were eluted from FTA cards (1) and subjected to reverse transcription (RT)-PCR with primers (PVY/Y4A and PVY/Y3S) specific to the coat protein of PVY (3). Samples infected with PVY ordinary strain (PVYO), tuber necrosis strain (PVYNTN), tobacco veinal necrosis strains (PVYEU-N and PVYNA-N), and a recombinant strain (PVYN:O) were included as references to validate RT-PCR results. A single DNA product of approximately 480 bp was amplified from potato samples that tested positive with PVY-specific immunostrips. The amplified fragments from two samples from Dushanbe and six from Buston areas were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA) and two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 90 to 100% identity among the cloned amplicons (GenBank Accession Nos. JQ743609 to JQ743616) and 90 to 100% with corresponding nucleotide sequence of reference PVY strains (GenBank Accession Nos. JQ743617 to JQ743621). A global phylogenetic analysis of sequences revealed the presence of PVYO in both samples from Dushanbe and one sample from Buston regions and presence of PVYNTN in the remaining five samples from the Buston region. Because of the possible occurrence of mixed infections of PVY strains (2), further studies are needed to determine the presence of mixed infections of two or more strains of PVY and their specificity to potato cultivars. To our knowledge, this study represents the first confirmed report of two distinct strains of PVY in potato in Tajikistan. The occurrence of PVYNTN, a quarantine pathogen in many countries (2), warrants additional investigations to improve sanitary status of potato fields and to facilitate the availability of virus-free seed in clean plant programs for significant yield increases in Tajikistan. References: (1) O. J. Alabi et al. J. Virol. Methods 154:111, 2008. (2) S. Gray et al. Plant Dis. 94:1384, 2010. (3) R. P. Singh et al. J. Virol. Methods 59:189, 1996.

First Report of Grapevine fleck virus in Idaho Grapevines.

Idaho has a growing viticulture industry, with nearly 1,600 acres of wine grapes (Vitis vinifera L.). Production is largely concentrated in two locations, the Snake River valley, which includes Canyon County in the southwest, and the Clearwater River valley, primarily Nez Perce County in the northwest. Grapevine fleck virus (GFkV) belongs to the genus Maculavirus, family Tymoviridae, comprising positive-sense, single-stranded RNA viruses with ca. 7.6-kb genome (3). It is one of five non-mechanically transmitted viruses associated with the fleck disease complex and has been previously documented to occur in the neighboring state of Washington (2). Main sources of wine grape nursery material imported to Idaho reside in Washington or in California, and it is important to monitor virus status of the planting material brought to the state. However, no information was available on the occurrence and prevalence of GFkV in wine grapes in Idaho. During three growing seasons in 2009 through 2011, random grapevine samples were collected in 14 vineyards in Canyon, Elmore, Ada, and Nez Perce counties. A total of 434 samples were tested by one step RT-PCR using GFkV-specific primers, GFkVf: 5'-TGACCAGCCTGCTGTCTCTA-3' and GFkVr: 5'-TGGACAGGGAGGTGTAGGAG-3' designed to amplify a fragment of the GFkV capsid protein gene (1). Twenty-four samples tested positive for GFkV by RT-PCR and produced the expected 179-bp DNA fragment. These samples came from five vineyards sampled across all surveyed counties, and represented seven wine grape cultivars, including Pinot Noir, Cabernet Sauvignon, Syrah, Lemberger, Riesling, Chardonnay, Pinot Gris, and one unknown table grape cultivar. Twelve PCR products were cloned into the pGEM-T Easy plasmid vector (Promega), sequenced (numbered ID1 to 12, available upon request), and confirmed to represent fragments of the GFkV CP gene between positions 6,453 and 6,631 in the genome of GFkV isolate MT48 (GenBank Accession No. AJ309022.1). Eight of the Idaho GFkV sequences (ID2, ID3, ID7 to 11, and ID12) matched closely with other GFkV sequences from Washington State, Italy, India, and South America, showing 97 to 99% identity at the nucleotide level in pair-wise comparisons. Four GFkV sequences from Idaho (ID1 and ID4 to 6) showed only modest (90 to 92%) identity in pair-wise comparisons with GFkV sequences available in GenBank. Consequently, in phylogenetic reconstructions eight Idaho GFkV sequences clustered in the same lineage with the six GFkV sequences deposited in GenBank, and four other GFkV sequences were placed outside of this main clade. It is possible that this phylogeny of the Idaho GFkV reflects different sources of the virus-infected planting material brought to the state. In the absence of symptoms expressed in wine grape cultivars infected with GFkV, laboratory methods remain the only tool to detect the virus. To our knowledge, this is the first report of GFkV found in wine grapes in Idaho demonstrating its substantial presence in production areas. References: (1) G. Gambino and I. Gribaudo. Phytopathology 96:1223, 2006. (2) R. A. Naidu et al. Plant Dis. 94:784, 2010. (3) S. Sabanadzovic et al. J. Gen. Virol. 82:2009, 2001.

Banana bunchy top virus in sub-Saharan Africa: investigations on virus distribution and diversity.

Banana bunchy top virus (BBTV) was first reported from sub-Saharan Africa (SSA) from Democratic Republic of Congo (DRC) in the 1950s, has become invasive and spread into 11 countries in the region. To determine the potential threat of BBTV to the production of bananas and plantains (Musa spp.) in the sub-region, field surveys were conducted for the presence of banana bunchy top disease (BBTD) in the DRC, Angola, Cameroon, Gabon and Malawi. Using the DNA-S and DNA-R segments of the virus genome, the genetic diversity of BBTV isolates was also determined from these countries relative to virus isolates across the banana-growing regions around the world. The results established that BBTD is widely prevalent in all parts of DRC, Malawi, Angola and Gabon, in south and western part of Cameroon. Analysis of the nucleotide sequences of DNA-S and DNA-R indicate that BBTV isolates from these countries are genetically identical forming a unique clade within the 'South Pacific' phylogroup that includes isolates from Australia, Egypt, South Asia and South Pacific. These results imply that farmers' traditional practice of transferring vegetative propagules within and between countries, together with virus spread by the widely prevalent banana aphid vector, Pentalonia nigronervosa, could have contributed to the geographic expansion of BBTV in SSA. The results provided a baseline to explore sanitary measures and other 'clean' plant programs for sustainable management of BBTV and its vector in regions where the disease has already been established and prevent the spread of the virus to as yet unaffected regions in SSA.

Occurrence of Grapevine leafroll-associated virus 1 in Two Ornamental Grapevine Cultivars in Washington State.

Roger's Red, an interspecific hybrid between wild grape (Vitis californica, native to northern California) and the V. vinifera cv. Alicante Bouschet (1), and Claret Vine (V. vinifera cv. Purpurea Nana) are grown for their ornamental value in home gardens and other settings. We collected potted grapevines of Roger's Red and Claret Vine showing dull green-to-scarlet red leaves from two different retail nurseries in the Richland-Kennewick area and Prosser, WA, respectively. Since these symptoms 'mimic' grapevine leafroll disease, we tested petiole samples from four grapevines per cultivar for a panel of grapevine-infecting viruses by single-tube one-step reverse transcription (RT)-PCR (4). All samples tested positive only for Grapevine leafroll-associated virus 1 (GLRaV-1; genus Ampelovirus, family Closteroviridae). To further confirm these results, total RNA was subjected to RT-PCR to amplify a portion of the heat shock protein 70 homolog (HSP70h), coat protein duplicate 2 (CPd2), and ORF 9 (p24) of GLRaV-1. RT was performed at 52°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 5 min final extension step at 72°C. Primers specific to HSP70h (HSP70h/416F: 5'-CAGGCGTCGTTTGTACTGTG and HSP70h/955R: 5'-TCGGACAGCGTTTAAGTTCC), CPd2 (CPd2/F: 5'-GTTACGGCCCTTTGTTTATTATGG and CPd2/R: 5'-CGACCCCTTTATTGTTTGAGTATG) and ORF 9 (p24/F: 5'-CGATGGCGTCACTTATACCTAAG and p24/R (5'-CACACCAAATTGCTAGCGATAGC) were designed based on GLRaV-1 sequence (GenBank Accession No. AF195822) to amplify 540, 398, and 633 base pair (bp) DNA fragments, respectively. To verify that the amplified products were specific to the genome of GLRaV-1, the amplicons were cloned into pCR2.1 vector (Invitrogen Corp, Carlsbad, CA) and three independent clones for each amplicon were sequenced in both directions. Pairwise comparison of HSP70h (Accession Nos. HQ833472 and HQ833473), CPd2 (Accession Nos. HQ833474 and HQ833475), and p24 (Accession Nos. HQ833476 and HQ833477) sequences from Roger's Red and Claret Vine showed 100, 96, and 99% identities, respectively, between them, and 86 to 100, 80 to 97, and 86 to 90% nucleotide sequence identities, respectively, with corresponding sequences of GLRaV-1 isolates deposited in GenBank. We further confirmed the presence of GLRaV-1 in these two ornamental grape cultivars by double antibody sandwich-ELISA using commercially available antibodies (Bioreba AG, Reinach, Germany). Previous studies have reported the presence of GLRaV-2 and -3 (1,3) and Grapevine virus A and B (2,3) in Roger's Red. To our knowledge, this study represents the first report of the occurrence of GLRaV-1 in two Vitis species distributed as ornamental grapes. It is important to prevent virus spread via the supply of virus-tested ornamental grapevines by commercial nurseries. References: (1) G. S. Dangl et al. Am. J. Enol. Vitic. 61:266, 2010. (2) D. A. Golino et al. Phytopathology (Abstr.) 99(suppl.):S44, 2009. (3) V. Klaassen et al. Online publication. doi:10.1094/PDIS-09-10-0621. Plant Dis., 2011. (4) T. A. Mekuria et al. Phytopathology (Abstr.) 99(suppl.):S83, 2009.

First Report of Grapevine fleck virus from Washington Vineyards.

Grapevine fleck virus (GFkV) is a positive-sense, single-stranded RNA virus with a genome size of 7,564 nucleotides (3). The virus is present in many grape-growing regions (1,2,4). GFkV is phloem-limited and graft transmissible, but a biological vector is not yet known (4). It causes latent infections in Vitis vinifera cultivars, but induces specific foliar symptoms in the indicator host, V. rupestris. While testing samples from wine grape cultivars, samples from cv. Chardonnay tested positive for GFkV in single-tube one-step reverse transcription (RT)-PCR assay using forward primer GFkV585F (5'-CTCAGCCTCCACCTTGCCCCGT-3') and reverse primer GFkV1117R (5'-CAATTTGGCTGGGCGAGAAGTACA-3'). The forward primer is identical to nt 585 to 606 and the reverse primer is complementary to nt 1094 to 1117 in the GFkV genome (Accession No. AJ309022) and encompass a portion of the RNA-dependent RNA polymerase. The primer pair amplified a 533-nt fragment from 15 of 37 individual grapevines in the Chardonnay block. The amplicons obtained from five grapevines were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Three independent clones per amplicon were sequenced in both orientations. Sequences were edited and assembled using ContigExpress project in the Vector NTI Advance 11 sequence analysis software packages (Invitrogen). Pairwise comparisons of these sequences (Accession Nos. GU372367 to GU372371) with a corresponding sequence of a GFkV isolate deposited in the GenBank (Accession No. AJ309022) showed 89 to 97% identity at the nucleotide and 95 to 98% identity at the amino acid level. To further support these results, we amplified a 714-nt fragment specific to the complete coat protein (CP) gene of GFkV from three of the five isolates sequenced above using primers GFkV-6351F (5'-CTCTCCGCCTCGTCTGATGA-3') and GFkV-7064R (5'-TCGGTTCATGACGAGGGAGT-3'). The amplicons were cloned and sequenced as described above. A comparison of these sequences (Accession Nos. GU372372 to GU372374) with the CP sequence of GFkV available in the GenBank (Accession No. AJ309022) showed 94 to 95% and 98 to 100% identity, respectively, at the nucleotide and amino acid level. ELISA with GFkV-specific antibodies (BIOREBA AG, Reinach, Switzerland) further confirmed the presence of the virus in samples that were positive in RT-PCR. ELISA results validated the data described above and confirmed the presence of GFkV in Chardonnay samples that tested positive by RT-PCR assay. Previously, GFkV was documented in grapevines in California and Missouri (2), Australia (4) and Europe (1). To our knowledge, this is the first confirmed report of the occurrence of GFkV in Washington vineyards. The results expand our current knowledge on the distribution of GFkV and help to prevent its dissemination through the supply of grapevine cuttings by 'clean' plant programs. References: (1) G. P. Martelli et al. Arch Virol. 147:1847, 2002. (2) B. N. Milkus and R. N. Goodman. Am. J. Enol. Vitic. 50:133, 1999. (3) S. Sabanadzovic et al. J. Gen. Virol 82:2009, 2001. (4) B. J. Shi et al. Ann. Appl. Biol. 142:349, 2003.

First Report of Grapevine Virus Sequences Highly Similar to Grapevine Syrah virus-1 from Washington Vineyards.

Grapevine Syrah virus-1 (GSyV-1), a tentative member of the genus Marafivirus in the family Tymoviridae, has recently been found in a declining Syrah grapevine in California vineyards (1). To determine if GSyV-1 is present in grapevines grown in Washington State vineyards, extracts prepared from individual grapevines of six cultivars (Merlot, Chardonnay, Pinot Noir, Lemberger, Cabernet Sauvignon, and Syrah/Shiraz) were tested by single-tube reverse transcription (RT)-PCR using the primer pair GSyV-1 Det-F (5'-CAAGCCATCCGTGCATCTGG-3') and GSyV-1 Det-R (5'-GCCGATTTGGAACCCGATGG-3'). The primer GSyV-1 Det-F is identical to nucleotides (nt) 1125 to 1144 and GSyV-1 Det-R complementary to nt 1401 to 1420 of the GSyV-1 genome (GenBank Accession No. NC_012484) in the putative movement protein encoding gene (1). DNA fragment of approximately 296 base pairs (bp) was amplified only from 7 of 60 and 2 of 20 individual grapevines of cv. Syrah/Shiraz and Chardonnay, respectively, obtained from geographically separate vineyards. The 296-bp fragments from three Syrah/Shiraz and two Chardonnay grapevines were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Three independent clones derived from each DNA fragment were sequenced from both orientations and the sequences edited and assembled using ContigExpress project in the Vector NTI Advance 11 sequence analysis software packages (Invitrogen). Pairwise comparison of four of these sequences (Accession Nos. GU372349-52) showed 99 to 100% amino acid (aa) sequence identity among themselves and with corresponding sequences of GSyV-1. Because of the lack of antibodies, an additional 611-bp fragment specific to the capsid protein (CP) gene of GSyV-1 was amplified from six isolates (five from cv. Syrah/Shiraz, and one from cv. Chardonnay) (Accession Nos. GU372353-66) using primers GSyV-1-F (5'-TGTCGACGCTCCAATGTCTGA-3') and GSyV-1-R (5'-CATTGCTGCGCTTTGGAGGCTTTA-3'). GSyV-1-F is identical to nt 5775 to 5795 and GSyV-1-R is complementary to nt 6385 to 6408 of the GSyV-1 genome. The amplicons were cloned and sequenced as described above. Comparison of these sequences among themselves and with corresponding sequences of GSyV-1 showed 96 to 99% aa sequence identity, further complementing the results obtained above. To our knowledge, this is the first report of the occurrence of viral sequences closely related to GSyV-1 in Washington vineyards. Together with other reports (1,2), this study suggests that viruses similar to GSyV-1 could be widely distributed in wine grape cultivars across grape-growing regions. References: (1) M. Rwahnih et al. Virology 387:395, 2009. (2) S. Sabanadzovic. Virology 394:1, 2009.

The Occurrence of Bean common mosaic virus and Cucumber mosaic virus in Yardlong Beans in Indonesia.

Yardlong bean (Vigna unguiculata subsp. sesquipedalis) is extensively cultivated in Indonesia for consumption as a green vegetable. During the 2008 season, a severe outbreak of a virus-like disease occurred in yardlong beans grown in farmers' fields in Bogor, Bekasi, Subang, Indramayu, and Cirebon of West Java, Tanggerang of Banten, and Pekalongan and Muntilan of Central Java. Leaves of infected plants showed severe mosaic to bright yellow mosaic and vein-clearing symptoms, and pods were deformed and also showed mosaic symptoms on the surface. In cv. 777, vein-clearing was observed, resulting in a netting pattern on symptomatic leaves followed by death of the plants as the season advanced. Disease incidence in the Bogor region was approximately 80%, resulting in 100% yield loss. Symptomatic leaf samples from five representative plants tested positive in antigen-coated plate-ELISA with potyvirus group-specific antibodies (AS-573/1; DSMZ, German Resource Center for Biological Material, Braunschweig, Germany) and antibodies to Cucumber mosaic virus (CMV; AS-0929). To confirm these results, viral nucleic acids eluted from FTA classic cards (FTA Classic Card, Whatman International Ltd., Maidstone, UK) were subjected to reverse transcription (RT)-PCR using potyvirus degenerate primers (CIFor: 5'-GGIVVIGTIGGIWSIGGIAARTCIAC-3' and CIRev: 5'-ACICCRTTYTCDATDATRTTIGTIGC-3') (3) and degenerate primers (CMV-1F: 5'-ACCGCGGGTCTTATTATGGT-3' and CMV-1R: 5' ACGGATTCAAACTGGGAGCA-3') specific for CMV subgroup I (1). A single DNA product of approximately 683 base pairs (bp) with the potyvirus-specific primers and a 382-bp fragment with the CMV-specific primers were amplified from ELISA-positive samples. These results indicated the presence of a potyvirus and CMV as mixed infections in all five samples. The amplified fragments specific to potyvirus (four samples) and CMV (three samples) were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 93 to 100% identity among the cloned amplicons produced using the potyvirus-specific primers (GenBank Accessions Nos. FJ653916, FJ653917, FJ653918, FJ653919, FJ653920, FJ653921, FJ653922, FJ653923, FJ653924, FJ653925, and FJ653926) and 92 to 97% with a corresponding nucleotide sequence of Bean common mosaic virus (BCMV) from Taiwan (No. AY575773) and 88 to 90% with BCMV sequences from China (No. AJ312438) and the United States (No. AY863025). The sequence analysis indicated that BCMV isolates from yardlong bean are more closely related to an isolate from Taiwan than with isolates from China and the United States. The CMV isolates (GenBank No. FJ687054) each were 100% identical and 96% identical with corresponding sequences of CMV subgroup I isolates from Thailand (No. AJ810264) and Malaysia (No. DQ195082). Both BCMV and CMV have been documented in soybean, mungbean, and peanut in East Java of Indonesia (2). Previously, BCMV, but not CMV, was documented on yardlong beans in Guam (4). To our knowledge, this study represents the first confirmed report of CMV in yardlong bean in Indonesia and is further evidence that BCMV is becoming established in Indonesia. References: (1) J. Aramburu et al. J. Phytopathol. 155:513, 2007. (2) S. K. Green et al. Plant Dis. 72:994, 1988. (3) C. Ha et al. Arch. Virol. 153:25, 2008. (4) G. C. Wall et al. Micronesica 29:101, 1996.

Occurrence of Banana Bunchy Top Disease Caused by the Banana bunchy top virus on Banana and Plantain (Musa sp.) in Cameroon.

Banana bunchy top virus (BBTV; genus Babuvirus, family Nanoviridae) is a serious pathogen of banana (AAA genome) and plantain (AAB genome) (Musa sp.). It is transmitted by the banana aphid (Pentalonia nigronervosa) in a persistent manner (1). In recent years, BBTV has emerged as a major constraint to banana and plantain production in several countries of Africa and had been previously confirmed in viz., Burundi, Central African Republic, Republic of Congo, Democratic Republic of Congo, Egypt, Equatorial Guinea, Gabon, Malawi, and Rwanda (1) and more recently in Mozambique and Zambia (2) and Angola (3). To assess the potential threat of BBTV in West-Central Africa, we conducted surveys in August and September 2008 in 36 major banana- and plantain-producing regions of Littoral, South, Southwest, and Western Provinces of Cameroon. DNA was extracted from 520 plants and tested by PCR with primers specific for a conserved domain of BBTV DNA-R segment (4). A 240-bp DNA fragment specific to the virus was amplified in 31 samples from 18 plantain and 13 banana plants from Southwest, Western, and Southern Cameroon. Among virus-positive samples, symptoms (upright leaf growth, small leaves with pale chlorotic margins that choked the throat of the plant creating the bunchy appearance at the top) typical of bunchy top disease were observed only in banana (cv. Cavendish Williams) from Muea in the Southwest Province. PCR products obtained from the symptomatic and asymptomatic banana (Cavendish Williams) from Muea and Abang, respectively, were cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and two independent clones from each isolate were sequenced in both directions. Pairwise comparison of these sequences showed 100% sequence homology. A comparison of these sequences (Accession No. F580970) with corresponding sequences in GenBank showed 99% nt sequence identity with a BBTV isolate from Angola (Accession No. EU851977) and 96 to 98% identity with BBTV isolates belonging to the South Pacific group (Australia, Africa, South Asia, and South Pacific). However, the BBTV isolate from Cameroon showed 85 to 90% sequence identity with isolates belonging to the Asian group (China, Indonesia, Japan, Taiwan, Philippines, and Vietnam). To further confirm the virus identity, complete nucleotide sequence of the DNA-SCP segment that encodes for the virus coat protein was determined using PCR amplification of viral DNA (1), cloning of products into pCR2.1 vector, and sequencing. The derived sequence (1,075 nt; Accession No. GQ249344) in BLAST search at NCBI database revealed 98% nt sequence identity with coat protein gene of BBTV isolate from Burundi (Accession No. AF148943). These results, together with phylogenetic analysis, indicate that BBTV isolates from Cameroon have greater affinity to the South Pacific group. To our knowledge, this is the first report of BBTV in West-Central Africa. The occurrence of BBTV in the Western and Southern provinces of Cameroon, neighboring north of Gabon, suggests a possible spread of the virus from Gabon. This report also underscores the need to monitor other countries of West Africa for BBTV and enforce quarantine measures to prevent further spread through infected suckers from endemic areas of West and Central Africa. References: (1) I. Amin et al. Virus Genes 36:191, 2008. (2) W. T. Gondwe et al. InfoMusa 16:38, 2007. (3) P. L. Kumar et al. Plant Pathol. 58:402, 2009. (4) S. Mansoor et al. Mol. Biotechnol. 30:167, 2005.

First Report of Grapevine leafroll-associated virus-3 in Six Wine Grape Cultivars in Idaho.

In recent years, wine grape (Vitis vinifera) acreage in Idaho has expanded because of favorable climatic conditions for premium wine production. Nearly 95% of the 491.7 ha (1,215 acres) of wine grapes are in the Snake River Valley with Canyon County accounting for 81% of the vines. Previous studies have shown that grapevine leafroll disease (GLD) is the most widespread and economically significant virus disease in wine grapes in Washington and Oregon (1,2). However, little is known about the incidence and economic impact of GLD on wine grapes in Idaho. During the 2008 growing season, leaf samples were collected from approximately 25 individual grapevines of red-berried cultivars (Cabernet Sauvignon, Merlot, Syrah, and Petit Syrah) showing GLD symptoms and white-berried (Chardonnay) cultivars with suspected GLD symptoms growing in 10 geographically separate vineyards in Canyon County. An additional five samples were collected from a Lemberger block in Elmore County. Petiole extracts from these samples were tested by single-tube reverse transcription (RT)-PCR with primers LC 1 (5'-CGC TAG GGC TGT GGA AGT ATT-3') and LC 2 (5'-GTT GTC CCG GGT ACC AGA TAT-3') specific for the heat shock protein 70 homologue (HSP-70 gene) of Grapevine leafroll-associated virus-3 (GLRaV-3) (3). All samples, except the Petit Syrah, produced a single band of the expected size of 546 bp. ELISA with GLRaV-3-specific antibodies (BIOREBA AG, Reinach, Switzerland) confirmed the presence of the virus in samples that were positive in RT-PCR. GLRaV-3-specific amplicons were cloned in pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA) and 2 to 3 independent clones per isolate were sequenced in both orientations. A pairwise comparison of 22 sequences, six from Chardonnay (GenBank Accessions GQ344810, GQ344811, GQ344823, GQ344824, GQ344825, and GQ344826), five from Cabernet Sauvignon (GQ344807, GQ344808, GQ344809, GQ344827, and GQ344828), four each from Merlot (GQ344815, GQ344816, GQ344817, and GQ344818) and Syrah (GQ344819, GQ344820, GQ344821, and GQ344822), and three from Lemberger (GQ344812, GQ344813, and GQ344814) showed 87 to 100% identity at the nucleotide level and 92 to 100% identity at the amino acid level. A pairwise comparison of HSP-70 sequences of GLRaV-3 isolates from Idaho with corresponding sequences of GLRaV-3 isolates from GenBank showed nucleotide sequence identities between 88% (AJ748519) and 100% (DQ780885). Phylogenetic analysis of HSP-70 sequences from Idaho and GenBank showed clustering of Idaho sequences into five groups, with 12 sequences clustering with a Washington isolate (DQ780885), six sequences in a second group clustering with an isolate from Tunisia (AJ748522), two sequences in a third group clustering with an isolate from Austria (AJ748513), and one sequence each in groups four and five clustering with isolates from Italy (AJ748520) and Washington (DQ780889), respectively. The clustering was not cultivar- or vineyard-specific, suggesting separate introductions of different GLRaV-3 isolates in planting materials. To our knowledge, this is the first report of GLRaV-3 in grapevines grown in Idaho. These and previous results (1,2), indicate the wide distribution of GLRaV-3 in several grapevine cultivars in the Pacific Northwest Region. References: (1) R. R. Martin et al. Plant Dis. 89:763, 2005. (2) R. A. Naidu et al. (Abstr.) Phytopathology 96(suppl.):S83, 2006. (3) M. J. Soule et al. Plant Dis. 90:1461, 2006.

First Report of the Occurrence of African cassava mosaic virus in a Mosaic Disease of Soybean in Nigeria.

African cassava mosaic virus (ACMV; genus Begomovirus, family Geminiviridae) is one of six viruses documented in cassava (Manihot esculenta Crantz.) plants showing cassava mosaic disease in sub-Saharan Africa (SSA). In addition to cassava, the natural host range of ACMV includes a few wild Manihot species, Jatropha multifida, and Ricinus communis L. in Euphorbiaceae, and Hewittia sublobata in Convolvulaceae. The experimental host range of ACMV includes Nicotiana sp. and Datura sp. in the Solanaceae (2). Recently, natural occurrence of ACMV was reported in Combretum confertum (Benth.), Leucana leucocephala (Lam.) De Witt, and Senna occidentalis (L.) Link belonging to Leguminasae from Nigeria (1,3). During reconnaissance studies conducted on soybean (Glycine max L. Merr.) in September and October of 2007 in the Ibadan (N = 19) and Benue (N = 23) regions and in February of 2008 in Ibadan (N = 16), we observed soybean showing yellow mosaic and mottling symptoms. Samples from these plants (N = 58) were tested by indirect ELISA and symptomatic leaves tested negative to Cucumber mosaic virus, Cowpea mottle virus, Southern bean mosaic virus, Tobacco ringspot virus, Soybean dwarf virus, Cowpea aphid-borne mosaic virus, Blackeye cowpea mosaic virus, Peanut mottle virus, and Broad bean mosaic virus, which have been documented in soybean in SSA. However, 8.6% of these samples (5 of 58) (one each from Ibadan and Benue in the 2007 survey and three from Ibadan in the 2008 survey) tested positive in triple-antibody sandwich-ELISA with a monoclonal antibody (SCR33) to ACMV. ELISA results were further confirmed by PCR with ACMV specific primers AL1/F and AR0/R that amplified a 987-bp DNA fragment corresponding to the intergenic region, AC-4 and AC-1 genes of DNA-A segment (4). The PCR product was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and three independent clones were sequenced in both orientations. Pairwise comparison of the derived consensus sequence (GenBank Accession No. EU367500) with corresponding ACMV sequence of ACMV isolate from Nigeria (GenBank Accession No. X17095) showed 98% identity at the nucleotide level. To further confirm the virus identity, complete nucleotide sequence of the DNA-A segment was determined by PCR amplification of viral DNA with four primers, cloning of overlapping products into pCR2.1 vector and sequencing. The derived sequence (2,781 nucleotides; GenBank Accession No. EU685385) was compared with the DNA sequences available at NCBI database using BLAST. This revealed 97% nucleotide sequence identity with ACMV-[NG:Ogo:90] (Accession No. AJ427910) and ACMV-[NG] (Accession No. X17095) from Nigeria. These results confirm the presence of ACMV in symptomatic soybean leaves. To our knowledge, this is the first report of soybean as a natural host of ACMV in SSA. On the basis of previous reports (1) and the results currently presented it seems that ACMV has a wide host range. References: (1) O. J. Alabi et al. Phytopathology (Abstr.) 97(suppl.):S3, 2007. (2) A. A. Brunt et al., eds. Plant viruses online: Descriptions and lists from the VIDE database. Version 20. Online publication, 1996. (3) F. O. Ogbe et al. Plant Dis. 90:548, 2006; (4) X. Zhou et al. J. Gen. Virol. 78:2101, 1997.

First Report of the Occurrence of Grapevine fanleaf virus in Washington State Vineyards.

Grapevine fanleaf virus (GFLV; genus Nepovirus, family Comoviridae), responsible for fanleaf degeneration disease, is one of the most important viruses of grapevines worldwide (1). During our reconnaissance studies during 2007, dormant wood cuttings from individual grapevines of wine grape cv. Chardonnay were collected randomly from two geographically separate vineyards in eastern Washington State. Extracts made from cambial scrapings of these cuttings were tested separately for different viruses by single-tube reverse transcription (RT)-PCR using virus-specific primers. Two of the thirty-one grapevines in one vineyard tested positive for GLFV as mixed infection with Grapevine leafroll-associated virus (GLRaV)-3. In another vineyard, six of the twenty-six grapevines tested positive for GFLV as mixed infection with GLRaV-1, GLRaV-3, and Grapevine virus A (GVA) A forward primer (5'-ACCGGATTGACGTGGGTGAT, corresponding to nucleotides [nt] 2231-2250) and reverse primer (5'-CCAAAGTTGGTTTCCCAAGA, complementary to nt 2533-2552) specific to RNA-2 of GFLV-F13 isolate (GenBank Accession No. X16907) were used in RT-PCR assays for the detection of GFLV (4). Primers used for RT-PCR detection of GLRaV-1, GLRaV-2, and GVA were described in Martin et al (2) and Minafra et al (3). The RT-PCR results indicated mixed infection of GFLV with GLRaV-1, GLRaV-3, and GVA. To confirm the presence of GFLV, the 322-bp sequence representing a portion of the coat protein encoded by RNA-2 genomic segment was cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Amplicons obtained from six individual grapevines in the two vineyards were used for cloning. Three independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 99 to 100% nucleotide sequence identity among themselves, indicating that GFLV isolates from the two vineyards may be identical. A comparison of the consensus sequence (GenBank Accession No. EU573307) with corresponding sequences of other GFLVs deposited in GenBank showed 89 to 91% identity at the nucleotide level and 95 to 99% identity at the amino acid level. However, mixed infection of GFLV with different viruses in the two vineyards suggests separate introduction of the planting material. ELISA with GFLV-specific antibodies further confirmed the presence of the virus in samples that were positive in RT-PCR. To our knowledge, this is the first report of GFLV in grapevines grown in the Pacific Northwest states of the United States. Further investigations are being carried out on the distribution, symptoms, molecular variability, and nematode vector transmission of GFLV. References: (1) P. Andret-Link et al. J. Plant Pathol. 86:183, 2004. (2) R. R. Martin et al. Plant Dis. 89:763, 2005. (3) A. Minafra et al. Arch. Virol. 142:417, 1997 (4) A. Rowhani et al. Phytopathology 83:749, 1993.

First Report of Cucumber mosaic virus in Yams (Dioscorea spp.) in Ghana, Togo, and Republic of Benin in West Africa.

Yam (Dioscorea spp., family Dioscoreaceae) is one of the most important food crops cultivated in the West African yam zone comprising the forest and savannah areas of Nigeria, Ghana, Côte d'Ivoire, Republic of Benin, and Togo, which account for more than 90% of the 4.59 million ha of yam cultivation worldwide (1). A survey was conducted in 2005 to document viruses in yams in Ghana, Togo, and the Republic of Benin. Samples (1,405) from five species of yam showing mosaic, chlorosis, and stunting as well as asymptomatic plants were tested for Dioscorea bacilliform virus (DBV, genus Badnavirus), Yam mosaic virus (YMV, genus Potyvirus), and Yam mild mosaic virus (YMMV, genus Potyvirus), the three most common viruses infecting yams. In addition, samples were tested for Cucumber mosaic virus (CMV), since CMV was previously reported to infect yams in Côte d'Ivoire (2) and Nigeria (3). In protein-A sandwich-ELISA with polyclonal antibodies to a cowpea isolate of CMV, 23 of the 1,405 samples (6 of 218 samples from Togo, 13 of 628 samples from Ghana, and 4 of 559 samples from Republic of Benin) tested positive for CMV. The CMV-positive samples were from D. alata (N = 16) and D. rotundata (N = 7), whereas all samples from D. cayenensis, D. dumetorum, and D. bulbifera tested negative. CMV was detected as mixed infections with DBV, YMV, or YMMV in 21 of 23 samples. Some of these samples showed puckering, chlorosis, mottling, and crinkling, whereas some plants infected by two or more viruses were asymptomatic. Only two samples from D. rotundata had a single infection of CMV and they showed mild chlorotic symptoms in young leaves that were inconspicuous in mature leaves. In sap inoculations, the virus induced systemic mosaic in Nicotiana glutinosa. The presence of CMV in ELISA-positive yam samples was further confirmed by immunocapture-reverse transcription (IC-RT)-PCR using CMV antibodies as trapping antibody and oligonucleotide primers specific for a 485 nt corresponding to 3' end of the coat protein gene and C-terminal noncoding region of RNA-3 (4). To confirm the specificity of IC-RT-PCR, the 485-bp amplicons from an isolate from the Republic of Benin was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and three independent clones were sequenced from both orientations. Pairwise comparison of a consensus sequence (Accession No. EU274471) with corresponding sequences of other CMV isolates deposited in GenBank showed 99% identity at the nucleotide sequence level (Accession No. U22821) and revealed that the CMV isolate from yam belongs to sub-Group IA. To our knowledge, this is the first report of CMV infection in yams (D. alata and D. rotundata) in Ghana, Togo, and the Republic of Benin. Together with a previous documentation of CMV in D. alata and D. trifida in Côte d'Ivoire and Nigeria (2,3), this report adds to existing knowledge on distribution of CMV in yams with implications for yam production and germplasm distribution in the West Africa Region. References: (1) FAO. Online publication. FAOSTAT, 2007. (2) C. Fauquet and J. C. Thouvenel. Plant Viral Diseases in the Ivory Coast. ORSTROM: Documentation Techniques. Paris, 1987. (3) Jd'A. Hughes et al. Phytopathology 87:S45, 1997. (4) S. Wylie et al. Aus. J. Agric. Res. 44:41, 1993.

First Report of Grapevine leafroll-associated virus-9 in Washington State Vineyards.

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.

First Report of the Natural Infection of Coreopsis auriculata 'Nana' with Lettuce mosaic virus in the United States.

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.