First Report of Aster Yellow Phytoplasmas ('Candidatus Phytoplasma asteris') in Canadian Grapevines.

In North America, elm yellows, aster yellows (AY), and X-disease phytoplasmas have been detected in American grapevines (1), and recently, Bois noir was detected in Canadian vineyards from British Columbia (BC) and Ontario (ON) (2). Typical symptoms of grapevine yellows (GY) include leaf rolling and chlorosis, uneven or total lack of lignification of canes, flower abortion or berry withering, and stunting. In 2006 and 2007, independent surveys were conducted by the Canadian Food Inspection Agency (CFIA) and Agriculture and Agri-Food Canada (AAFC) to detect phytoplasmas in Canadian vineyards containing different cultivars in BC, ON, Québec (QC), Nova Scotia, New Brunswick, and Prince Edward Island. The CFIA collected and tested 651 fresh leaf samples from recently imported grapevines and older grapevines in the same or neighboring blocks displaying symptoms typical of those associated with disease caused by phytoplasmas. Many vineyards were surveyed only once. AAFC collected and tested 3,485 samples from symptomatic and asymptomatic grapevines from established vineyards in ON, BC, and QC. The same vineyards were sampled in ON and BC both years; QC vineyards were only sampled in 2007. AAFC-collected leaf samples were freeze dried and stored at -20°C before processing. CFIA samples were tested by a modified real-time PCR assay and TaqMan probe targeting the 16S ribosomal RNA gene that detects a wide range of known phytoplasmas (2). Positive samples were confirmed by conventional PCR using the phytoplasma-specific primers P1/P7 (3) and the resulting ~1,800-bp fragment was cloned and sequenced as previously described (2). DNA extracted by AAFC was amplified by nested PCR technology with universal phytoplasma specific primer pairs P1/P6 and R16R2/R16F2 (3) and the resulting 1,200-bp fragment was cloned and sequenced. Two plants, one located in ON in 2006 and the other in BC in 2007, were found to be infected with an AY-like phytoplasma by the CFIA. The phytoplasmas detected in both infected plants had a 99.9% nt sequence identity with AY phytoplasma sequences from GenBank (Accession Nos. AF222063 and AY665676, respectively), with the BC isolate also showing 100% identity to a strain of AY, ash witches'-broom phytoplasma (GenBank Accession No. AY566302). AAFC detected phytoplasma DNA in both years in a total of 17 symptomatic plants and 21 asymptomatic plants from different vine varieties in ON, BC, and QC. Positive samples were found to have a 99.0% nt sequence identity to AY subgroup 16SrI-A (GenBank Accession No. AY180956). Sequences were exchanged for confirmation of phytoplasma identity and were deposited in Genbank under Accession Nos. FJ659844 and FJ824597. Phytoplasma strains were identified for all plants in which phytoplasmas were detected. Results show that AY is present in vineyards in the provinces of ON, BC, and QC. To our knowledge, this is the first report of AY being detected in grapevines in Canada. References: (1) E. Boudon-Padieu. Bull. O I V, 79:299, 2003. (2) M. Rott et al. Plant Dis. 91:1682, 2007. (3) E. Tanne et al. Phytopathology 91:741, 2001.

Distribution of Plum pox virus in Residential Sites, Commercial Nurseries, and Native Plant Species in the Niagara Region, Ontario, Canada.

Extensive surveys of native weed populations in peach orchards heavily infected with Plum pox virus strain D (PPV-D) in the Niagara Region quarantine area, Ontario, Canada, failed to identify natural infection in any of the species examined. Surveys of rural and urban residential properties within areas of high PPV incidence did not detect widespread infection of susceptible hosts, with infected Prunus glandulosa (dwarf flowering almond) being found only at one site. The prominent color-breaking observed in blossoms of PPV-infected P. glandulosa would make this an excellent sentinel species for early detection of virus in Prunus orchards. Surveys of susceptible ornamental Prunus spp. in Niagara nurseries failed to demonstrate PPV infection in any of the nursery field plantings.

First Report of Onion Yellow Dwarf Virus in Ontario.

Onion yellow dwarf virus (OYDV) (1) was identified in a commercial planting of garlic (Allium sativum L.) near Delhi, Ontario, in 1998. Infected plants exhibited mild mosaic symptoms that became less noticeable by mid-July. Many plants were co-infected with garlic latent virus (GLV) (4), which was mechanically transmitted to leek (A. porrum L.). Since leek is not susceptible to OYDV (3), it is an appropriate host for the differentiation of GLV and OYDV. OYDV was transmitted nonpersistently by Myzus persicae (Schultz) from garlic to garlic and onion (A. cepa L.). Infected onion exhibited yellow striping, leaf curling, and pronounced stunting. Necrotic lesions were not present on inoculated leaves of Chenopodium amaranticolor Coste & Reyn. and C. quinoa Willd., which are often associated with co-infection with leek yellow dwarf virus (2). The isolate reacted strongly in enzyme-linked immunosorbent assay with OYDV antisera (from M. Fukami, Chiba Prefectural Agric. Exp. Stn., Chiba 266, Japan; D. Z. Maat, DLO Research Instit., Plant Prot., Wageningen, The Netherlands). Electron microscopic observations of negatively stained preparations of infected leaf tissues revealed virus particles averaging 765 ± 45 nm with typical pinwheel inclusions. References: (1) L. Bos. CMI/AAB Descrip. Plant Viruses no. 158, 1976. (2) K. Graichen. Nachrichtenbl. Pflanzenschutz DDR 32: 245, 1978. (3) K. Graichen and H. U. Leistner. Arch. Phytopathol. Pflanzenschutz 23:165, 1987. (4) L. W. Stobbs et al. Plant Dis. 80:343, 1996.

Occurrence of Turnip Yellow Mosaic Virus on Oriental Cruciferous Vegetables in Southern Ontario, Canada.

Turnip yellow mosaic virus (TYMV) has been reported throughout Europe, New Zealand, and Australia. In 1994, this virus was identified in two field plantings of Bok Choi and one planting of Pak Choi (Brassica campestris Chinensis group var. communis) in Durham and Haldimand-Norfolk counties, respectively. In early October, approximately 25% of the plants were infected at each site. Both the striped flea beetle (Phyllotreta striolata (F.)) and the crucifer flea beetle (P. Cruciferae(Goeze)), reported vectors of the virus (1), were present at each site. Infected plants exhibited bright yellow to yellow-green mosaic mottling and often showed chlorotic lesions on the lower leaves. Vein clearing was also seen on several plants. Plants were often coinfected with turnip mosaic virus. Four symptomatic plants were taken from each field site for testing. Spherical virus particles (28 nm) were identified as TYMV by electron microscopy following post-antibody decoration and enzyme-linked immunosorbent assay with the TYMV Agdia test kit. Symptoms were reproduced on both Bok and Pak Choi by mechanical inoculation into healthy plants. Extended host range susceptibility tests with 14 differential hosts were consistent with those reported in the VIDE database (1). This virus, in the presence of the flea beetle vectors, may pose a threat to susceptible traditional cruciferous vegetables grown extensively in this area. Reference: (1) A. A Brunt et al., eds. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 16th January 1997.

Construction of a simple, inexpensive multiple enzyme-linked immunosorbent assay microdilution plate washer.

In this paper, plans are given for the construction of an inexpensive enzyme-linked immunosorbent assay plate washer from readily available materials. The wash unit uses an intermittent wash cycle based on a wash manifold cycling over the microdilution plates for a predetermined time. Laboratory tests showed that the unit provided reliable, rapid washing of plates with tap water, with no detectable contamination between wells. Substrate absorbance values for test samples from machine-washed plates were equal to or greater than absorbance values for corresponding samples from plates washed manually by an accepted protocol, by using either enzyme-linked immunosorbent assay wash buffer or tap water.