Epidemiology and Genetic Diversity of Grapevine Leafroll-Associated Viruses in British Columbia.

Grapevine leafroll disease (GLD) is a complex associated with one or more virus species belonging to the family Closteroviridae. The majority of viruses in this complex are vectored by one or more species of mealybugs (Pseudococcidae) and/or scale insects (Coccidae). Grape-growing regions of British Columbia (BC), including Okanagan, Similkameen, and Fraser valleys and Kamloops (BC central interior), Vancouver, and Gulf islands, were surveyed during the 2014 and 2015 growing seasons for the presence of four major grapevine leafroll-associated viruses, including Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, and GLRaV-4. In total, 3,056 composite five-vine samples were collected from 153 Vitis vinifera and three interspecific hybrid vineyard blocks. The results showed GLRaV-3 to be the most widespread, occurring in 16.7% of the composite samples, followed by GLRaV-4 (3.9%), GLRaV-1 (3.8%), and GLRaV-2 (3.0%). Mixed infections of two or more GLRaVs were found in 4.1% of the total samples. The relative incidence of GLRaVs differed among regions and vineyard blocks of a different age. Characterization of partial CO1 region from a total of 241 insect specimens revealed the presence of Pseudococcus maritimus, Parthenolecanium corni, and other Pulvinaria sp. in BC vineyards. Spatial patterns of GLRaV-3 infected grapevines in three vineyard blocks from three different regions in the Okanagan Valley showed variable degrees of increase in disease spread ranging from 0 to 19.4% over three growing seasons. Regional differences in the relative incidence and spread of GLD underline the need for region-based management programs for BC vineyards.

Host plant suitability and feeding preferences of the grapevine pest Abagrotis orbis (Lepidoptera: Noctuidae).

Thirteen plant species were tested for their suitability as hosts for Abagrotis orbis (Grote), a climbing cutworm pest of grapevines in British Columbia. Choice tests were also conducted to investigate larval feeding preferences for the Brassicaceae species joi choi, Brassica rapa variety. Chinensis L., spring draba; Draba verna L.; and shepherd's purse, Capsella bursa-pastoris (L.) Medik; compared with postdormant buds of grape, Vitis vinifera L. (Vitaceae), and leaves of nine other plant species from several families. Results showed that tah tsai, Brassica rapa L. variety rosularis (M. Tsen & S. H. Lee) Hanelt (Brassicaceae), is a superior host for A. orbis based on shorter time to adult eclosion, heavier pupae, and higher rates of survival. Later-instar larvae died when fed draba, whereas those reared on shepherd's purse did not survive beyond the third instar. White clover, Trifolium repens L. (Fabaceae), and grape leaves were unsuitable hosts throughout development. Fifth-instar A. orbis preferred plants of the Brassicaceae family, dandelion, Taraxacum officinale Weber (Asteraceae), and strawberry, Fragaria sp. L. (Rosaceae), compared with postdormant grape buds. The results of this study suggest that the winter annual mustards draba and shepherd's purse that often grow abundantly in vine rows might help reduce climbing cutworm damage to the buds of grapevines.

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.

Nicotiana occidentalis: A New Herbaceous Host for Blueberry scorch virus.

Blueberry scorch virus(BlScV) is a carlavirus that causes a serious disease of blueberries (Vaccinium corymbosum L.) in North America (2). In aphid-transmission studies of BlScV using blueberry as host and test species, we found the rate of transmission to be low, and a lengthy incubation period was required before BlScV could be detected. For sequencing studies, RNA extraction from blueberry using standard methods was unreliable and inefficient. These problems prompted a search for alternate hosts. Of 12 herbaceous hosts screened for BlScV transmission using the blueberry aphid, Ericaphis fimbriata Richards, with mechanical transmission, only Nicotiana occidentalis (Wheeler) became infected. After 3 to 4 weeks, infection of N. occidentalis with BlScV resulted in mild symptoms that included pronounced leaf twisting and swollen leaf veins. Infection with BlScV was confirmed using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) kit (Agdia Inc., Elkhart, IN), polyclonal antibodies to BlScV from the antiserum collection at the Pacific Agri-Food Research Center, Summerland, British Columbia, Canada, and reverse transcription-polymerase chain reaction (RT-PCR). Forward (5'-NTAAACACTCCCGAATATAC-3') and reverse (5'-CAGATTGCTTATCCGGCTTC-3') primers were designed with the published sequence of BlScV isolate NJ-02 (GenBank Accession No. NC003499). An amplicon of the expected size was generated and sequenced. BLAST analysis indicated that the nucleotide sequence of the amplified fragment was 87% identical to the corresponding sequence in NJ-02 (1). N. occidentalis was readily infected with BlScV following aphid or mechanical inoculations from blueberry. With E. fimbriata as the aphid vector, the transmission rate from blueberry to N. occidentalis was approximately 26%, compared with 70% for mechanical inoculations. Mechanical transfer of BlScV between infected N. occidentalisplants resulted in a 100% transmission rate. Recently, with N. occidentalis, we have completely sequenced two strains of BlScV from British Columbia, Canada and identified several aphid vector species. The identification of N. occidentalis as an herbaceous host of BlScV greatly facilitates future studies on the virus. References: (1) T. D. Cavileer et al. J. Gen. Virol. 75: 711, 1994. (2) R. R. Martin and P. R. Bristow. Phytopathology 78:1636, 1988.

Antifeedant activity of extracts from neem,Azadirachta indica, to strawberry aphid,Chaetosiphon fragaefolii.

Leaf disk choice test bioassays demonstrated that formulated neem seed oil (NSO) was equally deterrent to first- and third-instar nymphs and adult strawberry aphids,Chaetosiphon fragaefolii (Cockerell). Concentrations of NSO resulting in 50% feeding deterrence were approximately 1.1% for this species. The rapid disruption of aphid feeding (<1 hr) was not related to the presence of the limonoid azadirachtin, and deterrence likely results from the combined activity of several compounds. Activity toC. fragaefolii disappeared within 12-24 hr following application to strawberry in the greenhouse. NSO was deterrent to only half of the six aphid species tested. The antifeedant properties of neem do not appear to contribute significantly to the control of aphids and the viruses they transmit.