J. ENTOMOL. SOC. BRIT. COLUMBIA 105, DECEMBER 2008 27 Identification of new aphid vector species of Blueberry scorch virus D. THOM AS LOW ERY1, M ICHAEL G. BERNARDY1, ROBYN M . DEYOUNG1 and CHRIS J. FRENCH1 ABSTRACT Transmission of Blueberry scorch virus (BlScV) by the aphid species Ericaphis fimbri- ata (Richards), Aphis spiraecola (Patch), Aphis pomi DeGeer, Acyrthosiphon pisum (Harris), Myzus ornatus Laing, Aphis helianthi Monell, Myzus persicae (Sulzer), and Rhopalosiphum padi (L.), was studied in the laboratory using timed aphid acquisition feeding periods and known numbers of aphid vectors. Successful infection of Nicotiana occidentalis W heeler (Solanaceae), a newly identified herbaceous host, and highbush blueberry, Vaccinium corymbosum L. (Ericaceae), following brief virus-acquisition feeds lasting less than 5 min, demonstrated that BlScV was transmitted in a non- persistent, non-circulative manner. Based on transfer of 10 aphids per plant, the most efficient vector of BlScV from infected to healthy N. occidentalis was M. ornatus. Com- pared with this herbaceous host, infection rates for blueberry were much lower even though higher numbers of aphids (25/plant) were used. The highest rate of infection for blueberry (20%) was achieved when the green colour form of E. fimbriata was used to transmit the virus. The relatively low rate of transmission from infected to healthy blue- berry suggests that BlScV would spread slowly in the field. Planting of certified virus- free nursery material and aggressive removal of infected plants should help control this economically important disease of highbush blueberries. Key W ords: Blueberry scorch virus, aphid vectors, virus transmission INTRODUCTION Blueberry scorch virus (BlScV) was sis of new leaves, twigs and flower clusters first reported in New Jersey in the late and almost complete loss of yield over time 1970's as Sheep Pen Hill disease of (Martin and Bristow 1988, Catlin and highbush blueberry, Vaccinium corymbo- Schloemann 2004, W egener et al. 2006). sum (L.) (Ericaceae) (Podleckis and Davis The latent period between infection and 1989). Several distinct strains infect development of symptoms for established highbush blueberry in the northeastern and plants is thought to be one to two years northwestern United States and southwest- (Caruso and Ramsdell 1995). ern British Columbia (Cavileer et al. 1994, There are relatively few previous studies Catlin and Schloemann 2004, Bernardy et on BlScV; these mostly relate to detection, al. 2005, W egener et al. 2006). BlScV has symptomology and strain differentiation. also been recently reported from Europe Although little is currently known about the (Ciuffo et al. 2005). Depending on the virus insect vectors of BlScV, carlaviruses as a strain and blueberry cultivar, infection can group are transmitted primarily by aphids in result in a wide range of symptoms. W hile a non-persistent, non-circulative manner some varieties are tolerant to certain strains (Ng and Perry 2004). Non-persistent virus and display no visible symptoms, infection transmission is characterized by short ac- with other strains can result in severe necro- quisition and inoculation feeding times, 1 Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, 4200 Hwy 97, Box 5000, Sum- merland, British Columbia, Canada V0H 1Z0 28 J. ENTOMOL. SOC. BRIT. COLUMBIA 105, DECEMBER 2008 lasting from several seconds to a few min- studies were not designed to determine if utes in duration (Raccah 1986). In uncon- BlScV was transmitted by aphids in a semi- trolled cage studies, Hillman et al. (1995) persistent or non-persistent manner. Two were the first to demonstrate aphid trans- carlaviruses vectored by aphids are thought mission of BlScV. An unidentified aphid to be transmitted in a semi-persistent man- collected from blueberry and placed on ner (Bristow et al. 2000). infected Chenopodium quinoa W illd. A better understanding of BlScV epide- (Chenopodiaceae), an alternate herbaceous miology will aid in the development of ef- host for the New Jersey strain of BlScV, fective control measures. To this end, the was shown to transmit the virus to unin- purpose of our study was to determine the fected C. quinoa. In a similar manner, Bris- mode of transmission of BlScV and com- tow et al. (2000) were able to demonstrate pare aphid transmission efficiencies of E. infection of containerized highbush blue- fimbriata, a species that colonizes blue- berry plants in cages supplied with diseased berry, with transmission by several non- blueberry leaves infested with Ericaphis colonizing aphid species. Identification of fimbriata (Richards). In the same study, effective aphid vectors will also assist in transfer of individual aphids from infected future laboratory investigations to deter- blueberry leaves to containerized potted test mine biological differences between the plants resulted in a very low rate of infec- various strains of BlScV. tion, less than one percent. These previous M ATERIALS AND M ETHODS Plant and aphid culture. Large Oregon) and grown in the greenhouse in highbush blueberry plants from two com- 3.8-litre plastic pots under the same condi- mercial fields near Abbotsford, British Co- tions. lumbia (BC), that had previously tested Aphids were maintained in vented, positive for BlScV by ELISA using poly- Plexiglas ® cages (50 cm x 50 cm x 33 cm clonal antibodies (Agdia, Elkhart, Indiana) wide) in a growth room (18 EC, 16-h photo- were potted into large (~ 60 cm x 43 cm phase) on suitable host plants as follows: deep) plastic pots and moved to a green- red and green forms of E. fimbriata on house at the Pacific Agri-Food Research strawberry, Fragaria x ananassa Duchesne Centre, Summerland, BC. These plants also (Rosaceae); spirea aphid, Aphis spiraecola formed the basis for the isolation and mo- (Patch) and apple aphid, A. pomi DeGeer, lecular characterization of two major strains on apple, Malus domestica L. (Rosaceae); of BlScV (Bernardy et al. 2005). pea aphid, Acyrthosiphon pisum (Harris), Nicotiana occidentalis W heeler, re- on garden pea, Pisum sativum L. cently identified as a herbaceous host for (Fabaceae); violet aphid, Myzus ornatus BlScV (Lowery et al. 2005), was grown in Laing, and Aphis helianthi Monell on sun- the greenhouse in 20-cm plastic containers flower, Helianthus annuus L. (Asteraceae); in a 1:1:5 mixture of steam-sterilized field green peach aphid, Myzus persicae (Sulzer), soil, perlite, and commercial potting soil on bok-choi, Brassica rapa L. (Pro-Mix BX, Premier Horticulture Ltd., (Brassicaceae); and the bird cherry-oat Dorval, Quebec). Temperatures were vari- aphid, Rhopalosiphum padi (L.), on barley, able and ranged from daytime highs of 25 E Hordeum vulgare L. (Poaceae). Host plants C to nighttime lows of 15 EC, with supple- were reared in the greenhouse under condi- mental lighting supplied by sodium vapour tions outlined above. lamps to provide a 16-h photophase. Plants Except for E. fimbriata that were origi- were used at the four- or five-true-leaf nally collected from commercial fields of stage. Small BlScV-free blueberry plants cv highbush blueberry in the Fraser Valley and ”Berkeley‘ were acquired from a commer- provided by Dr. D.A. Raworth (Agriculture cial supplier (Fall Creek Nurseries, Lowell, and Agri-Food Canada, Pacific Agri-Food J. ENTOMOL. SOC. BRIT. COLUMBIA 105, DECEMBER 2008 29 Research Centre, Agassiz, BC), all of the Clark and Adams (1977). All reagents were aphid species used in these studies, other added at 100 Fl per well in microtitre than A. pisum, were collected in Summer- plates. Microtitre plates (EIA Microplate, land, BC, from the hosts on which they ICN Biomedicals, Irvine, California) were were reared. Acyrthosiphon pisum was col- coated with purified immunoglobulin (IgG) lected from garden peas in Armstrong, BC. (Agdia, Elkhart, Indiana) diluted (5 Fl ml-1) Aphids were identified by Dr. R.G. Foottit in phosphate-buffered saline (PBS) for 4 h (Agriculture and Agri-Food Canada, East- at 37 EC. Plates were washed three times ern Cereals and Oilseeds Research Centre, with PBS. Plant samples (0.25 g) were thor- Ottawa, Ontario). oughly ground in Bioreba bags (Bioreba Aphid transmission studies. Fourth AG, Reinach, Switzerland) with 1.5 ml instar and adult apterous aphids from the borate buffer (0.1 M boric acid, 0.01 M laboratory colonies were placed in small sodium borate, 2% polyvinylpyrolidine self-sealing petri dishes containing mois- (PVP 44,000), 0.2% non-fat milk powder, tened filter paper for a 2- to 3-h pre- 0.05% Tween-20, 0.5% nicotine), and the acquisition starvation period. Aphids were bags briefly centrifuged at 2000 rpm to aid allowed to feed for 5 min on BlScV- pipetting. The liquid extract (25 Fl) and infected leaf pieces in groups of 10 aphids/ borate buffer (75 Fl) were added to the mi- petri dish, and then transferred, 25 aphids/ crotitre plates, which were covered in cello- plant for blueberry and 10 aphids/plant for phane and placed overnight on an orbital N. occidentalis, to BlScV-free test plants, shaker at 600 rpm. After washing the plates which were then sealed in plastic bags to with PBS-Tween and adding a dilute (5 Fl prevent the aphids from escaping. Fine, ml-1) IgG-enzyme conjugate in PBS- moistened natural fibre brushes were used Tween-BSA-polyvinylpyrolidine, plates to transfer aphids. At least 1 h after the final were incubated at 37 EC for 2 h. After transfer, plants were sprayed with the aphi- plates were washed with buffer, a dilute cide pirimicarb (Pirimor 50W P, Chipman (0.5 mg ml-1) solution of p-nitrophenyl Chemicals Ltd., Stoney Creek, Ontario) to phosphate buffer was added. Plates were kill any remaining aphids. Plants were held incubated at room temperature on an orbital in the bags for a further 24 h to ensure that shaker (600 rpm) for about 1 hr and absorb- all aphids were dead.