New Variant of Little cherry virus Associated with Little Cherry Disease of Sweet Cherry in British Columbia, Canada.

Little cherry disease (LChD) occurs in most cherry growing areas in the world. Infection of sensitive cultivars results in small fruit with poor color, angular shape, and insipid flavor. Three viruses associated with LCD have been described: (i) Little cherry virus-1 (LChV-1) first found and described in Germany (4); (ii) LChV-2 an isolate obtained from the United States (2); and (iii) LChV-3 first found and described in British Columbia (1). Despite similarities in symptom development in orchard trees and woody indexing, the three viruses have distinct molecular sequences (3). LChV-2 and -3 share greater homology with each other than either does with LChV-1. For many years, the British Columbia Ministry of Agriculture, Fisheries and Food (BCMAFF) in conjunction with Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre (AAFC-PARC) has conducted a survey to monitor the incidence and spread of LChD in the Okanagan and Kootenay valleys of British Columbia, Canada. Until recently, testing for LChD used woody indexing on indicator trees, Prunus avium cv. Lambert (fruit symptoms) and cvs. Canindex1 and Sam (foliar symptoms). Recently, incidence of LChD has been evaluated using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) developed at AAFC-PARC (3), and reverse transcription-polymerase chain reaction (RT-PCR) tests based on sequence data from LChV-3 (1). During the 1999 survey, orchard trees displaying symptoms typical of LChD tested negative for LChV-3 using ELISA and RT-PCR. Also, trees that formerly tested positive for LChD by woody indexing also tested negative for LChV-3 using RT-PCR and ELISA. Two hundred ninety-three trees were subsequently tested for LChV-1 by RT-PCR using the primer set LCV3EC/LCV16659 (4). A 276-bp fragment corresponding to the extreme 3' untranslated region (3' UTR) of the LChV-1 genome was amplified by RT-PCR from 140 of the trees. The RT-PCR amplicon from one sample (#99-68B from Peachland) was sequenced and using a BLASTn search, LChV-1 was identified as the most probable match (E value 4e-59). Sequence alignment using ClustalX identified two regions of high sequence homology; bases 2 to 70 (92%) and bases 99 to 239 (95%). The intervening region displayed much lower homology (61%). The overall homology of the amplicon was 88% compared to the corresponding region in LChV-1. Divergence between the published sequence of LChV-1 and the sequence of the new LChV isolate (tentatively named LChV-4) was investigated. Seven sets of primers constructed on the basis of sequence data from various regions of the LChV-1 genome (two sets from each of the RNA-dependent RNA polymerase, heat shock 70 protein homologue, and coat protein) failed to yield RT-PCR products when tested with the LChV-4 isolate. LChV-4 is clearly related to LChV-1 within the 3'-UTR but complete sequencing is required to determine the overall relationship with other viruses causing LChD. The discovery of a new isolate of LChV in British Columbia may require a reevaluation of the epidemiology of LChD and disease management strategy. References: (1) K. C. Eastwell and M. G. Bernardy. Phytopathology 91:268, 2001. (2) M. E. Rott, and W. Jelkmann. Phytopathology 91:261, 2001. (3) J. Theilmann et al. Phytopathology 92:87, 2002. (4) M. Vitushkina et al. Eur. J. Plant Pathol. 103;803, 1997.