First Report of Blackcurrant Reversion Virus in Ribes nigrum Germplasm in the United States

Abstract

Reversion disease, thought to be caused by the eriophyid mite transmitted blackcurrant reversion virus (BRV), is the most economically important disease of black currants (Ribes nigrum L.) and can be found nearly everywhere blackcurrants are cultivated except North America and Australia (Jones and McGavin 2002; Lemmetty et al. 1997; Susi 2004). BRV causes morphological changes to leaves and flowers, reduced flower bud hairiness, decreased plant vigor, sterility, and increased flower pigmentation (Jones and McGavin 2002; Susi 2004). Its host range is limited to the genus Ribes, and the virus rarely infects species other than R. nigrum. National quarantines have been used to prevent the movement of BRV and its vector, Cecidophyopsis ribis Westwood, into the U.S. However, BRV may have been introduced prior to the development of a reliable detection method (Jones and McGavin 2002). The R. nigrum cultivar Burga (PI 653058, from France) has abnormal leaf morphology and was evaluated using next generation sequencing (NGS) during the summer of 2016. During a preliminary analysis, a significant portion of the BRV genome was detected, warranting further investigation. In November 2016, RNA was extracted from leaves of ‘Burga’ and 11 other NCGR black currant accessions, several of which also exhibited suspicious leaf symptoms. RNA was extracted from two symptomatic leaves of each accession and bulked. These samples were assayed in triplicate by quantitative reverse transcription PCR (qRT-PCR) in Oregon for BRV using the iTaq One-Step SYBR green RT-PCR Kit (Bio-Rad Laboratories Inc, Hercules, CA). The primer pairs BRV1-8F/BRV1-8R, BRV1-10F/BRV1-10R, and BR1-F/BR3-R previously developed from BRV RNA-1 were used in conjunction with the recommended annealing conditions (Dolan et al. 2011; Jones and McGavin 2002). Four cultivars tested positive for BRV at the USDA-ARS NCGR: Bogatyr (PI 556413, from Germany); Burga; Minaj Shmyrev (PI 617766, from England); and an R. nigrum selection (PI 617781, from France). Amplicons generated from BR1-F/BR3-R from Bogatyr and Burga were sequenced (GenBank: MH078528-9) and showed greater than 96% identity to published BRV sequences when using BLASTx (ABL84271.1, ABL84276.1, AAO52686.1, AAO52685.1, ABL84275.1, AAO60043.1, AAO52687.1, NP_733982.2, AAO52684.1, and NP_612586.1). In summer 2018, total RNAs were extracted and sequenced again as described by Zheng et al. (2018) on an Illumina NextSeq 500 system. A total of 19,002,868 reads were generated and a de novo assembly was conducted with CLC genomics workbench. A BLASTx search was conducted using the contigs. Four contigs ranging from 178 to 335 bp assembled from 1,066 reads were found to match with the BRV genome with greater than 88% identity. Three of these contigs mapped to BRV RNA1 (MK302449) and one mapped to RNA2 (MK302450). The contigs account for 7% of the BRV genome. The vector, C. ribis, is not known to be present in North America and it is unknown if any mites endemic to North America could serve as vectors. Three of the infected clones have been growing at the NCGR for more than 20 years. These clones had been tested by a graft-bioassay when they entered the country and were thought to be BRV-free. At the time, this graft-bioassay was the only tool available for BRV detection, but it is not sufficient to enforce a BRV quarantine due to low virus concentrations in planta. Germplasm from the NCGR Ribes collection has been distributed to nurseries and research programs across North America as part of the National Plant Germplasm System. To ensure there has not been any spread and that other black currant clones in the NCGR collection are free of BRV, all 200 black currant clones in the collection will be tested for BRV. Distribution records of the four infected black currant accessions were used to identify where these clones have been shipped and the recipients of these materials have been notified.