Genomic and Biological Characterization of Blackberry Calico Virus

Over 18,000 metric tons of blackberries are produced in the United States, predominantly in Oregon. Blackberry calico disease (BCD) has been reported in many commercial cultivars including Marionberry, which accounts for over half the Oregon blackberry production. BCD has been observed in a wide range of Rubus spp. hosts: in blackberry-raspberry hybrids, California blackberry (Rubus ursinus Cham. & Schltdl.), and in the naturalized variety Himalaya (R. armeniacus Focke), among others. Blackberry plants with BCD symptoms were screened for viruses by high-throughput sequencing, and nine genomic ssRNA virus sequences, ca. 8 kb, were obtained. BLAST analysis revealed that these sequences share 75% identity with 49% query coverage at the nucleotide level with blackberry virus E, genus Allexivirus, family Alphaflexiviridae. Phylogenetic analysis also grouped the newly sequenced blackberry virus within the allexiviruses. RT-PCR- and RT-qPCR-based detection methods were developed for routine detection of the virus targeting the replicase and coat protein regions of the genome, respectively. Graft bioassay analysis was done to observe the symptom development and transmission of the virus in blackberry, black raspberry, and red raspberry. The tentatively named blackberry calico virus (BCV) is strongly associated with BCD and transmitted through grafting, fulfilling Koch’s postulates under controlled conditions, although symptom expression can be inconsistent and delayed.