Highly divergent isolates of chrysanthemum virus B and chrysanthemum virus R infecting chrysanthemum in Russia

Background: Chrysanthemum is a popular ornamental and medicinal plant that suffers from many viruses and viroids. Among them, chrysanthemum virus B (CVB, genus Carlavirus, family Betaflexiviridae) is widespread in all chrysanthemum-growing regions. Another carlavirus, chrysanthemum virus R (CVR), has been recently discovered in China. Information about chrysanthemum viruses in Russia is very scarce. The objective of this work was to study the prevalence and genetic diversity of CVB and CVR in Russia. Methods: We surveyed the chrysanthemum (Chrysanthemum morifolium Ramat.) germplasm collection in the Nikita Botanical Gardens, Yalta, Russia. To detect CVB and CVR, we used RT-PCR with virus-specific primers. To reveal the complete genome sequences of CVB and CVR isolates, metatransciptomic analysis of the cultivars Ribonette, Fiji Yellow, and Golden Standard plants, naturally co-infected with CVB and CVR, was performed using Illumina high-throughput sequencing. The recombination detection tool (RDP4) was employed to search for recombination in assembled genomes. Results: A total of 90 plants of 23 local and introduced chrysanthemum cultivars were surveyed. From these, 58 and 43% plants tested positive for CVB and CVR, respectively. RNA-Seq analysis confirmed the presence of CVB and CVR, and revealed tomato aspermy virus in each of the three transcriptomes. Six near complete genomes of CVB and CVR were assembled from the RNA-Seq reads. The CVR isolate X21 from the cultivar Golden Standard was 92% identical to the Chinese isolate BJ. In contrast, genomes of the CVR isolates X6 and X13 (from the cultivars Ribonette and Fiji Yellow, respectively), were only 76% to 77% identical to the X21 and BJ, and shared 95% identity to one another and appear to represent a divergent group of the CVR. Two distantly related CVB isolates, GS1 and GS2, were found in a plant of the cultivar Golden Standard. Their genomes shared from 82% to 87% identity to each other and the CVB genome from the cultivar Fiji Yellow (isolate FY), as well as to CVB isolates from Japan and China. A recombination event of 3,720 nucleotides long was predicted in the replicase gene of the FY genome. It was supported by seven algorithms implemented in RDP4 with statistically significant P-values. The inferred major parent was the Indian isolate Uttar Pradesh (AM765837), and minor parent was unknown. Conclusion: We found a wide distribution of CVB and CVR in the chrysanthemum germplasm collection of the Nikita Botanical Gardens, which is the largest in Russia. Six near complete genomes of CVR and CVB isolates from Russia were assembled and characterized for the first time. This is the first report of CVR in Russia and outside of China thus expanding the information on the geographical distribution of the virus. Highly divergent CVB and CVR isolates have been identified that contributes the better understanding the genetic diversity of these viruses.