First report of fig mosaic virus on fig in Russia

Fig mosaic virus (FMV) (genus Emaravirus in the family Fimoviridae) is considered the etiological agent of fig mosaic disease (FMD) that is recorded in most of the fig growing areas with an average global infection rate of 33%. The multipartite FMV genome is comprised of six negative monocistronic ssRNAs, each of which is separately encapsidated (Preising et al. 2020). Although FMD-like symptoms, which include mosaic, chlorotic ringspots, and oak leaf patterns, were observed in approximately a third of 400 fig accessions in the Nikita Botanical Gardens, Yalta, Russia (Mitrofanova et al. 2016), FMV has not been identified as the causal agent of the disease. In June of 2020, total RNA was isolated from symptomatic leaves of 59 thirty two-year-old trees representing 31 local and 27 introduced Ficus carica L. cultivars and a single F. pseudocarica Miq. tree using RNeasy Plant Mini kit (Qiagen, USA). FMV was tested by RT-PCR using primer sets E5 (Elbeaino et al. 2009) and EMARAVGP (Walia et al. 2009), which amplify a 302-bp fragment of RNA1 and a 468-bp fragment of RNA2, respectively. PCR products of the expected sizes were generated in all samples, indicating a high FMV incidence in the plantings. The genome sequences of FMV isolates from F. carica cvs. Bleuet, Kraps di Hersh, Smena, Temri, and F. pseudocarica (Fig. S1) were determined by high-throughput sequencing on MiSec Illumina platform. Double-stranded RNA was isolated from FMV-positive leaves using Viral Gene-spin™ Viral DNA/RNA Extraction Kit (iNtRON, Korea), followed by cDNA library preparation with the NEBNext® Ultra™ II RNA Library Prep Kit (New England Biolabs, USA). In average, 695,000 quality-filtered 150 bp pair-ended reads per a library were produced and used in a de novo assembly using metaSpades program version 3.14 (Nurk et al. 2017). In each of five samples, BLASTn analysis found six FMV-related contigs. The contigs spanned 99 to 100% of corresponding genomic segments of the most closely related isolates. In addition to FMV, fig cryptic virus-related contigs were also detected in some samples. The FMV contigs covering RNA1 to RNA6 had the highest identity to corresponding genomic segments of isolates AM941711 (96.5 to 96.6%), FM864225 (94.4 to 94.6%), FM991954 (97.9 to 98.2%), AB697863 (96.4 to 96.6%), AB697879 (93.3 to 93.4%), and AB697895 (95.4 to 97.0%), respectively. Five Russian isolates shared 99.2 to 100% nucleotide sequence identity, depending on the genomic segment. Their sequences were deposited in GenBank under accession numbers MW201216 to MW201230 and MW208662 to MW208676. Phylogenetic analysis of six ORFs showed that ORF1 to ORF3 and ORF6 of the Russian isolates clustered with FMV isolates from Italy while ORF4 grouped with the isolate JTT-Pa (AB697863) from Japan (Fig. S2). ORF5 of the Russian isolates formed a separate cluster with the isolates SB1 and SB2 from Serbia and JTT-Vi from Japan (AB697879 to AB697884). Incongruency of phylogenetic relationship among the genomic segments suggests reassortment among ancestors of the Russian FMV isolates. In addition, similar to the SB1, SB2 and JTT-Vi, ORF5 of the Russian isolates encodes a protein of 486 amino acid (aa) residues in contrast to the corresponding protein of Italian isolates consisting of 502 aa. To the best of our knowledge, this is the first report of FMV in Russia. This finding not only expands the information on the geographical distribution of FMV, but also extends knowledge on F. pseudocarica as a natural host of the virus.

High-Throughput Sequencing Reveals Cyclamen persicum Mill. as a Natural Host for Fig Mosaic Virus

In a search for viral infections, double-stranded RNA (dsRNA) were recovered from a diseased cyclamen (Cyclamen persicum Mill.) accession (Cic) and analyzed by high-throughput sequencing (HTS) technology. Analysis of the HTS data showed the presence of Fig mosaic emaravirus (FMV) in this accession. The complete sequences of six FMV-Cic RNA genomic segments were determined from the HTS data and using Sanger sequencing. All FMV-Cic RNA segments are similar in size to those of FMV from fig (FMV-Gr10), with the exception of RNA-6 that is one nucleotide longer. The occurrence of FMV in cyclamen was investigated through a small-scale survey, from which four plants (out of 18 tested) were found RT-PCR positive. To study sequence variations of cyclamen isolates of FMV, RT-PCR products generated through the amplification of the partially RNA-dependent RNA polymerase (RdRp, RNA-1), glycoprotein (GP, RNA-2), and nucleocapsid (NCP, RNA-3) genes were explored. The nucleotide sequence identities for cyclamen isolates ranged between 86% and 99% in RNA-1, 93% and 99% in RNA-2, and 98% and 99% in RNA-3, while lower identity levels were observed with the sequences of fig isolates. Based on the phylogenetic tree obtained with a 304-nt fragment of RNA3, all FMV isolates from cyclamens were assigned to a single cluster close to fig isolates from the Mediterranean. FMV was graft-transmitted to healthy cyclamens eliciting symptoms similar to those observed in the Cic accession, thus suggesting a causal role of FMV in the symptoms that prompted the investigation. This is the first report of FMV in a non-fig host, Cyclamen persicum, a finding that may help in the control of the mosaic and mosaic-like diseases of fig and cyclamen, respectively.

Prevalence and phylogenetic analysis of Fig mosaic virus and Fig badnavirus-1 in Iran

Fig mosaic virus (FMV) and Fig badnavirus-1 (FBV-1) are two of the most important fig infecting viruses. The incidence and distribution of FBV-1 and FMV were determined by testing in PCR 138 asymptomatic and symptomatic samples. These samples were collected from 60 fig gardens and agricultural fields in three provinces of Iran. The fig infecting viruses FBV-1 and FMV, respectively, were detected in 92 (66.6%) and 34 (24.6%) samples collected from all the surveyed fields. Overall, 24 out of 138 (17.3%) samples showed mixed infections. The sequence analysis of a genomic fragment of 922 nt, comprising the entire ORF-2 and part of the 5’ termini of the ORF-3 of 10 selected FBV-1 Iranian isolates from different provinces, and of the type member from GenBank (Acc. No: JF411989), showed a variation ranging from 1 to 3% at nucleotide level and 1% at the amino acid level. The phylogenetic analysis grouped the FBV-1 isolates into two groups, with the Iranian isolates clustered in two distinct subgroups of group I, according to their geographical origin. In our research, the prevalence and sequence analysis of FBV-1 as the only identified DNA virus infecting fig trees, was studied for the first time in Iran.