We identified a novel virus in diseased European ash (Fraxinus excelsior) and manna ash (F. ornus) trees exhibiting chlorotic ringspots, mottle and leaf deformation such as curling and shoestring symptoms. High-throughput sequencing (HTS, Illumina RNASeq) of total RNA isolated from diseased leaf material in combination with RT-PCR-based amplification techniques and Sanger sequencing determined five complete genome segments, each encoding a single open reading frame. Sequence analyses of RNA1–RNA5 revealed a genome organization typical for emaraviruses, i.e., (i) conserved and complementary terminal 5′ and 3′ termini of each genome segment (ii) proteins showing significant homologies to the RNA-dependent RNA polymerase (RdRP) encoded by RNA1, the glycoprotein precursor (GPP) encoded by RNA2, the viral nucleocapsid protein (N, RNA3), the movement protein (MP, RNA4), and a protein of 26 kDA (P26, RNA5) highly similar to proteins of unknown function encoded by other emaraviruses. Furthermore, we identified spherical particles (double-membrane bodies, DMB) of different sizes (70–80 nm in diameter) which are typical for emaraviruses exclusively in virus-infected leaf tissue exhibiting mottle and leaf deformation. Sequence comparison and phylogenetic analyses confirmed the identified novel virus as a new member of the genus Emaravirus. We established a species-specific RT-PCR detection protocol and could associate the observed disease symptoms with the infection of the novel emaravirus in F. excelsior and F. ornus. Therefore, we propose the name ash shoestring-associated emaravirus (ASaV). Investigation of ASaV-infected sample trees originating from different locations in Switzerland, Germany, Italy and Sweden provided a wide geographical distribution of the virus in affected ash species. To our knowledge, this is the first confirmation of an emaravirus affecting ash tree species with shoestring symptoms of leaves in Europe.

Ash shoestring-associated virus (ASaV; Emaravirus fraxini) is a five-segmented, negative-sense RNA virus associated with chlorosis and leaf deformation in Fraxinus species. This study investigated the genetic diversity and evolutionary dynamics of ASaV by analyzing nearly full-length RNA2–RNA5 and partial RNA1 sequences from isolates collected from different geographic regions and Fraxinus hosts. The sequence data uncovered that ASaV has a conserved genome, in which RNA3 and RNA5 showed more genetic divergence than other segments in the sequenced isolates. ASaV RNA3 and partial RNA1 were the most informative genomic regions for phylogenetic studies. There was a correlation between the clustering of the ASaV isolates and host species when the phylogenetic tree was constructed based on the RNA1 region. The ASaV genome is predominantly under purifying selection. Newly designed primers in this study facilitated robust amplification of genomic regions.