Characterization of a Novel Emaravirus Affecting Ash Species (Fraxinus spp.) in Europe

Abstract

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.

The Complex World of Emaraviruses – Challenges, Insights and Prospects

“…However, some studies have also revealed that emaraviruses can infect host plants in a latent way with plants remaining symptomless during early infection, enabling the virus to spread inconspicuously. Latent infection with emaraviruses have been reported, for instance in RRV-infected roses [28], EMARaV-infected rowan [29], pistachios infected by Pistachia virus B (PiVB) [30], pear chlorotic leaf spot-associated virus (PCLSaV)-infected pear [31] and ASaV-infected ash [21]. For emaraviruses infecting long-living woody hosts, visual inspection and sampling of big tree crowns is difficult and often requires technical support.…”

“…Additionally, an eriophyid mite of the family Diptilomiopidae was demonstrated to transmit a novel emaravirus to star anise [71]. New, yet unclassified eriophyid mite species are suspected to be vectors for other putative emaraviruses ( [21,33,53,56,57,64], see Table 1). However, vector transmission has so far not been demonstrated for all emaraviruses.…”

“…Since its first description in German rowan trees in 2005 [6], EMARaV has been reported in several European countries including Austria, the Czech Republic, Sweden, Finland, Norway, and the United Kingdom [108]. Novel emaraviruses affecting oak, sycamore, ash and aspen trees in forests and urban landscapes were detected in several Northern and Central European countries [17,[19][20][21].…”

“…For instance, a full-length PCR strategy was used to determine the first four complete genome segments of EMA-RaV [7] and later, a generic primer, PDAP213, was used to identify additional segments of RRV [47]. This primer was also used to amplify complete genome segments of other emaraviruses, such as RYRaV [51], EMARaV [14], AsMaV [17], CORaV [19], ASaV [21], and further species belonging to different clades. However, the RNA 1 with a size of more than 7 kb was usually not accessible using this approach.…”

“…[16], aspen mosaic-associated virus (AsMaV) in Populus tremula [17], common oak ringspot-associated virus (CORaV) in Quercus robur [18,19], maple mottle-associated virus (MaMaV) in Acer pseudoplatanus [20] and ash shoestring-associated virus (ASaV) in Fraxinus spp. [21]. Emaraviruses have thereby become the most prevalent group of viruses in long-living plants, causing diseases in key species of the temperate and boreal forests.…”