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

Thomas R. Gaskin,Carmen Büttner,Max Tischendorf,Ines Günther,Susanne von Bargen,Marius Rehanek

doi:10.3390/f12111574

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.

Highlights

In the high throughput sequencing (HTS) datasets obtained by RNAseq from pooled leaf samples of two different ash species (F. excelsior and F. ornus) exhibiting chlorotic ringspots and lines, vein yellowing and leaf deformations such as curling and shoestring (Figure 1), we could identify several scaffolds relating to five different genome segments of the plant infecting emaraviruses (Table 2)
Our results show a strong correlation between suspected virus symptoms and associated virus (ASaV) infection in Fraxinus spp
In the case of the putative N protein of ASaV, the aa sequence is nearly identical with the P3 of pea-associated emaravirus (PaEV), suggesting that the emaravirus identified in pea may be the same as the novel virus we identified in ash

Summary

Introduction

Our investigations of Fraxinus spp. were instigated by current interest in ash dieback, a disease that has decimated populations of European ash (Fraxinus excelsior L.) across the continent in recent years [5]. Ash (Fraxinus spp.; Family: Oleaceae) is a group of broad-leaved, mostly deciduous trees, with both the European ash (F. excelsior) and the manna ash (F. ornus) being native to Europe. Do both species have important roles in primary or secondary succession stages in forest ecosystems, they have a decorative, ornamental use in urban environments [6,7,8]. Manna ash trees appear to be less susceptible to ash dieback [11]

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Results

Conclusion