Unravelling the virome in birch: RNA-Seq reveals a complex of known and novel viruses

Abstract

25 High-throughput sequencing (HTS), combined with bioinformatics for de novo discovery and 26 assembly of plant virus or viroid genome reads, has promoted the discovery of abundant novel 27 DNA and RNA viruses and viroids. However, the elucidation of a viral population in a single plant is 28 rarely reported. In five birch trees of German and Finnish origin exhibiting symptoms of birch leaf-29 roll disease (BRLD), we identified in total five viruses, among which three are novel. The number of 30 identified virus variants in each transcriptome ranged from one to five. The novel species are 31 genetically -fully or partially -characterized, they belong to the genera Carlavirus, Idaeovirus and 32 Capillovirus and they are tentatively named birch carlavirus, birch idaeovirus, and birch 33 capillovirus, respectively. The only virus systematically detected by HTS in symptomatic trees 34 affected by the BRLD was the recently discovered birch leafroll-associated virus. The role of the 35 new carlavirus in BLRD etiology seems at best weak, as it was detected only in one of three 36 symptomatic trees. Continuing studies have to clarify the impact of the carlavirus to the BLRD. The 37 role of the Capillovirus and the Idaeovirus within the BLRD complex and whether they influence 38 plant vitality need to be investigated. Our study reveals the viral population in single birch trees and 39 provides a comprehensive overview for the diversities of the viral communities they harbor. 40 41 Keywords: Betula sp., virome, birch leaf-roll disease, birch carlavirus, birch idaeovirus, birch 42 capillovirus 3 44 Introduction 45 Symptoms in birch trees (Betula sp.) caused by various viruses and related to the birch 46 leaf-roll disease (BLRD) are observed throughout Europe [1-2]. Diseased birches exhibit foliar 47 disorders including vein banding, leaf roll, mottling, necrotic lesions and tip dieback. Based on 48 earlier studies on virus-diseased trees it is assumed that BLRD might significantly reduce the tree's 49 photosynthetic capacity and contribute to tree decline [3]. Due to lack of knowledge, risk analyses 50 and prevention measures, the disease has effectively spread throughout Europe and has until now 51 been reported in European countries with diverse climatic conditions such as Finland, Sweden, 52 Norway, Germany, Austria, UK and France, including stands of a Mediterranean island [2-5]. 53The initial hypothesis for the BLRD disease etiology implicated Cherry leaf roll virus (CLRV) 54 as the disease main causal agent [1-2; 4-5]. However, after long-lasting trials applying 55 conventional viral detection methods (virus purification, RT-PCR, double-stranded-RNA isolation, 56 virus mechanical transmission), this virus could not be convincingly associated with the 57 appearance of disease symptoms. To identify BLRD etiology, a birch metagenomic study was 58 therefore initiated. A RNA-Seq analysis revealed for the first time in diseased trees from Germany 59 and Finland birch leafroll-associated virus (BLRaV), the first reverse-tra...

Towards the Forest Virome: Next-Generation-Sequencing Drastically Expands our Understanding on Virosphere in Temperate Forest Ecosystems

“…This was already known before the development of NGS technology, but the deep sequencing methods profoundly revealed the complexity of plants and tree viromes. Novel data about the birch virome clearly demonstrate in holobionts mixed infections by multiple plant viruses as well as multiple variants of the same virus species [14,15,74]. This is the case also in other non-forest plant species like peach [155], or grapevine [156,157].…”

“…Among these, three virus genera are overwhelmingly represented. These are the genus Emaravirus with up to now four species (aspen mosaic-associated virus [26]; common oak ringspot-associated virus [27,28]; maple mottle-associated virus [29]; European mountain ash ringspot-associated virus [9]), the genus Badnavirus with two species (birch leafroll-associated virus [15]; chestnut mosaic virus [30]) and the genus Carlavirus with eight species (birch carlavirus [14]; blueberry scorch virus [31]; elderberry carlaviruses A, B, C, D, E [32][33]; elm carlavirus [34]). All the novel emara-and badnaviruses are found to be associated with the corresponding symptoms and are, consequently, plant pathogenic, while the role of the carlaviruses is not yet clarified.…”

“…However, the employment of NGS radically changed our concept regarding the causal agent of the BLRD. RNA-Seq revealed the presence of a novel badnavirus in affected birches [15], while a complex virome involving novel and known viruses was reaveled; birch idaeovirus, birch capillovirus, birch carla virus and cherry leaf roll virus arepresent in symptomatic trees [14]. Based on the metagenomic analysis of three birches that exhibited symptoms and two that did not exhibit symptoms and on further analysing from a considerable number of samples [74], it is suggested that the novel birch leafroll-associated badnavirusis (BLRaV) is the main causal agent of BLRD, however, the other viruses could possibly still contribute in symptom development in cases of mixed infection with BLRaV and/or CLRV.…”

“…RNA viruses, viroids and the RNA stages of actively replicating DNA viruses can be directly sequenced. Applying RNA-Seq, birch viromes in relation with BLRD were unraveled revealing a complex of novel and known viruses (Badna-, Nepo-and Capillovirus [14], while a novel badna virus was associated with the BLD symptoms and was genetically characterized [15]. Regarding EMARaV, it was initially reported only in European mountain ash, lately, however and with the use of NGS several emaraviruses were detected in new hosts as there are e.g.…”