Identification of the Coding-Complete Genome Sequence of a Novel Cytorhabdovirus in Tilia cordata Showing Extensive Leaf Chloroses.

Here, we report the coding-complete sequence (14,152 nucleotides [nt]) of a novel cytorhabdovirus detected in Tilia cordata and tentatively named cytorhabdovirus tiliae. The assumed genome organization is 3'-N-P-P3-M-G-p6-p6'-L-5'. The N gene encodes the putative nucleoprotein (59.1 kDa), P encodes the phosphoprotein (34.7 kDa), P3 encodes the movement protein (23.1 kDa), M encodes the matrix protein (23.1 kDa), G encodes a glycoprotein (64.4 kDa), and L encodes the viral RNA polymerase (247 kDa). P6 and P6' are overlapping open reading frames (ORFs), which may encode gene products of 7.9 and 9.5 kDa, respectively, of unknown functions.

Towards the Forest Virome: High-Throughput Sequencing Drastically Expands Our Understanding on Virosphere in Temperate Forest Ecosystems.

Thanks to the development of HTS technologies, a vast amount of genetic information on the virosphere of temperate forests has been gained in the last seven years. To estimate the qualitative/quantitative impact of HTS on forest virology, we have summarized viruses affecting major tree/shrub species and their fungal associates, including fungal plant pathogens, mutualists and saprotrophs. The contribution of HTS methods is extremely significant for forest virology. Reviewed data on viral presence in holobionts allowed us a first attempt to address the role of virome in holobionts. Forest health is dependent on the variability of microorganisms interacting with the host tree/holobiont; symbiotic microbiota and pathogens engage in a permanent interplay, which influences the host. Through virus-virus interplays synergistic or antagonistic relations may evolve, which may drastically affect the health of the holobiont. Novel insights of these interplays may allow practical applications for forest plant protection based on endophytes and mycovirus biocontrol agents. The current analysis is conceived in light of the prospect that novel viruses may initiate an emergent infectious disease and that measures for the avoidance of future outbreaks in forests should be considered.

Next-Generation Sequencing Reveals a Novel Emaravirus in Diseased Maple Trees From a German Urban Forest.

While the focus of plant virology has been mainly on horticultural and field crops as well as fruit trees, little information is available on viruses that infect forest trees. Utilization of next-generation sequencing (NGS) methodologies has revealed a significant number of viruses in forest trees and urban parks. In the present study, the full-length genome of a novel Emaravirus has been identified and characterized from sycamore maple (Acer pseudoplatanus) - a tree species of significant importance in urban and forest areas - showing leaf mottle symptoms. RNA-Seq was performed on the Illumina HiSeq2500 system using RNA preparations from a symptomatic and a symptomless maple tree. The sequence assembly and analysis revealed the presence of six genomic RNA segments in the symptomatic sample (RNA1: 7,074 nt-long encoding the viral replicase; RNA2: 2,289 nt-long encoding the glycoprotein precursor; RNA3: 1,525 nt-long encoding the nucleocapsid protein; RNA4: 1,533 nt-long encoding the putative movement protein; RNA5: 1,825 nt-long encoding a hypothetical protein P5; RNA6: 1,179 nt-long encoding a hypothetical protein P6). Two independent NGS sequencing runs from the same symptomatic maple tree detected the same genome segments. For one of these sequencing runs the cDNA library was prepared using a primer targeting the conserved genome terminal region, known to be shared between emaraviruses genome segments. We suggest, therefore, that the six identified genome segments represent the complete genome of a novel emaravirus from maple, which we tentatively name maple mottle-associated virus (MaMaV). Phylogenetic and sequence homology analyses place this virus on the distinct "subgroup a" clade within the Emaravirus genus along with - among others - rose rosette virus, Actinidia emaravirus 2, and fig mosaic virus. Validation RT-PCR assays performed on symptomatic and non-symptomatic trees suggest that MaMaV may be the symptom-inducing virus in the diseased trees. To our knowledge, this is the first time an Emaravirus is described from maple and is fully genetically characterized. With the discovery of MaMaV, the genus Emaravirus comprising negative-sense single-stranded viruses with very divergent genomes - that were until recently overlooked - has substantially increased counting 22 established and putative members.

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

To unravel the virome in birch trees of German and Finnish origin exhibiting symptoms of birch leaf-roll disease (BRLD), high-throughput sequencing (HTS) was employed. In total five viruses, among which three were so far unknown, were detected by RNAseq. One to five virus variants were identified in the transcriptome of individual trees. The novel viruses were genetically-fully or partially-characterized, belonging to the genera Carlavirus, Idaeovirus and Capillovirus and are tentatively named birch carlavirus, birch idaeovirus, and birch capillovirus, respectively. The recently discovered birch leafroll-associated virus was systematically detected by HTS in symptomatic seedlings but not in symptomless ones. The new carlavirus was detected only in one of the three symptomatic seedlings. The novel putative Capillovirus was detected in all seedlings-irrespective of their BLRD status-while the Idaeovirus was identified in a plant without leaf symptoms at the time of sampling. Further efforts are needed to complete Koch's postulates and to clarify the possible association of the detected viruses with the BLR disease. Our study elucidates the viral population in single birch seedlings and provides a comprehensive overview for the diversities of the viral communities they harbor, to date.

A novel badnavirus discovered from Betula sp. affected by birch leaf-roll disease.

In declining birches (Betula sp.) from different European stands affected by the "birch leaf-roll disease" (BLRD) a novel virus is identified by means of RNA-Seq virome analysis. The virus represents a new member in the genus Badnavirus, family Caulimoviridae, tentatively named Birch leaf roll-associated virus (BLRaV) and it is the first badnavirus found to infect birch. Complete genome sequences (7,862-7,864 nucleotides) of three viral isolates of Finnish and German origin have been determined. The virus sequences show a typical badnavirus organization with three major open reading frames (ORFs) and a fourth potential ORF overlapping with the end of ORF3. ORFs 1-2-3 show low level of amino acid identity to the corresponding proteins encoded by other badnaviruses, reaching a maximum of 44% identity (ORF3). Grapevine vein-clearing virus appears as the closest badnavirus when considering the polymerase region. So far, we can exclude evidence for presence of endogenous BLRaV elements in the birch genome, while evidence for the episomal activity of BLRaV is provided. The viral population holds significant haplotype diversity, while co-infection by different BLRaV variants are observed in single hosts. BLRaV presence is associated with the BLRD in both silver (B. pendula) and downy birch (B. pubescens). These results challenge the earlier hypothesis of a causal role of Cherry leaf roll virus in BLRD. Further work is now needed to finally prove that BLRaV is the causal agent for the BLRD.

A Framework for the Evaluation of Biosecurity, Commercial, Regulatory, and Scientific Impacts of Plant Viruses and Viroids Identified by NGS Technologies.

Recent advances in high-throughput sequencing technologies and bioinformatics have generated huge new opportunities for discovering and diagnosing plant viruses and viroids. Plant virology has undoubtedly benefited from these new methodologies, but at the same time, faces now substantial bottlenecks, namely the biological characterization of the newly discovered viruses and the analysis of their impact at the biosecurity, commercial, regulatory, and scientific levels. This paper proposes a scaled and progressive scientific framework for efficient biological characterization and risk assessment when a previously known or a new plant virus is detected by next generation sequencing (NGS) technologies. Four case studies are also presented to illustrate the need for such a framework, and to discuss the scenarios.

Particular Structure of Plasmopara viticola Populations Evolved Under Greek Island Conditions.

ABSTRACT Plasmopara viticola populations collected from three islands in the Ionian Sea-an arm of the Mediterranean Sea to the west of Greece-were analyzed with microsatellite molecular markers in order to investigate the pathogen population structure. Downy mildew populations from mainland regions previously studied were found to have high genotypic diversity and limited clonality; however, populations under Mediterranean island conditions mostly showed limited variation and the epidemics basically were driven by the multiple clonal infections of one or a few genotypes. Populations from different islands were differentiated from each other, whereas genetic divergence also was found among subpopulations of the same plot. Polyploid individuals and individuals that overwintered in asexual form were observed in some cases. The findings obtained by this population genetics study improve our understanding of the biology of the pathogen and lead to potential alternative control measures for the disease.

Complete nucleotide sequence of Cherry leaf roll virus (CLRV), a subgroup C nepovirus.

The complete nucleotide sequence of both genomic (+)ss RNAs of a rhubarb isolate of Cherry leaf roll virus (CLRV) was determined. The larger RNA1 is 7918 nucleotides and the shorter RNA2 6360 nucleotides in size, each genome component comprising a single open reading frame (ORF). The RNA1-encoded polyprotein (P1) is 2112 amino acids long (235.6 kDa) containing domains characteristic for a proteinase-cofactor (PCo), nucleotide-binding helicase (Hel), genome-linked protein (VPg), proteinase (Pro), and an RNA-dependent RNA polymerase (Pol). The RNA2-encoded polyprotein (P2) has a molecular mass of 174.9 kDa (1589 aa) encoding the putative movement protein (MP) and the coat protein (CP) of CLRV. The genome region upstream of the MP has a coding capacity of 77 kDa, however processing of P2 by the putative virus-encoded proteinase and protein-function encoded by this region is unknown. Furthermore, it could be demonstrated that the 5'-termini including the N-terminal region (208 aa) of P1 and P2 of the rhubarb isolate of CLRV are nearly identical among the two genome segments. The taxonomic position of CLRV as member of the genus Nepovirus was confirmed by phylogenetic analyses employing the amino acid sequences of the conserved Pro-Pol region of RNA1, the complete P2, and the CP. However, clustering of Nepovirus-species according to allocated subgroups was inconsistent and depended on the compared genome fragment.

Population genetic structure of Plasmopara viticola after 125 years of colonization in European vineyards.

SUMMARY To examine the within- and among-population genetic structure of Plamopara viticola oosporic populations in Europe, 8991 lesions from 32 vineyard plots were collected and analysed. Four multi-allelic microsatellite markers were used to genotype the pathogen. All populations had high levels of gene and genotypic diversity. Most populations were in Hardy-Weinberg equilibrium and thus randomly mating. Among P. viticola populations, significant low to moderate genetic differentiation was observed, even between geographically close populations. This genetic differentiation was also evident in the neighbour-joining phylogenetic genetic distance tree, showing clear substructure and distinguishing mainly five clusters based on geographical origin. Significant isolation by distance was found in central European P. viticola populations, suggesting a step-wise migration model. No significant isolation by distance was found within Greek populations, most probably owing to natural geographical barriers such as the sea and mountains, as well as the frequent population bottlenecks occurring in these populations, preventing natural migration among populations. The high variability of P. viticola provides explanation for its successful infestation of the heterogeneous European vineyards in the last 125 years after its introduction.