Aspen mosaic-associated virus (AsMaV) is a newly identified Emaravirus, in the family Fimoviridae, Bunyavirales, associated with mosaic symptoms in aspen trees (Populus tremula). Aspen trees are widely distributed in Europe and understanding the population structure of AsMaV may aid in the development of better management strategies. The virus genome consists of five negative-sense single-stranded RNA (-ssRNA) molecules. To investigate the genetic diversity and population parameters of AsMaV, different regions of the genome were amplified and analyzed and full-length sequence of the divergent isolates were cloned and sequenced. The results show that RNA3 or nucleoprotein is a good representative for studying genetic diversity in AsMaV. Developed RT-PCR-RFLP was able to identify areas with a higher number of haplotypes and could be applied for screening the large number of samples. In general, AsMaV has a conserved genome and based on the phylogenetic studies, geographical structuring was observed in AsMaV isolates from Sweden and Finland, which could be attributed to founder effects. The genome of AsMaV is under purifying selection but not distributed uniformly on genomic RNAs. Distant AsMaV isolates displayed amino acid sequence variations compared to other isolates, and bioinformatic analysis predicted potential post-translational modification sites in some viral proteins.
Plant diseases are mostly multicausal with several factors influencing the health status of affected hosts. Common ash (Fraxinus excelsior), a significant tree species of European forests, is currently mostly endangered by ash dieback, caused by the invasive fungus Hymenoscyphus fraxineus. However, contributing factors, including pathogenic viruses, are poorly understood. Here, we report the results of a virus screening conducted on selected special stands of F. excelsior. Over three consecutive years, ash trees from different origins were tested, including leaf material from mature seed trees, young trees and ash seedlings from the natural regeneration. Using RT-PCR, we screened for five viruses, including the generalist species ArMV (Nepovirus arabis) and CLRV (Nepovirus avii), as well as newly discovered viruses in ash, including the emaravirus ASaV (Emaravirus fraxini), the idaeovirus PrLBaV (Idaeovirus ligustri), and cytorhabdoviruses. The results revealed a high virus diversity in common ash. An association of ASaV detection with specific leaf symptoms, including shoestring, chlorotic ringspots, and vein yellowing, was documented. An analyses of relevant gene products of cytorhabdoviruses obtained from ashes of different sites revealed sequence diversities and two distinct phylogenetic groups present in ash populations. Signatures of novel viruses from different families have been identified by high-throughput sequencing. Together, our results provide insights into the virus diversity and distribution of viruses in ash and expand our knowledge about the virome of this endangered tree species.
Abstract We report the results of a virus screening conducted on selected special stands of common ash ( Fraxinus excelsior ) within the FraxVir project. This project among others deals with the recording and assessment of the virus diversity in common ash and the impact of the ash virome on ash dieback diseased trees. Ash dieback, a fungal disease caused by the invasive fungus Hymenoscyphus fraxineus is a major threat to Fraxinus excelsior populations across Europe. Over three consecutive years, ash trees from different origins were tested including leaf material from mother trees and seedlings from the natural regeneration as well as fruits to gain insight into possible modes of virus transmission. Using RT-PCR, we screened for five viruses, including the generalist species arabis mosaic virus and cherry leaf roll virus, as well as newly discovered viruses in ash including an emaravirus, an idaeovirus and viruses of the cytorhabdovirus group. Amplification and comparison of PCR amplicons comprising the nucleocapsid region revealed sequence diversities that confirm two distinct cytorhadovirus species present in different populations of common ash. Signatures of novel viruses from different families have been identified by high-throughput sequencing. Together our results provide insights into the virus diversity and distribution of viruses in ash and expand our knowledge about the virome of this endangered tree species.
Emaravirus (Order Bunyavirales; Family Fimoviridae) is a genus comprising over 20 emerging plant viruses with a worldwide distribution and economic impact. Emaraviruses infect a variety of host plants and have especially become prevalent in important long-living woody plants. These viruses are enveloped, with a segmented, single-stranded, negative-sense RNA genome and are transmitted by eriophyid mites or mechanical transmission. Emaraviruses have four core genome segments encoding an RNA-dependent RNA polymerase, a glycoprotein precursor, a nucleocapsid protein, and a movement protein. They also have additional genome segments, whose number varies widely. We report here that the proteins encoded by these segments form three main homology groups: a homolog of the sadwavirus Glu2 Pro glutamic protease; a protein involved in pathogenicity, which we named “ABC”; and a protein of unknown function, which we named “P55”. The distribution of these proteins parallels the emaravirus phylogeny and suggests, with other analyses, that emaraviruses should be split into at least two genera. Reliable diagnosis systems are urgently needed to detect emaraviruses, assess their economic and ecological importance, and take appropriate measures to prevent their spread (such as routine testing, hygiene measures, and control of mite vectors). Additional research needs include understanding the function of emaravirus proteins, breeding resistant plants, and clarifying transmission modes.
Abstract Since Emaraviruses have been discovered in 2007 several new species were detected in a range of host plants. Five genome segments of a novel Emaravirus from mosaic‐diseased Eurasian aspen ( Populus tremula ) have been completely determined. The monocistronic, segmented ssRNA genome of the virus shows a genome organisation typical for Emaraviruses encoding the viral RNA‐dependent RNA polymerase (RdRP, 268.2 kDa) on RNA1 (7.1 kb), a glycoprotein precursor (GPP, 73.5 kDa) on RNA2 (2.3 kb), the viral nucleocapsid protein (N, 35.6 kDa) on RNA3 (1.6 kb), and a putative movement protein (MP, 41.0 kDa) on RNA4 (1.6 kb). The fifth identified genome segment (RNA5, 1.3 kb) encodes a protein of unknown function (P28, 28.1 kDa). We discovered that it is distantly related to proteins encoded by Emaraviruses, such as P4 of European mountain ash ringspot‐associated virus. All proteins from this group contain a central hydrophobic region with a conserved secondary structure and a hydrophobic amino acid stretch, bordered by two highly conserved positions, thus clearly representing a new group of homologues of Emaraviruses. The virus identified in Eurasian aspen is closely associated with observed leaf symptoms, such as mottle, yellow blotching, variegation and chloroses along veins. All five viral RNAs were regularly detectable by RT‐PCR in mosaic‐diseased P . tremula in Norway, Finland and Sweden (Fennoscandia). Observed symptoms and testing of mosaic‐diseased Eurasian aspen by virus‐specific RT‐PCR targeting RNA3 and RNA4 confirmed a wide geographic distribution of the virus in Fennoscandia. We could demonstrate that the mosaic‐disease is graft‐transmissible and confirmed that the virus is the causal agent by detection in symptomatic, graft‐inoculated seedlings used as rootstocks as well as in the virus‐infected scions used for graft‐inoculation. Owing to these characteristics, the virus represents a novel species within the genus Emaravirus and was tentatively denominated aspen mosaic‐associated virus.
We report the first detection of European mountain ash ringspot-associated virus (EMARaV) in Karpatiosorbus × hybrida in Finland. The host species varies in morphology, containing primary diploid hybrids of Sorbus aria and Sorbus torminalis as well as stable apomictic and vegetative propagating forms (Sennikov & Kurtto, 5). Due to the decorative flowers, berries and leaf colouration in the autumn, the hybrid is planted in urban areas as a woody ornamental in the same way as other Sorbus species. We sampled leaf material from six trees of Karpatiosorbus × hybrida cultivated in a public park in the city of Helsinki, Finland, showing chlorotic ringspots, mottle, line patterns, sometimes accompanied by leaf deformation and decline (Figs. 1-4, Table 1). The observed disease resembled symptoms caused by EMARaV in Sorbus spp. (von Bargen et al., 6) and related hybrid species (Grimová et al., 3). Additionally, we collected samples from a rowan tree (S. aucuparia) with chlorotic ringspots on leaves growing adjacently. The rowan was infested by the pear blister gall mite, Phytoptus pyri, which is considered to be the vector of EMARaV. To confirm the presence of EMARaV, we performed RT-PCR from extracted total RNA. We could demonstrate that six Karpatiosorbus × hybrida and the S. aucuparia were affected by an emaravirus by amplification of a 360 bp fragment from the sampled leaf material using generic primers targeting RNA1 (Elbeaino et al., 1). Additionally, EMARaV-specific RT-PCRs (von Bargen et al., 6) detected all tested genome segments (RNA2-RNA4 and RNA6) of the virus in the corresponding samples, while none of the genome segments were detectable in a sample taken from a tree of Karpatiosorbus × hybrida without leaf symptoms. By sequencing PCR products amplified from viral RNA1 and RNA4 we could confirm that all seven sampled trees with leaf symptoms were infected by EMARaV. We compared the nucleotide sequences of the partial RNA1 (348 bp) and the complete coding region of RNA4 (699 bp) with reference sequences from GenBank (Table 1). The minimum nucleotide identity was 97.4% (RNA1) and 98.1% (RNA4), respectively, (Table 1) confirming the virus as EMARaV according to the current species demarcation criteria for the genus (Elbeaino et al., 2). Sequences have been deposited in the European Nucleotide Archive (ENA) and are available under the accession numbers LR811990-LR812003. This is the first record of EMARaV affecting Karpatiosorbus × hybrida in Finland. The rowan tree growing adjacently was infected by the virus, with the putative vector P. pyri also being found on the tree. Kallinen et al. (4) confirmed the virus to be widespread in rowan in Finland and Grimová et al. (3) demonstrated that EMARaV is graft-transmissible within species of the Rosaceae. It is therefore possible that the virus was transmitted from the rowan to the Karpatiosorbus × hybrida population by root grafting. However, how the trees in the park in Helsinki acquired the virus remains unknown as their origin and history could not be determined. We thank Dipl. Ing. Renate Junge for skilled technical assistance and the Einstein Foundation Berlin for financial support (project number EGP-2018-476). Financial support from the European Cooperation in Science and Technology (COST action FA1407 "DIVAS") enabled the scientific collaboration and is also kindly acknowledged.
: Emaravirus (Order Bunyavirales; Family Fimoviridae) is a genus comprising over 20 emerging plant viruses with a worldwide distribution and economic impact. Emaraviruses infect a variety of host plants and have especially become prevalent in important long-living woody plants. These viruses are enveloped, with a segmented, single-stranded, negative-sense RNA genome and are transmitted by eriophyid mites or mechanical transmission. Emaraviruses have four core genome segments encoding an RNA-dependent RNA polymerase, a glycoprotein precursor, a nucleocapsid protein, and a movement protein. They also have additional genome segments, whose number varies widely. We report here that the proteins encoded by these segments form three main homology groups: a homolog of the sadwavirus Glu2 Pro glutamic protease; a protein involved in pathogenicity, which we named “ABC”; and a protein of unknown function, which we named “P55”. The distribution of these proteins parallels the emaravirus phylogeny and suggests, with other analyses, that emaraviruses should be split into at least two genera. Reliable diagnosis systems are urgently needed to detect emaraviruses, assess their economic and ecological importance, and take appropriate measures to prevent their spread (such as routine testing, hygiene measures, and control of mite vectors). Additional research needs include understanding the function of emaravirus proteins, breeding resistant plants and clarifying transmission modes.
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