Raspberry Leaf Blotch Virus Research Articles

The species, density, and intra-plant distribution of mites on red raspberry (Rubus idaeus L.).

The adoption of the European Green Deal will limit acaricide use in high value crops like raspberry, to be replaced by biological control and other alternative strategies. More basic knowledge on mites in such crops is then necessary, like species, density, and their role as vectors of plant diseases. This study had four aims, focusing on raspberry leaves at northern altitude: (1) identify mite species; (2) study mite population densities; (3) investigate mite intra-plant distribution; (4) investigate co-occurrence of phytophagous mites, raspberry leaf blotch disorder and raspberry leaf blotch virus (RLBV). Four sites in south-eastern Norway were sampled five times. Floricanes from different parts of the sites were collected, taking one leaf from each of the upper, middle, and bottom zones of the cane. Mites were extracted with a washing technique and processed for species identification and RLBV detection. Mites and leaves were tested for RLBV by reverse transcription polymerase chain reaction (RT-PCR) with virus-specific primers. Phytophagous mites, Phyllocoptes gracilis, Tetranychus urticae, and Neotetranychus rubi, and predatory mites, Anystis baccarum and Typhlodromus (Typhlodromus) pyri were identified. All phytophagous mites in cultivated raspberry preferred the upper zone of floricanes, while in non-cultivated raspberry, they preferred the middle zone. The presence of phytophagous mites did not lead to raspberry leaf blotch disorder during this study. RLBV was detected in 1.3% of the sampled plants, none of them with leaf blotch symptoms, and in 4.3% of P. gracilis samples, and in some spider mite samples, implying that Tetranychids could also be vectors of RLBV.

Population density of eriophyid mites (Acari: Eriophyidae) on raspberry (Rubus idaeus) and their association with leaf blotch symptoms

Red raspberry, Rubus idaeus, is known to be infested by at least six species of eriophyid mites. Among them, the raspberry leaf and bud mite, Phyllocoptes gracilis, (Figure 1, A–B) is the only known vector of a raspberry virus, namely the raspberry leaf blotch virus (RLBV) (Dong et al. 2016; McGavin et al. 2012; Tan et al. 2022). Raspberry leaf blotch (Figure 1, C), a leaf disorder displaying as leaf chlorosis, distortion and patchy necrosis, yellowing and thinning on lateral branches, has been attributed to the feeding of P. gracilis until RLBV was also found to be associated with these symptoms (McGavin et al. 2012). Previous sampling of eriophyid mites was often based on the presence of the leaf blotch symptom, and there is a reasonable doubt if the symptom was caused by RLBV infection or mite infestation. It could also be hypothesized that eriophyid mites are attracted to RLBV-infected plants as viruses could make host plants more attractive to vectors (Donnelly & Gilligan 2020; Shi et al. 2019). It is therefore important to improve the detection of both mites and RLBV to efficiently manage the virus. In addition, knowledge on the dominant infestation area of eriophyid mites on raspberry canes is essential to develop an effective pest management approach. Gordon and Taylor (1976) reported that P. gracilis on primocanes during late summer (mid-August) was dominantly found on the upper leaves, due to the presence of predatory mites on the lower and middle leaves. But mite behavior on floricanes is also important to study if the goal is to prevent mite migration and RLBV transmission to primocanes. This study aimed to investigate the population density of eriophyid mites on raspberry floricanes and the association of eriophyid mites, RLBV, and leaf blotch symptoms.

Development of primer sets for detection of Raspberry leaf blotch virus and Raspberry leaf mottle virus by multiplex RT-PCR

More than 40 viruses and virus-like agents have been confirmed to infect Rubus species including raspberry (R. idaeus and R. occidentalis) and blackberry. The ones of most distributed viruses among cultivated raspberries are Raspberry leaf blotch virus (RLMV) and Raspberry leaf mottle virus (RLBV). The influence of these viruses on the raspberry crop yield is large and losses count in 30-50%. To detect the infected stock planting material or plants carrying infection in orchards, the sets of primer have been developed in this work. Capsid protein gene and nucleocapsid protein gene of RLMV and RLBV, respectively, were considered for design of primers. The specificity analysis confirmed absence of any off-target complementary of primers considering the nucleotide collection of NCBI. Eighty-five samples of raspberry were tested by developed primers. Four cultivars of pomological garden and one raspberry plant of wild population were positive for RLMV and RLBV, respectively. The sequencing analysis of RLMV CP and RLBV NP revealed, that homology with isolates from Bosnia and Herzegovina is higher compared to other isolates in NCBI.

Known and Potential Invertebrate Vectors of Raspberry Viruses.

The estimated global production of raspberry from year 2016 to 2020 averaged 846,515 tons. The most common cultivated Rubus spp. is European red raspberry (Rubus idaeus L. subsp. idaeus). Often cultivated for its high nutritional value, the red raspberry (Rubus idaeus) is susceptible to multiple viruses that lead to yield loss. These viruses are transmitted through different mechanisms, of which one is invertebrate vectors. Aphids and nematodes are known to be vectors of specific raspberry viruses. However, there are still other potential raspberry virus vectors that are not well-studied. This review aimed to provide an overview of studies related to this topic. All the known invertebrates feeding on raspberry were summarized. Eight species of aphids and seven species of plant-parasitic nematodes were the only proven raspberry virus vectors. In addition, the eriophyid mite, Phyllocoptes gracilis, has been suggested as the natural vector of raspberry leaf blotch virus based on the current available evidence. Interactions between vector and non-vector herbivore may promote the spread of raspberry viruses. As a conclusion, there are still multiple aspects of this topic that require further studies to get a better understanding of the interactions among the viral pathogens, invertebrate vectors, and non-vectors in the raspberry agroecosystem. Eventually, this will assist in development of better pest management strategies.

Raspberry leaf blotch virus

This datasheet on Raspberry leaf blotch virus covers Identity, Distribution, Hosts/Species Affected, Further Information.

PHYLOGENETIC ANALYSIS OF UKRAINIAN ISOLATE OF RASPBERRY LEAF BLOTCH VIRUS

Raspberry leaf blotch virus (RLBV) is a new member of the Emaravirus genus (Fimoviridae, Bunyavirales). Representatives of this family are characterized by a segmented "-" RNA genome, helical nucleocapsid and enveloped spherical or pleomorphic virions 80-120nm in diameter. Transmission of these viruses is carried out by eriophyid mites. The virus is widely distributed in Europe. In this paper, the phylogenetic relationships between the Ukrainian RLBV isolate and isolates from the other countries from the GenBank database were investigated. Samples of symptomatic raspberry plants were selected for the work. Total RNA was isolated and RT-PCR was performed using primers to the region of the nucleocapsid protein (P3) gene. The resulting amplicon with a length of about 500bp was sequenced. Sequences were analyzed using BLAST and MEGA7 programs. As a result of BLAST analysis, it was shown that the Ukrainian isolate of RLBV has a high similarity to some Finnish, British, Serbian and Slovak isolates (93-99% similarity). Nevertheless, the dendrogram constructed in MEGA7 did not distribute these isolates in a separate cluster. Interestingly, sequences of isolates from Finland, Britain, and the Balkans were segregated into different parts of the phylogenetic tree. This pattern can be explained by the low divergence of the virus population or small number of isolates in the database.

First report of Raspberry leaf mottle virus infecting raspberry in Poland.

Raspberry leaf mottle virus (RLMV) in Europe has been associated with raspberry mosaic disease (Martin et al. 2013) and crumbly fruit disease (Quito-Avila et al. 2014) in red raspberry (Rubus idaeus L.) beside Rubus yellow net virus (RYNV) and Black raspberry necrosis virus (BRNV). RLMV has also been reported infecting blackberry (Jones and McGavin 1998; Thekke-Veetil et al. 2016). A survey of virus diseases was conducted from 2016 to 2018 in 34 raspberry commercial plantations in Central and southeastern Poland. Some of the plants showed chlorotic spots, vein yellows, mosaic patterns and deformation of leaves and fruits. Leaf samples from 146 plants were tested using RT-PCR for RLMV, RYNV, BRNV, Raspberry vein chlorosis virus (RVCV), Raspberry leaf blotch virus (RLBV), and Raspberry bushy dwarf virus (RBDV). The pairs of primers CPhF and CPhR (Tzanetakis et al. 2007) as well as 1436F and 1437R (McGavin and MacFarlane 2010) were used to amplify a 452-bp fragment of the coat protein homologue (CPh) gene and a 681-bp of the 1 a helicase gene, respectively. RT-PCR products were obtained for five samples from cultivars: Malling Promise (MPrOK1 and MPrOk2; plantation A), Canby (CanKop; plantation B), Polka (PolKop; plantation B) and Malling Seedling (MSeWa; plantation C) grown in southeastern Poland. MPrOK1 and MSeWa were co-infected with RYNV while the other three samples were mixed infected with RBDV. Each of these plants showed chlorotic spots and mosaic symptoms of different intensity, leaf deformation and reduced growth. The RLMV-specific amplicons obtained for all five positive samples were directly sequenced at Genomed company, Warsaw, Poland. The obtained sequences were aligned using ClustalW of the Lasergene 7.1 software (DNASTAR Inc., USA) and were compared with sequences available in GenBank using the BLASTn algorithm. The identity of nucleotide (nt) sequences of Cph gene of RLMV isolates found in Poland was revealed. They shared 97.7% and 99% nt sequence similarity with 'HCRL Glen Clova' from USA (DQ357218) and 'SCRI stock' isolate from United Kingdom (FN391551) strains respectively The nucleotide sequence of isolates from Poland indicates a closer relationship with the strain found in UK. However, the amino acid (aa) sequence of both reference strains was identical and shared 99.2% identity with the corresponding fragment of the isolates from Poland. The representative sequence of PolKop isolate was deposited in GenBank under accession No. MT241265. The similarity of nucleotide sequence of the 1 a helicase gene ranged from 98.5% (between MseWa and four other isolates) to 100% (between MPrOk1 and MPrOk2). The amino acids sequences of MPrOk1, MPrOk2 and MSeWa isolates were identical whereas they shared 99.5% similarity with CanKop and PolKop isolates. The nt and aa sequences within this region of all five isolates were in 98% homologous with the sequences of 'HCRL Glen Clova' strain. Nucleotide sequences of the 1 a helicase gene of the CanKop, PolKop, MseWa, MPrOk1, MPrOk2 isolates were deposited in GenBank (acc. Nos. MT847633-MT8347637). Considering the low incidence of RLMV (five out of 146 positively tested), it can be assumed that there is no emergency of the RLMV large-scale occurrence in Poland. However, as RLMV is transmitted by Amphorophora idaei aphids found in our country, infected plants near raspberry plantations may play an important role in virus spread. This is the first report of RLMV infecting red raspberry in Poland. Acknowledgments This work was performed in the frame of multiannual programme 2015-2020 "Actions to improve the competitiveness and innovation in the horticultural sector with regard to quality and food safety and environmental protection" (task 2.1), financed by the Polish Ministry of Agriculture and Rural Development. I would like to thank Mrs. Dorota Starzec for technical assistance. References Jones, A. T, and McGavin, W. J. 1998. Ann. Appl. Biol. 132:239. https://doi.org/10.1111/j.1744-7348.1998.tb05200.x. Martin, R. R. et al. 2013. Plant Dis. 98:176. http://dx.doi.org/10.1094/PDIS-04-12-0362-FE. McGavin, W. J. and MacFarlane, S. A. 2010. Ann. Appl. Biol. 156:439. doi:10.1111/j.1744-7348.2010.00401.x. Quito-Avila, D. F., et al. 2014. Plant Dis. 98:176. http://dx.doi.org/10.1094/PDIS-05-13-0562-RE. Thekke-Veetil, T., et al. 2016. Plant Dis. 101:265. https://doi.org/10.1094/PDIS-07-16-1014-PDN Tzanetakis, I. E., et al. 2007. Virus Res. 127:26. doi:10.1016/j.virusres.2007.03.010.

Raspberry leaf blotch emaravirus in Bosnia and Herzegovina: population structure and systemic movement.

Raspberry leaf blotch virus (RLBV) is the putative agent of the homonymous disease and even though Bosnia and Herzegovina is a major producer worldwide there is no report of the virus presence in the country. We studied the virus population structure and assessed its ability to move systemically. RLBV is widespread in production areas and has a homogeneous population structure; leading to the hypothesis that the primary mode of dissemination is propagation material. The ability of the virus to move systemically eliminates propagation of root cuttings as a viable option to obtain RLBV-free plants, leaving RT-PCR screening as the better option to propagate RLBV- free plants in the absence of clean-up facilities or certification programs in the country.

High proportion of mixed virus infections in raspberry plantations in Serbia

Samples of red raspberry (Rubus idaeus L.) from the most important growing areas in Serbia were analyzed for the presence of four most important raspberry viruses: raspberry leaf blotch virus (RLBV), raspberry leaf mottle virus (RLMV), black raspberry necrosis virus (BRNV) and Rubus yellow net virus (RYNV), and their incidence in individual and mixed infected samples. Seventy-four samples of eight raspberry cultivars, selected from 32 main cultivation sites were tested, of which 62 leaf samples showed symptoms that could be caused by viruses and 12 samples were asymptomatic. The presence of viruses was tested by appropriate RT-PCR and PCR methods, using virus-specific primers. All four viruses were detected with highly significant differences of incidence in a total of 61 infected symptomatic samples. Infection with one of the four tested viruses was detected in 59.0% of all infected samples. The most prevalent was RLBV, which was associated with raspberry leaf blotch disorder in coexistence with its vector raspberry leaf and bud mite P. gracillis, and it was detected in 70.5% of the infected samples. It was followed by RYNV with an incidence of 42.6%, BRNV with 36.1% and RLMV with 9.8% incidence, considering all infected samples. The obtained results for the first time in Serbia showed a high proportion (41.0%) of mixed infections involving between two and four viruses. The most common were infections with two viruses, 68.0% of all mixed-infected samples, and among them coinfections with RLBV and RYNV (44.0%). Viruses of the raspberry mosaic complex, BRNV, RYNV and RLMV, including coinfections of 2-3 listed viruses along with RLBV, were found in a high joined proportion of 14.7% of all infected samples, i.e. 36.0% of mixed infected samples.

Identification and Characterization of a Novel Emaravirus Associated With Jujube (Ziziphus jujuba Mill.) Yellow Mottle Disease.

A previously unreported disease affecting jujube (Ziziphus jujuba Mill.) trees was observed in China (Liaoning province) in 2015 and named jujube yellow mottle disease (JYMD), due to prevalent symptoms on the leaves. Diseased plants produced also malformed and discolored fruits. In an attempt to identify the possible causal agent of JYMD, high-throughput sequencing of small RNA libraries was performed and a novel virus, tentatively named jujube yellow mottle-associated virus (JYMaV), was identified and characterized. Six genomic RNA segments of JYMaV were completely sequenced. Each one contains a single open reading frame in the viral complementary strand and two untranslated regions with complementary 5′ and 3′ terminal ends, thus showing typical features of other negative-stranded RNA viruses. RNA1 (7.1 kb), RNA2 (2.2 kb) and RNA3 (1.2 kb) encode putative proteins that, based on their conserved motifs, have been identified as the RNA dependent RNA polymerase, the glycoprotein and the nucleocapsid protein, respectively. These proteins share significant sequence identity (52.1–70.4%) with proteins encoded by raspberry leaf blotch virus (RLBV). RNA4 (1.5 kb) and RNA5 (1.2 kb) code for two putative 30 K movement proteins also related to the homologous RLBV protein. The functional role of the protein encoded by JYMaV RNA6 remains unknown. These data together with the phylogenetic relationships of JYMaV with other recognized emaraviruses support the proposal that JYMaV is the representative member of a novel species in the genus Emaravirus. In agreement with this proposal, virus-like particles and double-membrane-bound bodies, similar to those previously reported for other emaraviruses, were observed by transmission electron microscopy in extracts and tissues from symptomatic leaves, respectively. A specific RT-PCR-based detection method has been developed and used in a preliminary field survey that provided results strongly supporting the close association of JYMaV with the novel disease.

Occurrence and genetic diversity ofRaspberry leaf blotch virus(RLBV) infecting cultivated and wildRubusspecies in Finland

Raspberry leaf blotch virus (RLBV) is a recently described (−)ssRNA virus (genus Emaravirus) associated with the long-known, severe raspberry leaf blotch disorder (RLBD). The virus is presumably transmitted by the raspberry leaf and bud mite (Phyllocoptes gracilis; Eriophyidae). Cultivated and wild raspberries (Rubus idaeus) displaying RLBD or yellowing symptoms were sampled in 14 districts in Finland and tested for RNA3 and RNA5 of RLBV by reverse transcription PCR (RT-PCR) and dot blot hybridization. A total of 59 samples were tested for RLBV, including 36 plants of cultivated raspberry, 20 wild raspberry plants, one ornamental purple flowering raspberry plant (Rubus odoratus), and two samples of P. gracilis (20 mites pooled per sample) collected from RLBD-affected Glen Ample. Fifty-three (93%) of the 57 plants tested were RLBV-positive, including seven raspberry cultivars (Balder, Glen Ample, Jenkka, Malling Minerva, Maurin Makea, Muskoka, Ottawa) and purple flowering raspberry, which is a new host for RLBV. RLBV was also detected by RT-PCR in mites (P. gracilis) collected from buds of RLBV-positive raspberry plants. The partial nucleotide (nt) sequence of the RLBV NP gene (nt 835–1284 of RNA3) was determined for 21 isolates obtained from 10 districts in Finland. Identical isolates were detected in distant districts, and distinctly different isolates were found in the same raspberry cultivation. Furthermore, eight different NP sequence variants were detected in the nine plants of a single raspberry cultivar (Glen Ample) tested from two districts. The nt and deduced amino acid sequences of the 21 isolates were 92.0–100% and 89.3–100% identical, respectively. Phylogenetic analysis revealed two main clusters. One cluster included three isolates, of which two isolates were from one farm in Finland and one isolate was previously characterised in Scotland. The other cluster contained the remaining 19 isolates characterised in this study. The results indicate that RLBV is widely distributed in cultivated and wild raspberries in Finland and shows considerable genetic variability.

Newly identified RNAs of raspberry leaf blotch virus encoding a related group of proteins.

Members of the genus Emaravirus, including Raspberry leaf blotch virus (RLBV), are enveloped plant viruses with segmented genomes of negative-strand RNA, although the complete genome complement for any of these viruses is not yet clear. Currently, wheat mosaic virus has the largest emaravirus genome comprising eight RNAs. Previously, we identified five genomic RNAs for RLBV; here, we identify a further three RNAs (RNA6-8). RNA6-8 encode proteins that have clear homologies to one another, but not to any other emaravirus proteins. The proteins self-interacted in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments, and the P8 protein interacted with the virus nucleocapsid protein (P3) using BiFC. Expression of two of the proteins (P6 and P7) using potato virus X led to an increase in virus titre and symptom severity, suggesting that these proteins may play a role in RLBV pathogenicity; however, using two different tests, RNA silencing suppression activity was not detected for any of the RLBV proteins encoded by RNA2-8.

Raspberry leaf blotch virus. [Distribution map

Abstract A new distribution map is provided for Raspberry leaf blotch virus. Emaravirus. Hosts: raspberry ( Rubus idaeus ) and tayberry ( Rubus hybrids). Information is given on the geographical distribution in Europe (Bulgaria, France, Serbia, UK, England and Wales and Scotland).

Emaravirus-specific degenerate PCR primers allowed the identification of partial RNA-dependent RNA polymerase sequences of Maize red stripe virus and Pigeonpea sterility mosaic virus

Emaravirus is a recently established viral genus that includes two approved virus species: European mountain ash ringspot-associated virus (EMARaV) and Fig mosaic virus (FMV). Other described but unclassified viruses appear to share biological characteristics similar to emaraviruses, including segmented, negative-single stranded RNA genomes with enveloped virions approximately 80-200nm in diameter. Sequence analysis of emaravirus genomes revealed the presence of conserved amino acid sequences in the RNA-dependent RNA polymerase gene (RdRp) denoted as pre-motif A, motifs A and C. Degenerate oligonucleotide primers were developed to these conserved sequences and were shown to amplify in reverse transcription-polymerase chain reaction assay (RT-PCR) DNA fragments of 276bp and 360bp in size. These primers efficiently detected emaraviruses with known sequences available in the database (FMV and EMARaV); they also detected viruses with limited sequence information such as Pigeonpea sterility mosaic virus (PPSMV) and Maize red stripe virus (MRSV). The degenerate primers designed on pre-motif A and motif A sequences successfully amplified the four species used as positive controls (276bp), whereas those of motifs A and C failed to detect only MRSV. The amino acid sequences obtained from PPSMV and MRSV shared the highest identity with those of two other tentative species of the Emaravirus genus, Rose rosette virus (RRV) (69%) and Redbud yellow ringspot virus (RYRV) (60%), respectively. The phylogenetic tree constructed with 92 amino acid-long portions of polypeptide putatively encoded by RNA1 of definitive and tentative emaravirus species clustered PPSMV and MRSV in two separate clades close to RRV and Raspberry leaf blotch virus (RLBV), respectively. The newly developed degenerate primers have proved their efficacy in amplifying new emaravirus-specific sequences; accordingly, they could be useful in identifying new emaravirus-like species in nature.

Emaravirus: A Novel Genus of Multipartite, Negative Strand RNA Plant Viruses

Ringspot symptoms in European mountain ash (Sorbus aucuparia L.), fig mosaic, rose rosette, raspberry leaf blotch, pigeonpea sterility mosaic (Cajanus cajan) and High Plains disease of maize and wheat were found to be associated with viruses that share several characteristics. They all have single-stranded multipartite RNA genomes of negative orientation. In some cases, double membrane-bound virus-like particles of 80 to 200 nm in diameter were found in infected tissue. Furthermore, at least five of these viruses were shown to be vectored by eriophyid mites. Sequences of European mountain ash ringspot-associated virus (EMARaV), Fig mosaic virus (FMV), rose rosette virus (RRV), raspberry leaf blotch virus (RLBV), pigeonpea sterility mosaic virus and High Plains virus strongly support their potential phylogenetic relationship. Therefore, after characterization of EMARaV, the novel genus Emaravirus was established, and FMV was the second virus species assigned to this genus. The recently sequenced RRV and RLBV are supposed to be additional members of this new group of plant RNA viruses.

First Report of Raspberry leaf blotch virus in Raspberries in Finland.

Raspberry (Rubus idaeus L.) is a valuable and widely grown softfruit that is a host for 40 viruses and virus-like agents, of which many are not characterized at the molecular level. Recently, Raspberry leaf blotch virus (RLBV, putative emaravirus species) was described from raspberries (cv. Glen Ample) in the United Kingdom and Serbia. Plants displayed conspicuous yellow blotches on leaves and abnormal development of leaf hairs in the corresponding areas of the abaxial side (3). Similar symptoms were observed in 'Glen Ample' grown in protective plastic tunnels and open fields in the main berry growing area in eastern Finland in June 2011. In three farms, leaves were sampled from symptomatic and symptomless plants of 'Glen Ample' and also cv. Polka displaying no symptoms. Total RNA was extracted using CTAB reagent. Equal amounts of RNA were pooled from 13 samples and subjected to small-RNA (sRNA) deep sequencing (Fasteris SA, Plan-les-Ouates, Switzerland) to detect viruses without advance information (2). Contigs were built on 21- to 24-nt sRNA reads using Velvet. Contigs larger than 50 nt were used to search homologous sequences in GenBank by BLAST and significant similarity (up to 99%) was observed with RLBV RNA3 and RNA4. Mapping sRNA reads to the genome of RLBV (1) by MAQ resulted in significant coverage of RNA1 (16%), RNA2 (37%), RNA3 (46%), RNA4 (65%), and RNA5 (27%). cDNA was synthesized on RNA of one symptom-expressing plant using random hexamer primers and the cDNA tested by PCR with a forward primer (RLBV-F 5'-TCAAATCCACTTGCATAGAACC-3', nt 723 to 744) and reverse primer (RLBV-R1 5'-CCTCAAACCTTGCAAACACA-3', nt 1,318 to 1,337) designed according to the nucleocapsid (NP) gene of the Scottish RLBV isolate (3). The sequence of the amplified partial NP gene (576 nt; GenBank Accession No. JQ684678) was 92.8% and 94.8% identical to the Scottish isolate at nt and amino acid levels, respectively. The forward primer RLBV-F and a new reverse primer (RLBV-R2 5'-GCCGAAAGTCAAACCTGGTG-3', nt 943 to 962) were used to test additional plants for RBLV and to make a probe (198 nt) to detect RLBV using digoxigen-labeled sense and antisense RNA probes, as described for European mountain ash ringspot associated virus (1). RLBV was detected in all tested plants of 'Glen Ample' with yellow leaf blotch symptoms in the three farms, but not in any symptomless plants of 'Glen Ample' and 'Polka.' The sense probes gave strong signals, in contrast to the antisense probes, which gave only weak or no detectable signals in the virus-positive plants, consistent with the negative RNA strand of RLBV being encapsidated in virus particles. The results show RLBV is associated with severe, distinct, and characteristic symptoms in raspberries of cv. Glen Ample grown in plastic tunnels and open fields in Finland and has an apparent negative impact on plant growth and yield. Our observations in 2011 also suggest that the incidence of diseased plants is much higher in plastic tunnels than in open fields, perhaps because the conditions for the vector of RLBV (raspberry leaf and bud mite, Phyllocoptes gracilis Nalepa) (1) are more favorable in plastic tunnels. These results clarify the etiology of raspberry leaf blotch disease in Finland and emphasize the need to inspect raspberry planting materials for RLBV for better control of the disease.

Raspberry leaf blotch virus, a putative new member of the genus Emaravirus, encodes a novel genomic RNA

A new, segmented, negative-strand RNA virus with morphological and sequence similarities to other viruses in the genus Emaravirus was discovered in raspberry plants exhibiting symptoms of leaf blotch disorder, a disease previously attributed to the eriophyid raspberry leaf and bud mite (Phyllocoptes gracilis). The virus, tentatively named raspberry leaf blotch virus (RLBV), has five RNAs that each potentially encode a single protein on the complementary strand. RNAs 1, 2 and 3 encode, respectively, a putative RNA-dependent RNA polymerase, a glycoprotein precursor and the nucleocapsid. RNA4 encodes a protein with sequence similarity to proteins of unknown function that are encoded by the genomes of other emaraviruses. When expressed transiently in plants fused to green or red fluorescent protein, the RLBV P4 protein localized to the peripheral cell membrane and to punctate spots in the cell wall. These spots co-localized with GFP-tagged tobacco mosaic virus 30K cell-to-cell movement protein, which is itself known to associate with plasmodesmata. These results suggest that the P4 protein may be a movement protein for RLBV. The fifth RLBV RNA, encoding the P5 protein, is unique among the sequenced emaraviruses. The amino acid sequence of the P5 protein does not suggest any potential function; however, when expressed as a GFP fusion, it localized as small aggregates in the cytoplasm near to the periphery of the cell.