Geographical variations, prevalence, and molecular dynamics of fastidious phloem-limited pathogens infecting sugar beet across Central Europe.

In Europe, two fastidious phloem-limited pathogens, 'Candidatus Phytoplasma solani' (16SrXII-A) and 'Candidatus Arsenophonus phytopathogenicus', are associated with rubbery taproot disease (RTD) and syndrome basses richesses (SBR) of sugar beet, respectively. Both diseases can significantly reduce yield, especially when accompanied by root rot fungi. This study investigates the presence, geographic distribution and genetic traits of fastidious pathogens and the accompanying fungus, Macrophomina phaseolina, found on sugar beet across four geographically separated plains spanning seven countries in Central Europe. The survey revealed variable incidences of symptoms linked to these fastidious pathogens in the Pannonian and Wallachian Plains, sporadic occurrence in the North European Plain, and no symptomatic sugar beet in the Bohemian Plain. Molecular analyses unveiled the occurrence of both 'Ca. P. solani' and 'Ca. A. phytopathogenicus' throughout Central Europe, with a predominance of the phytoplasma. These fastidious pathogens were detected in all six countries surveyed within the Pannonian and Wallachian Plains, with only a limited presence of various phytoplasmas was found in the North European Plain, while no fastidious pathogens were detected in Bohemia, aligning with observed symptoms. While 16S rDNA sequences of 'Ca. P. solani' remained highly conserved, multi-locus characterization of two more variable loci (tuf and stamp) unveiled distinct variability patterns across the plains. Notably, the surprising lack of variability of tuf and stamp loci within Central Europe, particularly the Pannonian Plain, contrasted their high variability in Eastern and Western Europe, corresponding to epidemic and sporadic occurrence, respectively. The current study provides valuable insights into the genetic dynamics of 'Ca. P. solani' in Central Europe, and novel findings of the presence of 'Ca. A. phytopathogenicus' in five countries (Slovakia, Czech Republic, Austria, Serbia, and Romania) and M. phaseolina in sugar beet in Slovakia. These findings emphasize the need for further investigation of vector-pathogen(s)-plant host interactions and ecological drivers of disease outbreaks.

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.

Molecular Characterization of a Novel Enamovirus Infecting Raspberry.

Raspberry plants, valued for their fruits, are vulnerable to a range of viruses that adversely affect their yield and quality. Utilizing high-throughput sequencing (HTS), we identified a novel virus, tentatively named raspberry enamovirus 1 (RaEV1), in three distinct raspberry plants. This study provides a comprehensive characterization of RaEV1, focusing on its genomic structure, phylogeny, and possible transmission routes. Analysis of nearly complete genomes from 14 RaEV1 isolates highlighted regions of variance, particularly marked by indel events. The evidence from phylogenetic and sequence analyses supports the classification of RaEV1 as a distinct species within the Enamovirus genus. Among the 289 plant and 168 invertebrate samples analyzed, RaEV1 was detected in 10.4% and 0.4%, respectively. Most detections occurred in plants that were also infected with other common raspberry viruses. The virus was present in both commercial and wild raspberries, indicating the potential of wild plants to act as viral reservoirs. Experiments involving aphids as potential vectors demonstrated their ability to acquire RaEV1 but not to successfully transmit it to plants.

Analysis of Virus-Derived siRNAs in Strawberry Plants Co-Infected with Multiple Viruses and Their Genotypes.

Plants can be infected with multiple viruses. High-throughput sequencing tools have enabled numerous discoveries of multi-strain infections, when more than one viral strain or divergent genomic variant infects a single plant. Here, we investigated small interfering RNAs (siRNAs) in a single strawberry plant co-infected with several strains of strawberry mottle virus (SMoV), strawberry crinkle virus (SCV) and strawberry virus 1 (StrV-1). A range of plants infected with subsets of the initial viral species and strains that were obtained by aphid-mediated transmission were also evaluated. Using high-throughput sequencing, we characterized the small RNA fractions associated with different genotypes of these three viruses and determined small RNA hotspot regions in viral genomes. A comparison of virus-specific siRNA (vsiRNA) abundance with relative viral concentrations did not reveal any consistent agreement. Strawberry mottle virus strains exhibiting considerable variations in concentrations were found to be associated with comparable quantities of vsiRNAs. Additionally, by estimating the specificity of siRNAs to different viral strains, we observed that a substantial pool of vsiRNAs could target all SMoV strains, while strain-specific vsiRNAs predominantly targeted rhabdoviruses, SCV and StrV-1. This highlights the intricate nature and potential interference of the antiviral response within a single infected plant when multiple viruses are present.

First report of 'Candidatus Phytoplasma rubi' associated with Rubus stunt disease of raspberry and blackberry in the Czech Republic.

Raspberries (Rubus idaeus L.), occurring in the temperate zone of the northern hemisphere and blackberries (R. fruticosus L.), cultivated and growing all over the world, are plant species of the family Rosaceae. These species are susceptible to phytoplasma infections, which cause Rubus stunt disease. It spreads uncontrolled by vegetative propagation of plants (Linck and Reineke 2019a) and by phloem-sucking insect vectors, especially Macropsis fuscula (Hemiptera: Cicadellidae) (de Fluiter and van der Meer, 1953; Linck and Reineke 2019b). During a survey in commercial field in June 2021, over 200 raspberry bushes cv Enrosadira exhibiting typical symptoms of Rubus stunt were observed in Central Bohemia. Symptoms included dieback, leaf yellowing/reddening, stunted growth, severe phyllody and fruit malformations. Most diseased plants were growing in the edge rows of the field (about 80%). No symptomatic plants were observed in the middle of the field. Similar symptoms were observed in private gardens in South Bohemia on raspberry cv Rutrago and blackberry (unknown cultivar) in June 2018 and August 2022, respectively. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) from flower stems and parts affected by phyllody of seven symptomatic plants as well as flower stems, leaf midribs, and petioles of five asymptomatic field plants. The DNA extracts were analyzed by a nested polymerase chain reaction assay using universal phytoplasma P1A/P7A primers followed by R16F2m/R1m and the group-specific R16(V)F1/R1 primers (Bertaccini et al. 2019). All samples from the symptomatic plants yielded an amplicon of expected size, while no product was amplified in asymptomatic plants. The P1A/P7A amplicons from three selected plants (two raspberries and one blackberry, each from different location) were cloned and bi-directionally Sanger sequenced (GenBank Accession Nos.OQ520100-2). The sequences spanned nearly full-length of 16S rRNA gene, 16S-23S rRNA intergenic spacer, tRNA-Ile gene, and a partial 23S rRNA gene. BLASTn search revealed the highest sequence identity (99.8-99.9%, query coverage 100%) to 'Candidatus Phytoplasma rubi' strain RS (GenBank Accession No. CP114006). To further characterize the 'Ca. P. rubi' strains, all these three samples were subjected to multigene sequence analysis. Sequences from a major portion of the tuf, rplV-rpsC, rpsH-rplR, uvrB-degV, and rplO-SecY-map genes (Acc. Nos. OQ506112-26) were obtained as described previously (Fránová et al. 2016). Comparison to GenBank sequences confirmed their highest identity (99.6-100%, query coverage 100%) with 'Ca. P. rubi' RS strain, regardless of their geographic location and host (raspberry or blackberry). Recently, Bertaccini et al. (2022) suggested the 98,65 % 'Ca. Phytoplasma' strain identity threshold within 16Sr RNA sequences. In this survey, all three strains sequenced shared ≥99.73% sequence identity of the analysed 16S rRNA gene sequences and the high identity in the other genes with the reference 'Ca. P. rubi' RS strain. To our knowledge, this is the first report of Rubus stunt disease in the Czech Republic as well as the first molecular identification and characterization of 'Ca. P. rubi' from raspberry and blackberry in our country. As Rubus stunt disease is of great economic importance (Linck and Reineke 2019a), the pathogen detection and prompt removal of the diseased shrubs are essential to mitigating the spread and impact of the disease.

Mixed Infection of Blackcurrant with a Novel Cytorhabdovirus and Black Currant-Associated Nucleorhabdovirus.

A virome screen was performed on a new breeding line, KB1, of blackcurrant. Rhabdovirus-like particles were observed by electron microscopy in ultrathin sections of flower stalks, and the complete genome sequence of a novel virus, provisionally named blackcurrant rhabdovirus 2 (BCRV2), was determined and verified using high-throughput sequencing. The genomic organization of BCRV2 was characteristic of cytorhabdoviruses (family Rhabdoviridae) and included seven genes: 3 ́- N-P´-P-P3-M-G-L -5 ́. BLASTP analysis revealed that the putative L protein had the highest amino acid sequence identity (75 %) with strawberry virus 2. BCRV2 was detected in Cryptomyzusgaleopsidis, but efficient transmission by this aphid was not confirmed. Of note, we observed coinfection of the KB1 line with blackcurrant-associated rhabdovirus (BCaRV) by RT-PCR. This is likely the first evidence of the presence of a cyto- and a nucleorhabdovirus in a single host.

Detection of Apple Hammerhead Viroid, Apple Luteovirus 1 and Citrus Concave Gum-Associated Virus in Apple Propagation Materials and Orchards in the Czech Republic and Hungary.

Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT-(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus.

Transmission of Diverse Variants of Strawberry Viruses Is Governed by a Vector Species.

Advances in high-throughput sequencing methods have boosted the discovery of multistrain viral infections in diverse plant systems. This phenomenon appears to be pervasive for certain viral species. However, our knowledge of the transmission aspects leading to the establishment of such mixed infections is limited. Recently, we reported a mixed infection of a single strawberry plant with strawberry mottle virus (SMoV), strawberry crinkle virus (SCV) and strawberry virus 1 (StrV-1). While SCV and StrV-1 are represented by two and three molecular variants, respectively, SmoV has three different RNA1 and RNA2 segments. In this study, we focus on virus acquisition by individual adult aphids of the Aphis gossypii, Aphis forbesi and Chaetosiphon fragaefolii species. Single-aphid transmission trials are performed under experimental conditions. Both different viruses and individual virus strains show varying performances in single aphid acquisition. The obtained data suggests that numerous individual transmission events lead to the establishment of multistrain infections. These data will be important for the development of epidemiological models in plant virology.

Identification and Characterization of a Novel Umbra-like Virus, Strawberry Virus A, Infecting Strawberry Plants.

A novel RNA virus infecting strawberry plants was discovered using high-throughput sequencing. The analyzed plant was simultaneously infected with three different genetic variants of the virus, provisionally named strawberry virus A (StrVA). Although StrVA is phylogenetically clustered with several recently discovered, unclassified plant viruses, it has a smaller genome and several unique features in its genomic organization. A specific and sensitive qPCR system for the detection of identified StrVA genetic variants was designed. A survey conducted in the Czech Republic revealed that StrVA was present in 28.3% of strawberry samples (n = 651) from various origins (plantations, gardens, and propagation material). Sequencing of 48 randomly selected StrVA-positive strawberry samples showed that two or all three StrVA genetic variants were present in 62.5% of the samples in various proportions. StrVA was found in mixed infections with other viruses (strawberry mild yellow edge virus, strawberry crinkle virus, strawberry mottle virus, strawberry polerovirus 1, or strawberry virus 1) in 57.1% of the samples, which complicated the estimation of its biological relevance and impact on the health status of the plants.

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.

High Incidence of Strawberry Polerovirus 1 in the Czech Republic and Its Vectors, Genetic Variability and Recombination.

In total, 332 strawberry plants from 33 different locations in the Czech Republic with or without disease symptoms were screened by RT-PCR for the presence of strawberry polerovirus 1 (SPV1) and five other viruses: strawberry mottle virus, strawberry crinkle virus, strawberry mild yellow edge virus, strawberry vein banding virus, and strawberry virus 1. SPV1 was detected in 115 tested strawberry plants (35%), including 89 mixed infections. No correlation between symptoms and the detected viruses was found. To identify potential invertebrate SPV1 vectors, strawberry-associated invertebrate species were screened by RT-PCR, and the virus was found in the aphids Aphis forbesi, A. gossypii, A. ruborum, A.sanquisorbae, Aulacorthum solani, Chaetosiphon fragaefolii, Myzus ascalonicus, and several other non-aphid invertebrate species. SPV1 was also detected in aphid honeydew. Subsequent tests of C. fragaefolii and A.gossypii virus transmission ability showed that at least 4 h of acquisition time were needed to acquire the virus. However, 1 day was sufficient for inoculation using C. fragaefolii. In conclusion, being aphid-transmitted like other tested viruses SPV1 was nevertheless the most frequently detected agent. Czech SPV1 isolates belonged to at least two phylogenetic clusters. The sequence analysis also indicated that recombination events influence evolution of SPV1 genomes.

Fragaria vesca-associated virus 1: a new virus related to negeviruses.

In this study, the complete genomic sequence of a novel virus was determined by next-generation sequencing of a sample from a symptomatic strawberry plant with severe yellow spots and mosaic on its leaves. Its genomic organization and sequence showed that this virus is related to members of the proposed insect-specific genus "Negevirus". The sample also contained sequences from the geranium aphid Acyrthosiphon malvae. Although the virus was detected repeatedly in the same plant during the three following years, no other positive samples were obtained from the surroundings or more-distant locations. Reverse transcription qPCR analysis revealed the presence of both genomic positive and complementary negative strands of the viral genome in the sample, with a 3- to 30-fold excess of the positive strand, indicating active viral replication. As the virus was not detected in any insect species collected at this location, the virus was provisionally named "Fragaria vesca-associated virus 1" (FVaV-1).

Genomic characterization of Malus domestica virus A (MdoVA), a novel velarivirus infecting apple.

Screening of apple samples using a high-throughput sequencing (HTS) approach led to the discovery of a novel virus, tentatively named "Malus domestica virus A" (MdoVA). Its genomic organisation and phylogenetic relationship showed relatedness to viruses of the genus Velarivirus in the family Closteroviridae. It is not clear whether MdoVA has any impact on its host, as the analysed apple tree contained other viruses and a viroid.

Molecular and Biological Characterization of a New Strawberry Cytorhabdovirus.

Virus diseases of strawberry present several complex problems. More than 25 viruses have been described in the genus Fragaria thus far. Here, we describe a novel rhabdovirus, tentatively named strawberry virus 1 (StrV-1), that infects F.ananassa and F.vesca plants. Genomic sequences of three distinct StrV-1 genotypes co-infecting a single F.ananassa host were obtained using combined Illumina and Ion Proton high-throughput sequencing. StrV-1 was transmitted to herbaceous plants via Aphisfabae and A.ruborum, further mechanically transmitted to Nicotianaoccidentalis 37B and sub-inoculated to N.benthamiana, N.benthamiana DCL2/4i, N.occidentalis 37B, and Physalisfloridana plants. Irregular chlorotic sectors on leaf blades and the multiplication of calyx leaves seem to be the diagnostic symptoms for StrV-1 on indexed F.vesca clones. StrV-1 was detected in asymptomatic grafted plants and in 49 out of 159 field strawberry samples via RT-PCR followed by Sanger sequencing. The bacilliform shape of the virions, which have a cytoplasm-limited distribution, their size, and phylogenetic relationships support the assignment of StrV-1 to a distinct species of the genus Cytorhabdovirus. Acyrthosiphonmalvae, A.fabae, and A.ruborum were shown to transmit StrV-1 under experimental conditions.

Muckle-Wells Syndrome Across Four Generations in One Czech Family: Natural Course of the Disease.

Background: Muckle-Wells syndrome (MWS) represents a moderate phenotype of cryopyrinopathies. Sensorineural hearing loss and AA amyloidosis belong to the most severe manifestations of uncontrolled disease. Simultaneous discovery of MWS in four generations of one large kindred has enabled us to document natural evolution of untreated disease and their response to targeted therapy. Methods: A retrospective case study, clinical assessment at the time of diagnosis and 2-year prospective follow-up using standardized disease assessments were combined. Results: Collaborative effort of primary care physicians and pediatric and adult specialists led to identification of 11 individuals with MWS within one family. Presence of p.Ala441Val mutation was confirmed. The mildest phenotype of young children suffering with recurrent rash surprised by normal blood tests and absence of fevers. Young adults all presented with fevers, rash, conjunctivitis, and arthralgia/arthritis with raised inflammatory markers. Two patients aged over 50 years suffered with hearing loss and AA amyloidosis. IL-1 blockade induced disease remission in all individuals while hearing mildly improved or remained stable in affected patients as did renal function in one surviving individual with amyloidosis. Conclusions: We have shown that severity of MWS symptoms gradually increased with age toward distinct generation-specific phenotypes. A uniform trajectory of disease evolution has encouraged us to postpone institution of IL-1 blockade in affected oligosymptomatic children. This report illustrates importance of close interdisciplinary collaboration.

Molecular diversity of "Candidatus Phytoplasma mali" strains associated with apple proliferation disease in Bulgarian germplasm collection.

A quarantine organism, "Candidatus Phytoplasma mali," is the causal agent of apple proliferation, one of the most important apple diseases in Europe. The genetic diversity of this pathogen in Central and Southern Europe has already been reported; however, almost no data exists from Eastern Europe. In this study, "Ca. P. mali" strains, which were identified in 14 apple trees from the Bulgarian germplasm collection, were characterized by restriction fragment length polymorphism (RFLP) and sequence analysis of four genomic loci. In total, nine distinct genetic lineages were recognized based on the combination of the following detected RFLP profiles: two profiles for the 16S-23S rDNA region (16SrX-A2, -A3), four profiles for the secY gene (one previously known: secY(X)-A, and three new: secY-C, secY-D, secY-E), three profiles for the rpl22-rps3 genes (rpX-A, rpX-B, rpX-F), and one profile for the nitroreductase- and rhodanese-like gene (AT-1). Phylogenetic analysis of the Bulgarian and other European "Ca. P. mali" strains based on 16S-23S rRNA gene sequences confirmed RFLP grouping, regardless of the phytoplasma origin. In a phylogenetic tree based on the secY data, only German strains formed separate clade from the other strains. The tree based on rp genes did not correspond to RFLP profiles. Unexpectedly, when using nitroreductase and rhodanese-like gene sequences, the Bulgarian strains clustered separately from the other European strains. Apart from the identification of different "Ca. P. mali" strains, the paper also recommends the unification of the rpX-subgroup nomenclature to avoid future confusions. Both aims of this paper provide valuable tools to understand the epidemiology of this quarantine pathogen.

Complete nucleotide sequence and genome organization of red clover associated virus 1 (RCaV1), a putative member of the genus Waikavirus (family Secoviridae, order Picornavirales).

Using high-throughput sequencing, a novel waikavirus was identified in a mixed virus infection of red clover (Trifolium pratense L.). Its complete genomic sequence was determined and characterized. The virus, tentatively named red clover associated virus 1 (RCaV1), is phylogenetically related to members of the genus Waikavirus (family Secoviridae, order Picornavirales).

Red clover-associated luteovirus - a newly classifiable member of the genus Luteovirus with an enamo-like P5 protein.

This study reports the complete genomic sequence of a novel virus isolated from red clover. According to its genomic organization, its similarity to luteoviruses, and a greater than 10% difference in all genes, this virus isolate likely represents a new luteovirus species. As seen in nectarine stem pitting-associated virus (NSPaV) and NSPaV-South Korea (SK) luteoviruses, it differs from typical luteoviruses through the absence of ORF3a and ORF4 encoding movement proteins. Furthermore, its P5 protein (responsible for aphid transmission) is more similar to the P5 of enamoviruses than that of luteoviruses. The virus isolate has been named red clover-associated luteovirus (RCaV).

Complete genome sequences of two divergent isolates of strawberry crinkle virus coinfecting a single strawberry plant.

Strawberry crinkle disease is one of the major diseases that threatens strawberry production. Although the biological properties of the agent, strawberry crinkle virus (SCV), have been thoroughly investigated, its complete genome sequence has never been published. Existing RT-PCR-based detection relies on a partial sequence of the L protein gene, presumably the least expressed viral gene. Here, we present complete sequences of two divergent SCV isolates co-infecting a single plant, Fragaria x ananassa cv. Cacanská raná.

Identification and molecular characterization of a novel varicosa-like virus from red clover.

During aetiological study of diseased red clover (Trifolium pratense L.) using high throughput sequencing, a novel virus with a 10 kb genome divided into two segments was discovered. The virus, tentatively named red clover associated varicosavirus (RCaVV), is phylogenetically related to classifiable members of the genus Varicosavirus (family Rhabdoviridae, order Mononegavirales). Analysis of mRNA levels from the individual RCaVV genes suggested possible differences in transcription regulation between rhabdoviruses with divided and undivided genomes.