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.

Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules.

Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, the preparation of high-quality samples has become a bottleneck in the cryo-EM structure-determination pipeline. Macromolecules can be damaged during the purification or preparation of vitrified samples for cryo-EM, making them prone to binding to the grid support, to aggregation or to the adoption of preferential orientations at the air-water interface. Here, it is shown that coating cryo-EM grids with a negatively charged polyelectrolyte, such as single-stranded DNA, before applying the sample reduces the aggregation of macromolecules and improves their distribution. The single-stranded DNA-coated grids enabled the determination of high-resolution structures from samples that aggregated on conventional grids. The polyelectrolyte coating reduces the diffusion of macromolecules and thus may limit the negative effects of the contact of macromolecules with the grid support and blotting paper, as well as of the shear forces on macromolecules during grid blotting. Coating grids with polyelectrolytes can readily be employed in any laboratory dealing with cryo-EM sample preparation, since it is fast, simple, inexpensive and does not require specialized equipment.

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.

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.

High-Throughput Sequencing Analysis of the Bacterial Community in Stone Fruit Phloem Tissues Infected by "Candidatus Phytoplasma prunorum".

"Candidatus Phytoplasma prunorum" (CPp) is a highly destructive phytopathogenic agent in many stone fruit-growing regions in Europe and the surrounding countries. In this work, we focused on documenting entire bacterial community in the phloem tissues of 60 stone fruit trees. Nested PCR and two real-time PCR assays were used to select CPp-positive (group A) and CPp-negative samples (group B). Afterwards, high-throughput amplicon sequencing was performed to assess bacterial community compositions in phloem tissues. The bacterial composition in phloem tissue consisted of 118 distinct genera, represented mainly by Pseudomonas, Acinetobacter, Methylobacterium, Sphingomonas, and Rhizobium. Statistics showed that CPp influenced the bacterial composition of infected plants (group A) and that the bacterial community depended on the geographical origin of the sample. This is the first work focusing on an analysis of the influence of CPp on the bacteria coexisting in the phloem tissues of stone fruit trees.