Diversity and Pathobiology of an Ilarvirus Unexpectedly Detected in Diverse Plants and Global Sequencing Data.

High-throughput sequencing (HTS) and sequence mining tools revolutionized virus detection and discovery in recent years, and implementing them with classical plant virology techniques results in a powerful approach to characterize viruses. An example of a virus discovered through HTS is Solanum nigrum ilarvirus 1 (SnIV1) (Bromoviridae), which was recently reported in various solanaceous plants from France, Slovenia, Greece, and South Africa. It was likewise detected in grapevines (Vitaceae) and several Fabaceae and Rosaceae plant species. Such a diverse set of source organisms is atypical for ilarviruses, thus warranting further investigation. In this study, modern and classical virological tools were combined to accelerate the characterization of SnIV1. Through HTS-based virome surveys, mining of sequence read archive datasets, and a literature search, SnIV1 was further identified from diverse plant and non-plant sources globally. SnIV1 isolates showed relatively low variability compared with other phylogenetically related ilarviruses. Phylogenetic analyses showed a distinct basal clade of isolates from Europe, whereas the rest formed clades of mixed geographic origin. Furthermore, systemic infection of SnIV1 in Solanum villosum and its mechanical and graft transmissibility to solanaceous species were demonstrated. Near-identical SnIV1 genomes from the inoculum (S. villosum) and inoculated Nicotiana benthamiana were sequenced, thus partially fulfilling Koch's postulates. SnIV1 was shown to be seed-transmitted and potentially pollen-borne, has spherical virions, and possibly induces histopathological changes in infected N. benthamiana leaf tissues. Overall, this study provides information to better understand the diversity, global presence, and pathobiology of SnIV1; however, its possible emergence as a destructive pathogen remains uncertain. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

First report of potato virus S and potato virus Y in tomatoes from Croatia.

Potato virus Y (PVY, genus Potyvirus) is an economically important aphid-transmissible virus with a very wide host range reported in many tomato-growing areas (Rivarez et al. 2021). Potato virus S (PVS, genus Carlavirus) has a limited host range (Lin et al. 2014) and occurs in tomato (Predajna et al. 2017), mostly in mixed infections with other viruses. In 2021, greenhouse tomatoes from Vidovec (46° 17' 3.4'' N, 16° 15' 37.0'' E) in the northwestern and Sedlarica (45° 54' 23.0'' N, 17° 12' 0.5'' E) in the eastern regions of Croatia were surveyed for virus-like diseases. In total, 30 plants were sampled (12 from Vidovec and 18 from Sedlarica) showing symptoms of mild mottling, leaf rugosity and mild bronzing followed by leaf necroses later in the season. Nucleic acids were extracted from leaves by adapted CTAB procedure (Murray and Thompson 1980) and DNase treated. Four representative samples from Vidovec and four from Sedlarica were pooled for high throughput sequencing (HTS). After rRNA depletion (RiboMinus™ Plant Kit for RNA-Seq, Invitrogen) and polyA tailing, two location specific libraries (PCR-cDNA sequencing kit, Oxford Nanopore Technologies) were prepared for nanopore HTS on MinION Mk1C device. From Vidovec samples, 459,285 raw reads (mean length 354 nt) were obtained and 206,718 (mean length 446 nt) from Sedlarica and mapped (Minimap2, v.2.17) against Kraken2 viral genome sequences database (https://benlangmead.github.io/aws-indexes/k2). The number of reads mapped to PVS genome was 1004 from Vidovec (coverage depth 1.56) and those mapped to PVY genome were 781 (coverage depth 0.99) and 57 (coverage depth 1), from Vidovec and Sedlarica, respectively. The PVS complete consensus genome from Vidovec (ON468562, 8485 nt) had 99.09% nucleotide identity (BLASTn) to a potato isolate from the Netherlands (MF418030). The PVY consensus genome sequences from Vidovec (ON505007, 9698 nt) and Sedlarica (ON505008, 9698 nt) had respectively 98.37% and 98.48% identities to a tomato isolate from Slovakia (MW685827). Reverse transcription polymerase chain reaction (RT-PCR) was performed for all 30 samples and amplicons were Sanger sequenced, with primers PVS-7773F/PVS-3'endR for a 720 nt PVS genome portion spanning the 3'-part of the CP and a complete 11K gene (Lin et al. 2014) and PVY-2F/2R primers for a 510 nt portion of PVY CP gene (Aramburu et al. 2006). Only one tomato out of 12 ('Borana') from Vidovec harbored PVS in the mixed infection with PVY. Two additional tomatoes from Vidovec and two from Sedlarica were infected solely by PVY. Amplicon sequences of PVS (ON651427) and PVY (ON707000-4, ON734067-8) had 100% identity with the HTS assembled sequences. The PVS isolate from Croatia grouped with PVSO (ordinary) strain in phylogenetic analysis and the PVY isolates from both sites grouped with the PVY-NTN strain (Cox and Jones 2010). Although PVY is considered to be widespread in tomato (Nikolic et al. 2018; Rivarez et al. 2021), this is its first report from Croatia. PVS, newly reported from Croatia here, is probably not associated with the symptoms recorded because the same symptomatology was observed in the singly and mixed infected 'Borana' tomato plants. The occurrence of PVY in the geographically distant (100 km apart) Vidovec and Sedlarica, suggests that it is widespread in the continental Croatia where tomatoes are commercially grown in plastic greenhouses. Further analyses are needed to elucidate PVY and PVS epidemiology and impact on the local tomato production.

Detection of Four New Tomato Viruses in Serbia Using Post Hoc High-Throughput Sequencing Analysis of Samples From a Large-Scale Field Survey.

Tomato production worldwide is affected by numerous plant virus species. The early and accurate detection of viruses is a critical step for disease control. However, the simultaneous detection of the most known tomato viruses can be difficult because of the high number and diversity of tomato-infecting viruses. Here, we have identified four new viruses in Serbia by applying target-independent small RNA high-throughput sequencing (HTS). HTS was applied on pools of samples and separate samples, in total comprising 30 tomato samples that exhibited (severe) virus-like symptoms and were collected in Serbia during three annual surveys (2011 to 2013). These samples had previously tested negative for the presence of 16 tomato viruses using targeted detection methods. Three divergent complete genome sequences of Physostegia chlorotic mottled virus were obtained from different localities, indicating for the first time that this virus is widespread in Serbia and might represent an emergent viral pathogen of tomato. The tomato torrado virus was detected at one locality with devastating yield losses. The southern tomato virus was detected at two localities, and the spinach latent virus was detected at one locality. In addition, we detected the presence of one already-known virus in Serbia, the tomato spotted wilt orthotospovirus. All the HTS results were subsequently confirmed by targeted detection methods. In this study, the successful application of post hoc HTS testing of a limited number of pooled samples resulted in the discovery of new viruses. Thus, our results encourage the use of HTS in research and diagnostic laboratories, including laboratories that have limited resources to resolve disease etiology.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Electrochemical inactivation of cyanobacteria and microcystin degradation using a boron-doped diamond anode - A potential tool for cyanobacterial bloom control.

Cyanobacterial blooms are global phenomena that can occur in calm and nutrient-rich (eutrophic) fresh and marine waters. Human exposure to cyanobacteria and their biologically active products is possible during water sports and various water activities, or by ingestion of contaminated water. Although the vast majority of harmful cyanobacterial products are confined to the interior of the cells, these are eventually released into the surrounding water following natural or artificially induced cell death. Electrochemical oxidation has been used here to damage cyanobacteria to halt their proliferation, and for microcystin degradation under in-vitro conditions. Partially spent Jaworski growth medium with no addition of supporting electrolytes was used. Electrochemical treatment resulted in the cyanobacterial loss of cell-buoyancy regulation, cell proliferation arrest, and eventual cell death. Microcystin degradation was studied separately in two basic modes of treatment: batch-wise flow, and constant flow, for electrolytic-cell exposure. Batch-wise exposure simulates treatment under environmental conditions, while constant flow is more appropriate for the study of boron-doped diamond electrode efficacy under laboratory conditions. The effectiveness of microcystin degradation was established using high-performance liquid chromatography-photodiode array detector analysis, while the biological activities of the products were estimated using a colorimetric protein phosphatase-1 inhibition assay. The results indicate potential for the application of electro-oxidation methods for the control of bloom events by taking advantage of specific intrinsic ecological characteristics of bloom-forming cyanobacteria. The applicability of the use of boron-doped diamond electrodes in remediation of water exposed to cyanobacteria bloom events is discussed.

Detection of Plant Viruses Using Nanopore Sequencing Based Metagenomic Approach.

Nanopore sequencing has proven to be a useful tool for the generic detection of plant viruses, especially in laboratories working with small number of samples. In this chapter, we describe the steps prior to library preparation as well as the library preparation itself, which we found provides comparable results to Illumina sequencing.

In-depth study of tomato and weed viromes reveals undiscovered plant virus diversity in an agroecosystem.

BACKGROUND: In agroecosystems, viruses are well known to influence crop health and some cause phytosanitary and economic problems, but their diversity in non-crop plants and role outside the disease perspective is less known. Extensive virome explorations that include both crop and diverse weed plants are therefore needed to better understand roles of viruses in agroecosystems. Such unbiased exploration is available through viromics, which could generate biological and ecological insights from immense high-throughput sequencing (HTS) data. RESULTS: Here, we implemented HTS-based viromics to explore viral diversity in tomatoes and weeds in farming areas at a nation-wide scale. We detected 125 viruses, including 79 novel species, wherein 65 were found exclusively in weeds. This spanned 21 higher-level plant virus taxa dominated by Potyviridae, Rhabdoviridae, and Tombusviridae, and four non-plant virus families. We detected viruses of non-plant hosts and viroid-like sequences and demonstrated infectivity of a novel tobamovirus in plants of Solanaceae family. Diversities of predominant tomato viruses were variable, in some cases, comparable to that of global isolates of the same species. We phylogenetically classified novel viruses and showed links between a subgroup of phylogenetically related rhabdoviruses to their taxonomically related host plants. Ten classified viruses detected in tomatoes were also detected in weeds, which might indicate possible role of weeds as their reservoirs and that these viruses could be exchanged between the two compartments. CONCLUSIONS: We showed that even in relatively well studied agroecosystems, such as tomato farms, a large part of very diverse plant viromes can still be unknown and is mostly present in understudied non-crop plants. The overlapping presence of viruses in tomatoes and weeds implicate possible presence of virus reservoir and possible exchange between the weed and crop compartments, which may influence weed management decisions. The observed variability and widespread presence of predominant tomato viruses and the infectivity of a novel tobamovirus in solanaceous plants, provided foundation for further investigation of virus disease dynamics and their effect on tomato health. The extensive insights we generated from such in-depth agroecosystem virome exploration will be valuable in anticipating possible emergences of plant virus diseases and would serve as baseline for further post-discovery characterization studies. Video Abstract.

Systematic Comparison of Nanopore and Illumina Sequencing for the Detection of Plant Viruses and Viroids Using Total RNA Sequencing Approach.

High-throughput sequencing (HTS) has become an important tool for plant virus detection and discovery. Nanopore sequencing has been rapidly developing in the recent years and offers new possibilities for fast diagnostic applications of HTS. With this in mind, a study was completed, comparing the most established HTS platform (MiSeq benchtop sequencer-Illumina), with the MinION sequencer (Oxford Nanopore Technologies) for the detection of plant viruses and viroids. Method comparisons were performed on five selected samples, containing two viroids, which were sequenced using nanopore technology for the first time and 11 plant viruses with different genome organizations. For all samples, sequencing libraries for the MiSeq were prepared from ribosomal RNA-depleted total RNA (rRNA-depleted totRNA) and for MinION sequencing, direct RNA sequencing of totRNA was used. Moreover, for one of the samples, which contained five different plant viruses and a viroid, three additional variations of sample preparation for MinION sequencing were also used: direct RNA sequencing of rRNA-depleted totRNA, cDNA-PCR sequencing of totRNA, and cDNA-PCR sequencing of rRNA-depleted totRNA. Whilst direct RNA sequencing of total RNA was the quickest of the tested approaches, it was also the least sensitive: using this approach, we failed to detect only one virus that was present in a sample at an extremely low titer. All other MinION sequencing approaches showed improved performance with outcomes similar to Illumina sequencing, with cDNA-PCR sequencing of rRNA-depleted totRNA showing the best performance amongst tested nanopore MinION sequencing approaches. Moreover, when enough sequencing data were generated, high-quality consensus viral genome sequences could be reconstructed from MinION sequencing data, with high identity to the ones generated from Illumina data. The results of this study implicate that, when an appropriate sample and library preparation are selected, nanopore MinION sequencing could be used for the detection of plant viruses and viroids with similar performance as Illumina sequencing. Taken as a balance of practicality and performance, this suggests that MinION sequencing may be an ideal tool for fast and affordable virus diagnostics.

Spider webs as eDNA samplers: Biodiversity assessment across the tree of life.

The concept of environmental DNA (eDNA) utilizes nucleic acids of organisms directly from the environment. Recent breakthrough studies have successfully detected a wide spectrum of prokaryotic and eukaryotic eDNA from a variety of environments, ranging from ancient to modern, and from terrestrial to aquatic. With their diversity and ubiquity in nature, spider webs might act as powerful biofilters and could thus represent a promising new source of eDNA, but their utility under natural field conditions is severely understudied. Here, we bridge this knowledge gap to establish spider webs as a source of eDNA with far reaching implications. First, we conducted a field study to track specific arthropod targets from different spider webs. We then used high-throughput amplicon sequencing of taxonomic barcodes to investigate the utility of spider web eDNA for biodiversity monitoring of animals, fungi and bacteria. Our results show that genetic remains on spider webs allow the detection of even the smallest target organisms. We also demonstrate that eDNA from spider webs is useful in research of community compositions across the different domains of life, with potentially highly detailed temporal and spatial information.

Interlaboratory Comparison Study on Ribodepleted Total RNA High-Throughput Sequencing for Plant Virus Diagnostics and Bioinformatic Competence.

High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data.

Viromics and infectivity analysis reveal the release of infective plant viruses from wastewater into the environment.

Viruses represent one of the most important threats to agriculture. Several viral families include highly stable pathogens, which remain infective and can be transported long distances in water. The diversity of plant viruses in wastewater remains understudied; however, their potential impact is increasing with the increased irrigation usage of reclaimed wastewater. To determine the abundance, diversity and biological relevance of plant viruses in wastewater influents and effluents we applied an optimized virus concentration method followed by high-throughput sequencing and infectivity assays. We detected representatives of 47 plant virus species, including emerging crop threats. We also demonstrated infectivity for pathogenic and economically relevant plant viruses from the genus Tobamovirus (family Virgaviridae), which remain infective even after conventional wastewater treatment. These results demonstrate the potential of metagenomics to capture the diversity of plant viruses circulating in the environment and expose the potential risk of the uncontrolled use of reclaimed water for irrigation.

High-Throughput Sequencing Facilitates Characterization of a "Forgotten" Plant Virus: The Case of a Henbane Mosaic Virus Infecting Tomato.

High-throughput sequencing has dramatically broadened the possibilities for plant virus research and diagnostics, enabling discovery of new or obscure viruses, and virus strains and rapid sequencing of their genomes. In this research, we employed high-throughput sequencing to discover a new virus infecting tomato, Henbane mosaic virus (Potyvirus, Potyviridae), which was first discovered at the beginning of 20th century in the United Kingdom in cultivated henbane. A field tomato plant with severe necrotic symptoms of unknown etiology was sampled in Slovenia and high-throughput sequencing analysis using small RNA and ribosomal RNA depleted total RNA approaches revealed a mixed infection with Potato virus M (Carlavirus, Betaflexiviridae), Southern tomato virus (Amalgavirus, Amalgamaviridae) and henbane mosaic virus in the sample. The complete genomic sequence of henbane mosaic virus was assembled from the sequencing reads. By re-inoculation of the infected material on selected test plants, henbane mosaic virus was isolated and a host range analysis was performed, demonstrating the virus was pathogenic on several plant species. Due to limited metadata in public repositories, the taxonomic identification of the virus isolate was initially putative. Thus, in the next step, we used small RNA sequencing to determine genomic sequences of four historic isolates of the virus, obtained from different virus collections. Phylogenetic analyses performed using this new sequence information enabled us to taxonomically position Henbane mosaic virus as a member of the Potyvirus genus within the chili veinal mottle virus phylogenetic cluster and define the relationship of the new tomato isolate with the historic ones, indicating the existence of at least four putative strains of the virus. The first detection of henbane mosaic virus in tomato and demonstration of its pathogenicity on this host is important for plant protection and commercial tomato production. Since the virus was initially present in a mixed infection, and its whole genome was not sequenced, it has probably been overlooked in routine diagnostics. This study confirms the applicability of a combination of high-throughput sequencing and classic plant virus characterization methods for identification and phylogenetic classification of obscure viruses and historical viral isolates, for which no or limited genome sequence data is available.

Next Generation Sequencing for Detection and Discovery of Plant Viruses and Viroids: Comparison of Two Approaches.

Next generation sequencing (NGS) technologies are becoming routinely employed in different fields of virus research. Different sequencing platforms and sample preparation approaches, in the laboratories worldwide, contributed to a revolution in detection and discovery of plant viruses and viroids. In this work, we are presenting the comparison of two RNA sequence inputs (small RNAs vs. ribosomal RNA depleted total RNA) for the detection of plant viruses by Illumina sequencing. This comparison includes several viruses, which differ in genome organization and viroids from both known families. The results demonstrate the ability for detection and identification of a wide array of known plant viruses/viroids in the tested samples by both approaches. In general, yield of viral sequences was dependent on viral genome organization and the amount of viral reads in the data. A putative novel Cytorhabdovirus, discovered in this study, was only detected by analysing the data generated from ribosomal RNA depleted total RNA and not from the small RNA dataset, due to the low number of short reads in the latter. On the other hand, for the viruses/viroids under study, the results showed higher yields of viral sequences in small RNA pool for viroids and viruses with no RNA replicative intermediates (single stranded DNA viruses).