Virome analysis of irrigation water sources provides extensive insights into the diversity and distribution of plant viruses in agroecosystems.

Plant viruses pose a significant threat to agriculture. Several are stable outside their hosts, can enter water bodies and remain infective for prolonged periods of time. Even though the quality of irrigation water is of increasing importance in the context of plant health, the presence of plant viruses in irrigation waters is understudied. In this study, we conducted a large-scale high-throughput sequencing (HTS)-based virome analysis of irrigation and surface water sources to obtain complete information about the abundance and diversity of plant viruses in such waters. We detected nucleic acids of plant viruses from 20 families, discovered several novel plant viruses from economically important taxa, like Tobamovirus and observed the influence of the water source on the present virome. By comparing viromes of water and surrounding plants, we observed presence of plant viruses in both compartments, especially in cases of large-scale outbreaks, such as that of tomato mosaic virus. Moreover, we demonstrated that water virome data can extensively inform us about the distribution and diversity of plant viruses for which only limited information is available from plants. Overall, the results of the study provided extensive insights into the virome of irrigation waters from the perspective of plant health. It also suggested that an HTS-based water virome surveillance system could be used to detect potential plant disease outbreaks and to survey the distribution and diversity of plant viruses in the ecosystem.

Cold plasma within a stable supercavitation bubble - A breakthrough technology for efficient inactivation of viruses in water.

Water scarcity, one of the most pressing challenges we face today, has developed for many reasons, including the increasing number of waterborne pollutants that affect the safety of the water environment. Waterborne human, animal and plant viruses represent huge health, environmental, and financial burden and thus it is important to efficiently inactivate them. Therefore, the main objective of this study was to construct a unique device combining plasma with supercavitation and to evaluate its efficiency for water decontamination with the emphasis on inactivation of viruses. High inactivation (>5 log10 PFU/mL) of bacteriophage MS2, a human enteric virus surrogate, was achieved after treatment of 0.43 L of recirculating water for up to 4 min. The key factors in the inactivation were short-lived reactive plasma species that damaged viral RNA. Water treated with plasma for a short time required for successful virus inactivation did not cause cytotoxic effects in the in vitro HepG2 cell model system or adverse effects on potato plant physiology. Therefore, the combined plasma-supercavitation device represents an environmentally-friendly technology that could provide contamination-free and safe water.

Benthic sediment as stores and sources of bacteria and viruses in streams: A comparison of baseflow vs. stormflow longitudinal transport and residence times.

The presence of bacteria and viruses in freshwater represents a global health risk. The substantial spatial and temporal variability of microbes leads to difficulties in quantifying the risks associated with their presence in freshwater. Fine particles, including bacteria and viruses are transported and accumulated into shallow streambed (i.e., benthic) sediment, delaying the downstream transmission during baseflow conditions but contributing to their resuspension and transport downstream during stormflow events. Direct measurements of pathogen accumulation in benthic sediments are rare. Until now, the dynamic role of benthic sediment as both a store and source of microbes, has not been quantified. In this study, we analyze microbial abundance in benthic sediment along a 1 km reach of an intermittent Mediterranean stream receiving inputs from the effluent of a wastewater treatment plant, a known point source of microbes in streams. We sampled benthic sediment during a summer drought when the wastewater effluent constituted 100 % of the stream flow, and thus, large accumulation and persistence of pathogens along the streambed was expected. We measured the abundance of total bacteria, Escherichia coli (as a fecal indicator), and presence of enteric rotavirus (RoV) and norovirus (NoV). The abundance of E. coli, based on qPCR detection, was high (4.99∙102 gc /cm2) along the first 100 m downstream of the wastewater effluent input and in general decreased with distance from the source, with presence of RoV and NoV along the study reach. A particle tracking model was applied, that uses stream water velocity as an input, and accounts for microbial exchange into, immobilization, degradation, and resuspension out of benthic sediment during baseflow and stormflow. Rates of exchange into benthic sediment were 3 orders of magnitude higher during stormflow, but residence times were proportionately lower, resulting in increased longitudinal connectivity from up to downstream during stormflow. Model simulations demonstrated mechanistically how the rates of exchange into and out of the benthic sediment resulted in benthic sediment to act as a store during baseflow and a source during stormflow.

Tomato brown rugose fruit virus in aqueous environments - survival and significance of water-mediated transmission.

Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the question of the significance of the detection of ToBRFV in water samples and experimental studies were performed to address this question. The data presented here confirm that the release of virus particles from the roots of infected plants is a source of infectious ToBRFV particles in water and that the virus can remain infective up to four weeks in water stored at room temperature, while its RNA can be detected for much longer. These data also indicate that irrigation with ToBRFV-contaminated water can lead to plant infection. In addition, it has been shown that ToBRFV circulated in drain water in commercial tomato greenhouses from other European countries and that an outbreak of ToBRFV can be detected by regular monitoring of drain water. A simple method for concentrating ToBRFV from water samples and a comparison of the sensitivity of different methods, including the determination of the highest ToBRFV dilution still capable of infecting test plants, were also investigated. The results of our studies fill the knowledge gaps in the epidemiology and diagnosis of ToBRFV, by studying the role of water-mediated transmission, and provide a reliable risk assessment to identify critical points for monitoring and control.

Inactivation of the enveloped virus phi6 with hydrodynamic cavitation.

The COVID -19 pandemic reminded us that we need better contingency plans to prevent the spread of infectious agents and the occurrence of epidemics or pandemics. Although the transmissibility of SARS-CoV-2 in water has not been confirmed, there are studies that have reported on the presence of infectious coronaviruses in water and wastewater samples. Since standard water treatments are not designed to eliminate viruses, it is of utmost importance to explore advanced treatment processes that can improve water treatment and help inactivate viruses when needed. This is the first study to investigate the effects of hydrodynamic cavitation on the inactivation of bacteriophage phi6, an enveloped virus used as a SARS-CoV-2 surrogate in many studies. In two series of experiments with increasing and constant sample temperature, virus reduction of up to 6.3 logs was achieved. Inactivation of phi6 at temperatures of 10 and 20 °C occurs predominantly by the mechanical effect of cavitation and results in a reduction of up to 4.5 logs. At 30 °C, the reduction increases to up to 6 logs, where the temperature-induced increased susceptibility of the viral lipid envelope makes the virus more prone to inactivation. Furthermore, the control experiments without cavitation showed that the increased temperature alone is not sufficient to cause inactivation, but that additional mechanical stress is still required. The RNA degradation results confirmed that virus inactivation was due to the disrupted lipid bilayer and not to RNA damage. Hydrodynamic cavitation, therefore, has the potential to inactivate current and potentially emerging enveloped pathogenic viruses in water at lower, environmentally relevant temperatures.

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.

A small bacteriophage protein determines the hierarchy over co-residential jumbo phage in Bacillus thuringiensis serovar israelensis.

Bacillus thuringiensis serovar israelensis is the most widely used biopesticide against insects, including vectors of animal and human diseases. Among several extrachromosomal elements, this endospore-forming entomopathogen harbors two bacteriophages: a linear DNA replicon named GIL01 that does not integrate into the chromosome during lysogeny and a circular-jumbo prophage known as pBtic235. Here, we show that GIL01 hinders the induction of cohabiting prophage pBtic235. The GIL01-encoded small protein, gp7, which interacts with the host LexA repressor, is a global transcription regulator and represses the induction of pBtic235 after DNA damage to presumably allow GIL01 to multiply first. In a complex with host LexA in stressed cells, gp7 down-regulates the expression of more than 250 host and pBtic235 genes, many of which are involved in the cellular functions of genome maintenance, cell-wall transport, and membrane and protein stability. We show that gp7 homologs that are found exclusively in bacteriophages act in a similar fashion to enhance LexA's binding to DNA, while likely also affecting host gene expression. Our results provide evidence that GIL01 influences both its host and its co-resident bacteriophage.

Evaluation of Methods and Processes for Robust Monitoring of SARS-CoV-2 in Wastewater.

The SARS-CoV-2 pandemic has accelerated the development of virus concentration and molecular-based virus detection methods, monitoring systems and overall approach to epidemiology. Early into the pandemic, wastewater-based epidemiology started to be employed as a tool for tracking the virus transmission dynamics in a given area. The complexity of wastewater coupled with a lack of standardized methods led us to evaluate each step of the analysis individually and see which approach gave the most robust results for SARS-CoV-2 monitoring in wastewater. In this article, we present a step-by-step, retrospective view on the method development and implementation for the case of a pilot monitoring performed in Slovenia. We specifically address points regarding the thermal stability of the samples during storage, screening for the appropriate sample concentration and RNA extraction procedures and real-time PCR assay selection. Here, we show that the temperature and duration of the storage of the wastewater sample can have a varying impact on the detection depending on the structural form in which the SARS-CoV-2 target is present. We found that concentration and RNA extraction using Centricon filtration units coupled with Qiagen RNA extraction kit or direct RNA capture and extraction using semi-automated kit from Promega give the most optimal results out of the seven methods tested. Lastly, we confirm the use of N1 and N2 assays developed by the CDC (USA) as the best performing assays among four tested in combination with Fast Virus 1-mastermix. Data show a realistic overall process for method implementation as well as provide valuable information in regards to how different approaches in the analysis compare to one another under the specific conditions present in Slovenia during a pilot monitoring running from the beginning of the pandemic.

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.

Inactivation of Pepper Mild Mottle Virus in Water by Cold Atmospheric Plasma.

Water scarcity is one of the greatest threats for human survival and quality of life, and this is increasingly contributing to the risk of human, animal and plant infections due to waterborne viruses. Viruses are transmitted through polluted water, where they can survive and cause infections even at low concentrations. Plant viruses from the genus Tobamovirus are highly mechanically transmissible, and cause considerable damage to important crops, such as tomato. The release of infective tobamoviruses into environmental waters has been reported, with the consequent risk for arid regions, where these waters are used for irrigation. Virus inactivation in water is thus very important and cold atmospheric plasma (CAP) is emerging in this field as an efficient, safe, and sustainable alternative to classic waterborne virus inactivation methods. In the present study we evaluated CAP-mediated inactivation of pepper mild mottle virus (PMMoV) in water samples. PMMoV is a very resilient water-transmissible tobamovirus that can survive transit through the human digestive tract. The efficiency of PMMoV inactivation was characterized for infectivity and virion integrity, and at the genome level, using test plant infectivity assays, transmission electron microscopy, and molecular methods, respectively. Additionally, the safety of CAP treatment was determined by testing the cytotoxic and genotoxic properties of CAP-treated water on the HepG2 cell line. 5-min treatment with CAP was sufficient to inactivate PMMoV without introducing any cytotoxic or genotoxic effects in the in-vitro cell model system. These data on inactivation of such stable waterborne virus, PMMoV, will encourage further examination of CAP as an alternative for treatment of potable and irrigation waters, and even for other water sources, with emphasis on inactivation of various viruses including enteric viruses.

Cold Plasma, a New Hope in the Field of Virus Inactivation.

Viruses can infect all cell-based organisms, from bacteria to humans, animals, and plants. They are responsible for numerous cases of hospitalization, many deaths, and widespread crop destruction, all of which result in an enormous medical, economical, and biological burden. Each of the currently used decontamination methods has important drawbacks. Cold plasma (CP) has entered this field as a novel, efficient, and clean solution for virus inactivation. We present recent developments in this promising field of CP-mediated virus inactivation, and describe the applications and mechanisms of the inactivation. This is particularly relevant because viral pandemics, such as COVID-19, highlight the need for alternative virus inactivation methods to replace, complement, or upgrade existing procedures.

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.

Generation and in Planta Functional Analysis of Potato Virus Y mutants.

Potato virus Y (PVY), the type member of the genus Potyvirus (family Potyviridae), is the most widespread virus affecting potato and is included in the top five most economically detrimental plant viruses. Recently, the structure of the PVY virion has been determined by cryo-electron microscopy, which has opened the doors to functional studies that explore the involvement of selected amino acids in different stages of the viral cycle. The only way to functionally challenge in planta the role of particular amino acids in the coat protein of PVY, or in other viral proteins, is by using cDNA clones. The use and manipulation of PVY cDNA clones, unlike those of other potyviruses, has been traditionally impaired by the toxicity that certain sequences within the PVY genome pose to Escherichia coli. Here, we describe the use of a published PVY cDNA clone, which harbours introns that overcome the aforementioned toxicity, to explore the effects of different coat protein modifications on viral infection. The protocol includes manipulation of the cDNA clone in E. coli, biolistic inoculation of plants with the constructed clones, observation of the biological effects on plants, quantification of cDNA clones by reverse transcription quantitative PCR, and confirmation of virion formation by transmission electron microscopy. Future possibilities involve the use of PVY cDNA clones tagged with fluorescent protein reporters to allow further insights into the effects of coat protein mutations on the cell-to-cell movement of PVY virions.

Structural basis for the multitasking nature of the potato virus Y coat protein.

Potato virus Y (PVY) is among the most economically important plant pathogens. Using cryoelectron microscopy, we determined the near-atomic structure of PVY's flexuous virions, revealing a previously unknown lumenal interplay between extended carboxyl-terminal regions of the coat protein units and viral RNA. RNA-coat protein interactions are crucial for the helical configuration and stability of the virion, as revealed by the unique near-atomic structure of RNA-free virus-like particles. The structures offer the first evidence for plasticity of the coat protein's amino- and carboxyl-terminal regions. Together with mutational analysis and in planta experiments, we show their crucial role in PVY infectivity and explain the ability of the coat protein to perform multiple biological tasks. Moreover, the high modularity of PVY virus-like particles suggests their potential as a new molecular scaffold for nanobiotechnological applications.

Cold Atmospheric Plasma as a Novel Method for Inactivation of Potato Virus Y in Water Samples.

While one of the biggest problems we are facing today is water scarcity, enormous quantities of water are still being used in irrigation. If contaminated, this water can act as an effective pathway for the spread of disease-causing agents, like viruses. Here, we present a novel, environmentally friendly method known as cold atmospheric plasma for inactivation of viruses in water used in closed irrigation systems. We measured the plasma-mediated viral RNA degradation as well as the plasma-induced loss of viral infectivity using potato virus Y as a model virus due to its confirmed water transmissibility and economic as well as biological importance. We showed that only 1 min of plasma treatment is sufficient for successful inactivation of viruses in water samples with either high or low organic background. The plasma-mediated inactivation was efficient even at markedly higher virus concentrations than those expected in irrigation waters. Obtained results point to reactive oxygen species as the main mode of viral inactivation. Our laboratory-scale experiments confirm for the first time that plasma has an excellent potential as the eukaryotic virus inactivation tool for water sources and could thus provide a cost-effective solution for irrigation mediated plant virus transmission. The outstanding inactivation efficiency demonstrated by plasma treatments in water samples offers further expansions of its application to other water sources such as reused wastewater or contaminated drinking waters, as well as other plant, animal, and human waterborne viruses, ultimately leading to the prevention of water scarcity and numerous human, animal, and plant infections worldwide.

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.

Water-Mediated Transmission of Plant, Animal, and Human Viruses.

Viruses represent the most abundant and diverse of the biological entities in environmental waters, including the seas and probably also freshwater systems. They are important players in ecological networks in waters and influence global biochemical cycling and community composition dynamics. Among the many diverse viruses from terrestrial environments found in environmental waters, some are plant, animal, and/or human pathogens. The majority of pathogenic viral species found in waters are very stable and can survive outside host cells for long periods. The occurrence of such viruses in environmental waters has raised concerns because of the confirmation of the infectivity of waterborne viruses even at very low concentrations. This chapter focuses mainly on the survival of human, animal, and plant pathogenic viruses in aqueous environments, the possibility of their water-mediated transmission, the ecological implications of viruses in water, the methods adapted for detecting such viruses, and how to minimize the risk of viruses spreading through water.

Surveillance of human enteric viruses in coastal waters using concentration with methacrylate monolithic supports prior to detection by RT-qPCR.

This is the first surveillance study using methacrylate monolithic supports to concentrate environmental coastal water samples, prior to molecular target detection by RT-qPCR. Rotaviruses (RoV) and Noroviruses (NoV) were monitored in a polluted area at the Bay of Koper (Gulf of Trieste, Northern Adriatic Sea) and at a nearby bathing area and mussel farm areas. RoV and NoV are released into the Bay of Koper, with higher rates close to the discharge of the wastewater treatment plant, however, they can be detected at recreational and mussel farming areas. Our results showed that water bodies considered safe based on FC concentrations, can still have low, yet potentially infective, concentrations of human viruses.

Monolith Chromatography as Sample Preparation Step in Virome Studies of Water Samples.

Viruses exist in aquatic media and many of them use this media as transmission route. Next-generation sequencing (NGS) technologies have opened new doors in virus research, allowing also to reveal a hidden diversity of viral species in aquatic environments. Not surprisingly, many of the newly discovered viruses are found in environmental fresh and marine waters. One of the problems in virome research can be the low amount of viral nucleic acids present in the sample in contrast to the background ones (host, eukaryotic, prokaryotic, environmental). Therefore, virus enrichment prior to NGS is necessary in many cases. In water samples, an added problem resides in the low concentration of viruses typically present in aquatic media. Different concentration strategies have been used to overcome such limitations. CIM monoliths are a new generation of chromatographic supports that due to their particular structural characteristics are very efficient in concentration and purification of viruses. In this chapter, we describe the use of CIM monolithic chromatography for sample preparation step in NGS studies targeting viruses in fresh or marine water. The step-by-step protocol will include a case study where CIM concentration was used to study the virome of a wastewater sample using NGS.

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).