Mottle: Accurate pairwise substitution distance at high divergence through the exploitation of short-read mappers and gradient descent.

Current tools for estimating the substitution distance between two related sequences struggle to remain accurate at a high divergence. Difficulties at distant homologies, such as false seeding and over-alignment, create a high barrier for the development of a stable estimator. This is especially true for viral genomes, which carry a high rate of mutation, small size, and sparse taxonomy. Developing an accurate substitution distance measure would help to elucidate the relationship between highly divergent sequences, interrogate their evolutionary history, and better facilitate the discovery of new viral genomes. To tackle these problems, we propose an approach that uses short-read mappers to create whole-genome maps, and gradient descent to isolate the homologous fraction and calculate the final distance value. We implement this approach as Mottle. With the use of simulated and biological sequences, Mottle was able to remain stable to 0.66-0.96 substitutions per base pair and identify viral outgroup genomes with 95% accuracy at the family-order level. Our results indicate that Mottle performs as well as existing programs in identifying taxonomic relationships, with more accurate numerical estimation of genomic distance over greater divergences. By contrast, one limitation is a reduced numerical accuracy at low divergences, and on genomes where insertions and deletions are uncommon, when compared to alternative approaches. We propose that Mottle may therefore be of particular interest in the study of viruses, viral relationships, and notably for viral discovery platforms, helping in benchmarking of homology search tools and defining the limits of taxonomic classification methods. The code for Mottle is available at https://github.com/tphoward/Mottle_Repo.

Correction: Haegeman et al. Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests. Plants 2023, 12, 2139.

In the original publication [...].

Managing the deluge of newly discovered plant viruses and viroids: an optimized scientific and regulatory framework for their characterization and risk analysis.

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties.

Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests.

High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.

Enhanced Apiaceous Potyvirus Phylogeny, Novel Viruses, and New Country and Host Records from Sequencing Apiaceae Samples.

The family Apiaceae comprises approximately 3700 species of herbaceous plants, including important crops, aromatic herbs and field weeds. Here we report a study of 10 preserved historical or recent virus samples of apiaceous plants collected in the United Kingdom (UK) import interceptions from the Mediterranean region (Egypt, Israel and Cyprus) or during surveys of Australian apiaceous crops. Seven complete new genomic sequences and one partial sequence, of the apiaceous potyviruses apium virus Y (ApVY), carrot thin leaf virus (CaTLV), carrot virus Y (CarVY) and celery mosaic virus (CeMV) were obtained. When these 7 and 16 earlier complete non-recombinant apiaceous potyvirus sequences were subjected to phylogenetic analyses, they split into 2 separate lineages: 1 containing ApVY, CeMV, CarVY and panax virus Y and the other CaTLV, ashitabi mosaic virus and konjac virus Y. Preliminary dating analysis suggested the CarVY population first diverged from CeMV and ApVY in the 17th century and CeMV from ApVY in the 18th century. They also showed the "time to most recent common ancestor" of the sampled populations to be more recent: 1997 CE, 1983 CE and 1958 CE for CarVY, CeMV and ApVY, respectively. In addition, we found a new family record for beet western yellows virus in coriander from Cyprus; a new country record for carrot torradovirus-1 and a tentative novel member of genus Ophiovirus as a co-infection in a carrot sample from Australia; and a novel member of the genus Umbravirus recovered from a sample of herb parsley from Israel.

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.

Development of simplex and multiplex RT-qPCR assays for the detection of three cryptic viruses of black-grass (Alopecurus myosuroides).

Simplex and multiplex RT-qPCR assays were developed for Alopecurus myosuroides partitivirus 1 (AMPV1), Alopecurus myosuroides partitivirus 2 (AMPV2) and Alopecurus myosuroides varicosavirus 1 (AMVV1), and compared to the existing conventional PCR assays. All assays had a high specificity and their sensitivity was increased compared to the conventional RT-PCR assays. As viral quantification is an important element in comparative experiments, the effect of high- and low-temperature drying treatments, prior to RNA extraction and analysis, was studied and optimised. AMVV1 detection was reduced by both drying treatments, but particularly by the high-temperature. AMPV1 and AMPV2 detection on the other hand was not impeded by the drying treatments, and enables standardisation of plant tissue prior to extraction, in particular for quantitative analysis.

A novel high-throughput sequencing approach reveals the presence of a new virus infecting Rosa: rosa ilarvirus-1 (RIV-1).

Roses are one of the most valuable ornamental flowering shrubs grown worldwide. Despite the widespread of rose viruses and their impact on cultivation, they have not been studied in detail in the United Kingdom (UK) since the 1980's. As part of a survey of rose viruses entering the UK, 35 samples were collected at Heathrow Airport (London, UK) and were tested by RT-qPCR for different common rose viruses. Of the 35 samples tested using RT-qPCR for prunus necrotic ringspot virus (PNRSV; genus Ilarvirus), 10 were positive. Confirmatory testing was performed using RT-PCR with both PNRSV-specific and ilarvirus-generic primers, and diverse results were obtained: One sample was exclusively positive when using the ilarvirus-generic primers, and subsequent sequencing of the RT-PCR product revealed homology to other ilarviruses but not PNRSV. Further work to characterise the virus was performed using high throughput sequencing, both the MinION Flongle and Illumina MiSeq. The sequencing confirmed the presence of a new virus within group 2 of the genus Ilarvirus and we propose the name "rosa ilarvirus-1″ (RIV-1). Here, we describe the identification of a novel virus using the low-cost Flongle flow cell and discuss its potential as a front-line diagnostic tool.

The Phylogeography of Potato Virus X Shows the Fingerprints of Its Human Vector.

Potato virus X (PVX) occurs worldwide and causes an important potato disease. Complete PVX genomes were obtained from 326 new isolates from Peru, which is within the potato crop's main domestication center, 10 from historical PVX isolates from the Andes (Bolivia, Peru) or Europe (UK), and three from Africa (Burundi). Concatenated open reading frames (ORFs) from these genomes plus 49 published genomic sequences were analyzed. Only 18 of them were recombinants, 17 of them Peruvian. A phylogeny of the non-recombinant sequences found two major (I, II) and five minor (I-1, I-2, II-1, II-2, II-3) phylogroups, which included 12 statistically supported clusters. Analysis of 488 coat protein (CP) gene sequences, including 128 published previously, gave a completely congruent phylogeny. Among the minor phylogroups, I-2 and II-3 only contained Andean isolates, I-1 and II-2 were of both Andean and other isolates, but all of the three II-1 isolates were European. I-1, I-2, II-1 and II-2 all contained biologically typed isolates. Population genetic and dating analyses indicated that PVX emerged after potato's domestication 9000 years ago and was transported to Europe after the 15th century. Major clusters A-D probably resulted from expansions that occurred soon after the potato late-blight pandemic of the mid-19th century. Genetic comparisons of the PVX populations of different Peruvian Departments found similarities between those linked by local transport of seed potato tubers for summer rain-watered highland crops, and those linked to winter-irrigated crops in nearby coastal Departments. Comparisons also showed that, although the Andean PVX population was diverse and evolving neutrally, its spread to Europe and then elsewhere involved population expansion. PVX forms a basal Potexvirus genus lineage but its immediate progenitor is unknown. Establishing whether PVX's entirely Andean phylogroups I-2 and II-3 and its Andean recombinants threaten potato production elsewhere requires future biological studies.

A Primer on the Analysis of High-Throughput Sequencing Data for Detection of Plant Viruses.

High-throughput sequencing (HTS) technologies have become indispensable tools assisting plant virus diagnostics and research thanks to their ability to detect any plant virus in a sample without prior knowledge. As HTS technologies are heavily relying on bioinformatics analysis of the huge amount of generated sequences, it is of utmost importance that researchers can rely on efficient and reliable bioinformatic tools and can understand the principles, advantages, and disadvantages of the tools used. Here, we present a critical overview of the steps involved in HTS as employed for plant virus detection and virome characterization. We start from sample preparation and nucleic acid extraction as appropriate to the chosen HTS strategy, which is followed by basic data analysis requirements, an extensive overview of the in-depth data processing options, and taxonomic classification of viral sequences detected. By presenting the bioinformatic tools and a detailed overview of the consecutive steps that can be used to implement a well-structured HTS data analysis in an easy and accessible way, this paper is targeted at both beginners and expert scientists engaging in HTS plant virome projects.

Development and Validation of Methodology for Estimating Potato Canopy Structure for Field Crop Phenotyping and Improved Breeding.

Traditional phenotyping techniques have long been a bottleneck in breeding programs and genotype- phenotype association studies in potato, as these methods are labor-intensive and time consuming. In addition, depending on the trait measured and metric adopted, they suffer from varying degrees of user bias and inaccuracy, and hence these challenges have effectively prevented the execution of large-scale population-based field studies. This is true not only for commercial traits (e.g., yield, tuber size, and shape), but also for traits strongly associated with plant performance (e.g., canopy development, canopy architecture, and growth rates). This study demonstrates how the use of point cloud data obtained from low-cost UAV imaging can be used to create 3D surface models of the plant canopy, from which detailed and accurate data on plant height and its distribution, canopy ground cover and canopy volume can be obtained over the growing season. Comparison of the canopy datasets at different temporal points enabled the identification of distinct patterns of canopy development, including different patterns of growth, plant lodging, maturity and senescence. Three varieties are presented as exemplars. Variety Nadine presented the growth pattern of an early maturing variety, showing rapid initial growth followed by rapid onset of senescence and plant death. Varieties Bonnie and Bounty presented the pattern of intermediate to late maturing varieties, with Bonnie also showing early canopy lodging. The methodological approach used in this study may alleviate one of the current bottlenecks in the study of plant development, paving the way for an expansion in the scale of future genotype-phenotype association studies.

Plant pest surveillance: from satellites to molecules.

Plant pests and diseases impact both food security and natural ecosystems, and the impact has been accelerated in recent years due to several confounding factors. The globalisation of trade has moved pests out of natural ranges, creating damaging epidemics in new regions. Climate change has extended the range of pests and the pathogens they vector. Resistance to agrochemicals has made pathogens, pests, and weeds more difficult to control. Early detection is critical to achieve effective control, both from a biosecurity as well as an endemic pest perspective. Molecular diagnostics has revolutionised our ability to identify pests and diseases over the past two decades, but more recent technological innovations are enabling us to achieve better pest surveillance. In this review, we will explore the different technologies that are enabling this advancing capability and discuss the drivers that will shape its future deployment.

Target-Site and Non-target-Site Resistance Mechanisms Confer Multiple and Cross- Resistance to ALS and ACCase Inhibiting Herbicides in Lolium rigidum From Spain.

Lolium rigidum is one the worst herbicide resistant (HR) weeds worldwide due to its proneness to evolve multiple and cross resistance to several sites of action (SoA). In winter cereals crops in Spain, resistance to acetolactate synthase (ALS)- and acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has become widespread, with farmers having to rely on pre-emergence herbicides over the last two decades to maintain weed control. Recently, lack of control with very long-chain fatty acid synthesis (VLCFAS)-inhibiting herbicides has been reported in HR populations that are difficult to manage by chemical means. In this study, three Spanish populations of L. rigidum from winter cereals were confirmed as being resistant to ALS- and ACCase-inhibiting herbicides, with broad-ranging resistance toward the different chemistries tested. In addition, reduced sensitivity to photosystem II-, VLCFAS-, and phytoene desaturase-inhibiting herbicides were confirmed across the three populations. Resistance to ACCase-inhibiting herbicides was associated with point mutations in positions Trp-2027 and Asp-2078 of the enzyme conferring target site resistance (TSR), while none were detected in the ALS enzyme. Additionally, HR populations contained enhanced amounts of an ortholog of the glutathione transferase phi (F) class 1 (GSTF1) protein, a functional biomarker of non-target-site resistance (NTSR), as confirmed by enzyme-linked immunosorbent assays. Further evidence of NTSR was obtained in dose-response experiments with prosulfocarb applied post-emergence, following pre-treatment with the cytochrome P450 monooxygenase inhibitor malathion, which partially reversed resistance. This study confirms the evolution of multiple and cross resistance to ALS- and ACCase inhibiting herbicides in L. rigidum from Spain by mechanisms consistent with the presence of both TSR and NTSR. Moreover, the results suggest that NTSR, probably by means of enhanced metabolism involving more than one detoxifying enzyme family, confers cross resistance to other SoA. The study further demonstrates the urgent need to monitor and prevent the further evolution of herbicide resistance in L. rigidum in Mediterranean areas.

Potato Virus A Isolates from Three Continents: Their Biological Properties, Phylogenetics, and Prehistory.

Forty-seven potato virus A (PVA) isolates from Europe, Australia, and South America's Andean region were subjected to high-throughput sequencing, and 46 complete genomes from Europe (n = 9), Australia (n = 2), and the Andes (n = 35) obtained. These and 17 other genomes gave alignments of 63 open reading frames 9,180 nucleotides long; 9 were recombinants. The nonrecombinants formed three tightly clustered, almost equidistant phylogroups; A comprised 14 Peruvian potato isolates; W comprised 37 from potato in Peru, Argentina, and elsewhere in the world; and T contained three from tamarillo in New Zealand. When five isolates were inoculated to a potato cultivar differential, three strain groups (= pathotypes) unrelated to phylogenetic groupings were recognized. No temporal signal was detected among the dated nonrecombinant sequences, but PVA and potato virus Y (PVY) are from related lineages and ecologically similar; therefore, "relative dating" was obtained using a single maximum-likelihood phylogeny of PVA and PVY sequences and PVY's well-supported 157 CE "time to most common recent ancestor". The PVA datings obtained were supported by several independent historical coincidences. The PVA and PVY populations apparently arose in the Andes approximately 18 centuries ago, and were taken to Europe during the Columbian Exchange, radiating there after the mid-19th century potato late blight pandemic. PVA's phylogroup A population diverged more recently in the Andean region, probably after new cultivars were bred locally using newly introduced Solanum tuberosum subsp. tuberosum as a parent. Such cultivars became widely grown, and apparently generated the A × W phylogroup recombinants. Phylogroup A, and its interphylogroup recombinants, might pose a biosecurity risk.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Monitoring and Surveillance of Aerial Mycobiota of Rice Paddy through DNA Metabarcoding and qPCR.

The airborne mycobiota has been understudied in comparison with the mycobiota present in other agricultural environments. Traditional, culture-based methods allow the study of a small fraction of the organisms present in the atmosphere, thus missing important information. In this study, the aerial mycobiota in a rice paddy has been examined during the cropping season (from June to September 2016) using qPCRs for two important rice pathogens (Pyricularia oryzae and Bipolaris oryzae) and by using DNA metabarcoding of the fungal ITS region. The metabarcoding results demonstrated a higher alpha diversity (Shannon-Wiener diversity index H' and total number of observed species) at the beginning of the trial (June), suggesting a higher level of community complexity, compared with the end of the season. The main taxa identified by HTS analysis showed a shift in their relative abundance that drove the cluster separation as a function of time and temperature. The most abundant OTUs corresponded to genera such as Cladosporium, Alternaria, Myrothecium, or Pyricularia. Changes in the mycobiota composition were clearly dependent on the average air temperature with a potential impact on disease development in rice. In parallel, oligotyping analysis was performed to obtain a sub-OTU identification which revealed the presence of several oligotypes of Pyricularia and Bipolaris with relative abundance changing during monitoring.

A pond-side test for Guinea worm: Development of a loop-mediated isothermal amplification (LAMP) assay for detection of Dracunculus medinensis.

Guinea worm Dracunculus medinensis causes debilitating disease in people and is subject to an ongoing global eradication programme. Research and controls are constrained by a lack of diagnostic tools. We developed a specific and sensitive LAMP method for detecting D. medinensis larval DNA in copepod vectors. We were able to detect a single larva in a background of field-collected copepods. This method could form the basis of a "pond-side test" for detecting potential sources of Guinea worm infection in the environment, in copepods, including in the guts of fish as potential transport hosts, enabling research, surveillance and targeting of control measures. The key constraint on the utility of this assay as a field diagnostic, is a lack of knowledge of variation in the temporal and spatial distribution of D. medinensis larvae in copepods in water bodies in the affected areas and how best to sample copepods to obtain a reliable diagnostic sample. These fundamental knowledge gaps could readily be addressed with field collections of samples across areas experiencing a range of worm infection frequencies, coupled with field and laboratory analyses using LAMP and PCR.

Complete Coding Sequence of Andean Potato Mottle Virus from a 40-Year-Old Sample from Peru.

A complete coding sequence of the type strain of Andean potato mottle virus from Peru (isolate Lm) was obtained. Comparison of its RNA1 and RNA2 sequences with variants of this virus isolated in Brazil revealed RNA1 and RNA2 nucleotide identities of 81 to 83% and 70 to 71%, respectively.

Genomic sequence and host range studies reveal considerable variation within the species Arracacha virus B.

Arracacha virus B type (AVB-T) and oca (AVB-O) strains from arracacha (Arracacia xanthorrhiza) and oca (Oxalis tuberosa) samples collected in 1975 and two additional isolates obtained from arracacha (AVB-PX) and potato (AVB-6A) in Peru in 1976 and 1978, respectively, were studied. In its host responses and serological properties, AVB-PX most resembled AVB-T, whereas AVB-6A most resembled AVB-O. Complete genomic sequences of the RNA-1 and RNA-2 of each isolate were obtained following high-throughput sequencing of RNA extracts from isolates preserved for 38 (AVB-PX) or 32 (the other 3 isolates) years, and compared with a genomic sequence of AVB-O obtained previously (PV-0082). RNA-2 was unexpectedly divergent compared to RNA-1, with the nucleotide (nt) sequence identity of different AVB isolates varying by up to 76% (RNA-2) and 89% (RNA-1). The coat protein amino acid sequences were the most divergent, with AVB-O and AVB-6A having only 68% identity to AVB-T and AVB-PX. Since the RNA2 sequence differences between the two isolate groupings also coincided with host range, symptom, and serological differences, AVB demonstrates considerable intraspecific divergence.

Rapid Detection of Monilinia fructicola and Monilinia laxa on Peach and Nectarine using Loop-Mediated Isothermal Amplification.

Monilinia laxa and M. fructicola are two causal agents of brown rot, one of the most important diseases in stone fruit. Two species cause blight on blossoms and twigs and brown rot on fruit in pre- and postharvest. Both species are distributed worldwide in North and South America, Australia, and Japan. In Europe, M. laxa is endemic, while M. fructicola was introduced in 2001 and it is now widespread in several countries. Currently, both species coexist in European stone fruit orchards. Monilinia spp. overwinter in cankers and mummified fruit. Mummy monitoring during winter permits growers to understand which species of Monilinia will be prevalent in an orchard during the following season, permitting planning of an appropriate crop protection. Traditionally, the identification has been carried out using morphological features and even with polymerase chain reaction (PCR)-based assays that requires time and well-equipped laboratories. In this study, two isothermal-based methods were designed to identify these pathogens in a faster way than using traditional methods. The loop-mediated amplification (LAMP) assays were validated on some isolates of Monilinia spp. coming from the mummy monitoring according to the international European and Mediterranean Plant Protection Organization standard (PM7/98), taking into account specificity, sensitivity, repeatability, and reproducibility. The sensitivity of both assays was checked by monitoring (at different time points) two nectarine varieties artificially inoculated and stored at two different temperatures. The reliability of both LAMP assays against the quantification of the inoculum was compared with previously published quantitative PCR assays. Both LAMP methods were able to detect a low number of cells. These LAMP methods could be a useful tool for monitoring brown rot causal agents in the field and during postharvest.

Biological and Molecular Properties of Wild potato mosaic virus Isolates from Pepino (Solanum muricatum).

In 1976, a virus with flexuous, filamentous virions typical of the family Potyviridae was isolated from symptomatic pepino (Solanum muricatum) plants growing in two valleys in Peru's coastal desert region. In 2014, a virus with similar-shaped virions was isolated from asymptomatic fruits obtained from pepino plants growing in six coastal valleys and a valley in Peru's Andean highlands. Both were identified subsequently as Wild potato mosaic virus (WPMV) by serology or high-throughput sequencing (HTS). The symptoms caused by two old and seven new isolates from pepino were examined in indicator plants. Infected solanaceous hosts varied considerably in their sensitivities to infection and individual isolates varied greatly in virulence. All seven new isolates caused quick death of infected Nicotiana benthamiana plants and more than half of them killed infected plants of Physalis floridana and S. chancayense. These three species were the most sensitive to infection. The most virulent isolate was found to be BA because it killed five of eight solanaceous host species whereas CA was the least severe because it only killed N. benthamiana. Using HTS, complete genomic sequences of six isolates were obtained, with one isolate (FE) showing evidence of recombination. The distances between individual WPMV isolates in phylogenetic trees and the geographical distances between their collection sites were found to be unrelated. The individual WPMV isolates displayed nucleotide sequence identities of 80.9-99.8%, whereas the most closely related virus, Potato virus V (PVV), was around 75% identical to WPMV. WPMV, PVV, and Peru tomato virus formed clusters of similar phylogenetic diversity, and were found to be distinct but related viruses within the overall Potato virus Y lineage. WPMV infection seems widespread and of likely economic significance to pepino producers in Peru's coastal valleys. Because it constitutes the fifth virus found infecting pepino and this crop is entirely vegetatively propagated, development of healthy pepino stock programs is advocated.