First Report of Melon Severe Mosaic Orthotospovirus infecting cucurbits in the United States.

Cucurbits (family Cucurbitaceae) includes globally important fruit and vegetable crops. Virus diseases pose a serious threat to cucurbits, limiting crop quality and yield (Regina et al. 2021). In fall 2023, leaf and fruit samples from two squash plants with chlorotic mosaic symptoms and fruit distortion from Monroe and Pope counties in Arkansas were received for diagnosis at the University of Arkansas Division of Agriculture Plant Clinic. Based on symptoms, samples were assessed for melon severe mosaic orthotospovirus (MeSMV) using the ImmunoStrip® developed for detection of the virus (Agdia® Inc., Elkhart, Indiana). The presence of MeSMV was also confirmed by RT-PCR using the Agdia Tospovirus group PCR primers. An amplicon was sequenced and showed 91% sequence identity to the MESMV type isolate (NC_033834, VE440-A). To further verify the results, nucleic acids from a squash sample from Pope County were extracted as described by Poudel et al. (2013), DNase treated, and sequenced on an Oxford Nanopore MinION as described by Liefting et al. (2021). A total of 25,914 raw reads were analyzed using VirFind (Ho and Tzanetakis 2014), which identified 112 reads mapping to the three segments of MeSMV. Primers for all three RNAs were developed and amplified 638, 650, and 1153 nt of the S, M, and L segments of the virus respectively. The amplicons were sequenced bidirectionally and show 89-93% identity to the type isolate from Mexico (GenBank accessions PP301332-4). MeSMV has only been identified in Mexico and can cause significant losses to honeydew melon, zucchini, and cucumber (Ciuffo et al. 2009). Thus, this is the first report of MeSMV outside Mexico. Given the severity of the symptoms observed in cucurbit crops, the virus poses a potential threat to the cucurbit industry in the United States. Growers should be aware of this virus and take the necessary precautions to prevent its spread in the field.

Integration of Rubus yellow net virus in the raspberry genome: A story centuries in the making.

Rubus yellow net virus (RYNV) belongs to genus Badnavirus. Badnaviruses are found in plants as endogenous, inactive or activatable sequences, and/or in episomal (infectious and active) forms. To assess the state of RYNV in Rubus germplasm, we sequenced the genomes of various cultivars and mined eight raspberry whole genome datasets. Bioinformatics analysis revealed the presence of a diverse array of endogenous RYNV (endoRYNV) sequences that differ significantly in their structure; some lineages have nearly complete, yet non-functional genomes whereas others have rudimentary, short sequence fragments. We developed assays to genotype the main lineages as well as the only known episomal lineage present in the United States. This study discloses the widespread presence of endoRYNVs in commercial raspberries, likely because breeding efforts have focused on a limited pool of germplasm that harbored endoRYNVs.

Phyllocoptes parviflori is a distinct species and a vector of the pervasive blackberry leaf mottle associated virus.

Several viruses are transmitted by eriophyid mites (Acariformes: Eriophyoidea) including blackberry leaf mottle-associated emaravirus (BLMaV) (Emaravirus rubi). BLMaV is transmitted by an unidentified eriophyid species and is involved in blackberry yellow vein, a devastating disease in the southeastern United States. In this study, we assessed the eriophyid mite Phylocoptes parviflori as a vector of BLMaV and clarified its taxonomic status as it was previously synonymized with Phyllocoptes gracilis. P. parviflori can efficiently transmit BLMaV. The virus was found to cause yellow vein disease symptoms on 'Ouachita' blackberry marking a paradigm shift as disease symptoms have always been associated with multiple virus infections. Therefore, we propose renaming the virus to blackberry leaf mottle virus. The occurrence of P. parviflori on wild and cultivated blackberries, as well as its ability to colonize other Rubus species, enhances its importance as a major contributor to the spread of yellow vein disease.

Development of Rapid and Affordable Virus-Mimicking Artificial Positive Controls.

A major bottleneck in the development of detection assays is the availability of positive controls. Their acquisition can be problematic, their maintenance is expensive, and without them, assays cannot be validated. Herein, we present a novel strategy for the development of virus-mimicking artificial positive controls (ViMAPCs). The time between design and application is less than 5 days, unlike alternatives which normally take several weeks to obtain and implement. The ViMAPCs provide a realistic representation of natural infection unlike alternatives and allow for an effortless recognition of laboratory-based contamination. The feasibility and adaptability of the strategy was evaluated using several RNA and DNA plant viruses. ViMAPCs can be used in diagnostics laboratories but also in the monitoring of pathogen outbreaks where rapid response is of utmost importance.

Genomic analyses of a widespread blueberry virus in the United States.

Screening of blueberry accessions using high throughput sequencing revealed the presence of a new virus. Genomic structure and sequence are similar to that of nectarine stem pitting associated virus (NSPaV), a member of the genus Luteovirus, family Tombusviridae. The full genome of the new luteovirus, tentatively named blueberry virus L (BlVL), was characterized and analyzed. Similar to NSPaV, BlVL does not contain readily identifiable movement proteins in any of the seven isolates sequenced. More than 600 samples collected from five states were screened and 79% were found infected, making BlVL the most widespread blueberry virus in the United States.

First report of constricta yellow dwarf virus infecting Lobelia in the United States and the world.

Blue cardinal (Lobelia siphilitica L., family: Campanulaceae) is a popular perennial ornamental plant. Lobelia spp. have been reported as hosts of economically important viruses including cucumber mosaic virus (Nameth and Fisher, 2001), turnip mosaic virus (Lockhart et al., 2002), and tomato spotted wilt virus (Brown, 1988). During fall 2022, in a garden in Fayetteville, Arkansas, USA, yellow speckling, chlorosis, and dwarfing were observed on several cardinal plants. Three symptomatic plants were sampled, and RNA was isolated as described in Poudel et al. (2013) and pooled. Material was sequenced using the MinION platform as described by Liefting et al. (2021). A total of 56,700 raw reads (mean-length 326) were analyzed using VirFind (Ho and Tzanetakis, 2014) revealing 23 contigs ranging from 209-12,776 nucleotides (nt) which showed 96.2-98.4% identity with constricta yellow dwarf virus (CYDV; genus Alphanucleorhabdovirus, KY549567) and 16 contigs ranging from 201-531 nt with 83.4-95.7% identity with hydrangea chlorotic mottle virus (HdCMV; genus Carlavirus, EU754720). A total of 6,387 reads were mapped to the CYDV genome (KY549567) with 181x average coverage per nucleotide, and the consensus sequence of 12773 nt shared 98.1% identity to KY549567. The results were verified by RT-PCR and sequencing of the amplicons using primers 8825F: 5'-ACCCTGAGACAGGCATTGTG-3' and L2 9760: 5'-GCCGTACTATGAGAAGGGGC-3' for CYDV and 6495F: 5'-CAAGTACGTCTGTGTGAGGT-3' and 6630R 5'-CTTTTTGATAGTGTCATTGCTACC-3' for HdCMV. RNA was subsequently extracted from eight symptomatic and 12 symptomless samples. All symptomatic samples tested positive for CYDV but none of the 20 was positive for HdCMV indicating that CYDV is possibly the causal agent of the observed symptoms. Amplicons were CYDV specific and showed 91-98% nt identity (99-100% amino acid identity) (accession No. OP998261-63) with the CYDV isolate from Solanum tuberosum (KY549567). To the best of our knowledge, this is the first report of CYDV infecting L. cardinalis in the USA and the world. CYDV has been reported as an important pathogen of potato in the USA and recent reports indicate that emergence and re-emergence of rhabdoviruses on new hosts worldwide may threaten agricultural production (Bejerman et. al., 2021). Given the lack of monitoring of cardinal plants for viruses, this could serve as a reservoir of CYDV for several vegetables and ornamental crops (Jang et al., 2017).

Identification of a Second Vector for Rose Rosette Virus.

Rose rosette devastates the ornamentals industry in the United States. The disease, caused by rose rosette emaravirus (RRV), is vectored by the eriophyoid mite Phyllocoptes fructiphilus (Acari: Eriophyoidea). In this communication, we investigate two other Phyllocoptes species, P. adalius and P. arcani, for their vector competency and transmission efficiencies in single and multiple mite transfer experiments. P. arcani was identified as a second vector of RRV, a finding of significance for the epidemiology of the disease, as the second vector may be present in plants where P. fructiphilus is absent.

A Novel, Divergent Member of the Rhabdoviridae Family Infects Strawberry.

Strawberry (Fragaria × ananassa) is the most important berry crop worldwide and viruses pose a constant threat to the industry. In this communication, we describe a novel virus in the family Rhabdoviridae referred to as strawberry virus 3 (StrV-3). The virus does not show significant homology when compared with recognized rhabdoviruses and, therefore, the establishment of a new genus should be considered. A triplex reverse-transcription PCR test was developed and successfully employed in a survey of the National Clonal Germplasm Repository Fragaria collection. A CRISPR-Cas-based protocol was also developed and shown to detect the virus in as little as 1 fg of total RNA, a protocol to be used in the detection of the virus in candidate G1 plants. The strawberry aphid (Chaetosiphon fragaefolii) was evaluated-alas, unsuccessfully-as a potential vector of the virus. This work broadens our understanding of the family Rhabdoviridae and assists in the quest of releasing plant material free of viruses.

Virus classification based on in-depth sequence analyses and development of demarcation criteria using the Betaflexiviridae as a case study.

Currently, many viruses are classified based on their genome organization and nucleotide/amino acid sequence identities of their capsid and replication-associated proteins. Although biological traits such as vector specificities and host range are also considered, this later information is scarce for the majority of recently identified viruses, characterized only from genomic sequences. Accordingly, genomic sequences and derived information are being frequently used as the major, if not only, criteria for virus classification and this calls for a full review of the process. Herein, we critically addressed current issues concerning classification of viruses in the family Betaflexiviridae in the era of high-throughput sequencing and propose an updated set of demarcation criteria based on a process involving pairwise identity analyses and phylogenetics. The proposed framework has been designed to solve the majority of current conundrums in taxonomy and to facilitate future virus classification. Finally, the analyses performed herein, alongside the proposed approaches, could be used as a blueprint for virus classification at-large.

Identification, Characterization, and Detection of a Novel Strawberry Cytorhabdovirus.

In 2020, a novel agent was discovered in strawberry, a rhabdovirus closely related to lettuce necrotic yellows virus. The new virus, named strawberry virus 2 (StrV-2), was discovered in an accession of the Fragaria virus collection of the National Clonal Germplasm Repository (NCGR), and for this reason, it was studied in-depth. The complete StrV-2 genome was obtained and investigated in silico. Transmission was assessed using two aphid species whereas a multiplex RT-PCR test targeting plant and virus genes was developed and used to screen the NCGR Fragaria virus collection.

Characterization of the first Rubus yellow net virus genome from blackberry.

Rubus yellow net virus (RYNV) is a badnavirus that infects Rubus spp. Mixed infections with black raspberry necrosis virus and raspberry leaf mottle virus cause raspberry mosaic, a disease that leads to significant losses and even plant death. RYNV has been reported in several European countries and the Americas yet there is substantial lack of knowledge, especially when it comes to virus diversity and the evolutionary forces that affect virus fitness outside its primary host, raspberry. Herein, we report the first RYNV episomal genome isolated from blackberry and this is the first report of the virus in Bosnia and Herzegovina. The isolate has five open reading frames (ORFs) and, when compared with other fully sequenced counterparts, showed 82-97% nucleotide pairwise identity. This communication adds to our limited knowledge on RYNV and addresses some of the gaps in RYNV genetics when it comes to the coding capacity of episomal isolates and the probability of the first fully sequenced isolate of the virus being integrated in the raspberry genome.

Proteome expansion in the Potyviridae evolutionary radiation.

Potyviridae, the largest family of known RNA viruses (realm Riboviria), belongs to the picorna-like supergroup and has important agricultural and ecological impacts. Potyvirid genomes are translated into polyproteins, which are in turn hydrolyzed to release mature products. Recent sequencing efforts revealed an unprecedented number of potyvirids with a rich variability in gene content and genomic layouts. Here, we review the heterogeneity of non-core modules that expand the structural and functional diversity of the potyvirid proteomes. We provide a family-wide classification of P1 proteinases into the functional Types A and B, and discuss pretty interesting sweet potato potyviral ORF (PISPO), putative zinc fingers, and alkylation B (AlkB)-non-core modules found within P1 cistrons. The atypical inosine triphosphate pyrophosphatase (ITPase/HAM1), as well as the pseudo tobacco mosaic virus-like coat protein (TMV-like CP) are discussed alongside homologs of unrelated virus taxa. Family-wide abundance of the multitasking helper component proteinase (HC-pro) is revised. Functional connections between non-core modules are highlighted to support host niche adaptation and immune evasion as main drivers of the Potyviridae evolutionary radiation. Potential biotechnological and synthetic biology applications of potyvirid leader proteinases and non-core modules are finally explored.

Population genetics of cycas necrotic stunt virus and the development of multiplex RT-PCR diagnostics.

Cycas necrotic stunt virus (CNSV) has an extensive host range and is detected in an accelerated pace around the globe in several agricultural crops. One of the plant species affected is peony (Paeonia lactiflora Pall.). The virus is asymptomatic in most peony cultivars, but there have been reports of symptoms in others. It is thus important to study CNSV and its population structure to gain insights into its evolution and epidemiology. The outputs of this study, in addition to the in-depth analysis of the virus population structure, include the development of a multiplex RT-PCR detection protocol that can amplify all published CNSV isolate sequences; allowing for accurate, reliable detection of the virus and safeguarding its susceptible, clonally-propagated hosts.

Molecular phylogeny of Phyllocoptes associated with roses discloses the presence of a new species.

There are few plant maladies as devastating as rose rosette, a disease caused by an eriophyoid -transmitted virus. Rosette annihilates roses across North America, and to date, there is a single verified vector of the virus, Phyllocoptes fructiphilus Keifer. In direct contrast to the importance of rose for the ornamental industry there is limited knowledge on the eriophyoids that inhabit roses in North America and even less information on their vectoring capacities. This study dissects the genetic diversity of the eriophyoid fauna in rosette-affected hotspots and provides evidence of the existence of an undescribed species named Phyllocoptes arcani sp. nov., that could potentially be a second vector of the rosette virus.

The population structure of the secovirid lychnis mottle virus based on the RNA2 coding sequences.

Lychnis mottle virus (LycMoV), family Secoviridae, is one of several viruses recently detected in peony. Given the high prevalence of the virus in the more than 300 samples tested, the population structure of the virus was studied using 48 isolates representing at least 20 cultivars and collected from major producing and propagating states in the United States. The homogeneity of the United States population, based on data from the RNA2 coding region, along with phylogenetic analyses of all publicly available sequences point to the dissemination of the virus through propagation material rather that active vector-mediated transmission.

Complete Nucleotide Sequence, Genome Organization, and Comparative Genomic Analyses of Citrus Yellow-Vein Associated Virus (CYVaV).

Citrus yellow-vein disease (CYVD) was first reported in California in 1957. We now report that CYVD is associated with a virus-like agent, provisionally named citrus yellow-vein associated virus (CYVaV). The CYVaV RNA genome has 2,692 nucleotides and codes for two discernable open reading frames (ORFs). ORF1 encodes a protein of 190 amino acid (aa) whereas ORF2 is presumably generated by a -1 ribosomal frameshifting event just upstream of the ORF1 termination signal. The frameshift product (717 aa) encodes the RNA-dependent RNA polymerase (RdRp). Phylogenetic analyses suggest that CYVaV is closely related to unclassified virus-like RNAs in the family Tombusviridae. Bio-indexing and RNA-seq experiments indicate that CYVaV can induce yellow vein symptoms independently of known citrus viruses or viroids.

Graft-Transmissible Diseases of Ribes - Pathogens, Impact, and Control.

This article provides an up-to-date review of disease-causing viruses and phytoplasmas of currants including symptoms, transmission, detection, economic impact, and control measures. Currants are widely cultivated in more than 30 countries in the temperate zones of Europe, Asia, South America, Australia, and New Zealand. Ribes spp. can be infected by more than 20 virus species and four Candidatus Phytoplasma species, with more to be described in the future. High-throughput sequencing opened a new era of deciphering virus variants and mixed infections, leading to the characterization of several new species. The use of clean propagation material is the basis for control of Ribes graft-transmissible diseases, but this has become a challenging task given the ever-growing number of newly discovered pathogens.

The population structure of Rose rosette virus in the USA.

Rose rosette virus (RRV) (genus Emaravirus) is the causal agent of the homonymous disease, the most destructive malady of roses in the USA. Although the importance of the disease is recognized, little sequence information and no full genomes are available for RRV, a multi-segmented RNA virus. To better understand the population structure of the virus we implemented a Hi-Plex PCR amplicon high-throughput sequencing approach to sequence all 7 segments and to quantify polymorphisms in 91 RRV isolates collected from 16 states in the USA. Analysis revealed insertion/deletion (indel) polymorphisms primarily in the 5' and 3' non-coding, but also within coding regions, including some resulting in changes of protein length. Phylogenetic analysis showed little geographical structuring, suggesting that topography does not have a strong influence on virus evolution. Overall, the virus populations were homogeneous, possibly because of regular movement of plants, the recent emergence of RRV and/or because the virus is under strong purification selection to preserve its integrity and biological functions.

Special Issue "Plant Virus Epidemiology and Control".

We recently completed the Special Issue on 'Plant Virus Epidemiology and Control'. As editors, we decided not to offer vouchers to scientists that submit to this issue. This action had an effect on the number of papers received and accepted. We received a total of 19 papers and we accepted four [...].