Identification and molecular characterization of two novel viruses from Allamanda cathartica in China.

Allamanda cathartica is an ornamental medicinal plant that grows widely in the tropics. In the present study, two novel viruses, Allamanda chlorotic virus A (AlCVA) and Allamanda chlorotic virus B (AlCVB), were identified in an A. cathartica plant with interveinal chlorosis by ribosomal RNA-depleted total-RNA sequencing. Phylogenetic analysis and sequence comparisons confirmed that AlCVA and AlCVB belong to the families Closteroviridae and Betaflexiviridae, respectively. Long, flexuous, filamentous virus particles approximately 12 nm in diameter and 784-2291 nm in length were observed using transmission electron microscopy. A specific RT-PCR assay was used to demonstrate a consistent association of viral infection with symptoms.

Molecular identification of a putative novel varicosavirus in Agastache rugosa in China.

A putative novel virus was identified in Agastache rugosa in China and tentatively named "Agastache rugosa-associated varicosavirus" (ARaVV). The nearly complete genome sequence of ARaVV was obtained through RNA sequencing (RNA-seq) and subsequent RT-PCR. The ARaVV genome consists of two negative-sense single-stranded RNA segments that are 6428 and 3862 nucleotides (nt) in size, respectively. RNA1 encodes a large polymerase (L), and RNA2 encodes a putative nucleocapsid protein (N), protein 2 (P2), and protein 3 (P3). The L protein shared the highest amino acid (aa) sequence identity (51.3%) with Erysimum virus 1 (EryV1, BK061766.1). The N, P2, and P3 shared the highest aa sequence identity (33.1%, 14.0%, and 24.2%) with Leucanthemum virus 1, Raphanus virus 1, and Spinach virus 1, respectively. Phylogenetic analysis based on amino acid sequences of the L protein showed that ARaVV clustered in a clade with the varicosaviruses, indicating that ARaVV is a putative new member of the genus Varicosavirus.

Genomic characterization of novel viruses associated with Olea europaea L. in South Africa.

South Africa has a small but growing olive industry. Until now, no virological research has been carried out on this crop locally. Seventeen samples were collected from various olive cultivars from a single producer in the Stellenbosch growing area of South Africa. RNAseq was performed on total RNA, and the compositions of the metaviromes were determined. Olive leaf yellowing-associated virus was detected for the first time in South Africa, as well as four novel viruses from the family Closteroviridae and one each from the families Tymoviridae and Solemoviridae.

Viromics unveils extraordinary genetic diversity of the family Closteroviridae in wild citrus.

Our knowledge of citrus viruses is largely skewed toward virus pathology in cultivated orchards. Little is known about the virus diversity in wild citrus species. Here, we used a metatranscriptomics approach to characterize the virus diversity in a wild citrus habitat within the proposed center of the origin of citrus plants. We discovered a total of 44 virus isolates that could be classified into species Citrus tristeza virus and putative species citrus associated ampelovirus 1, citrus associated ampelovirus 2, and citrus virus B within the family Closteroviridae, providing important information to explore the factors facilitating outbreaks of citrus viruses and the evolutionary history of the family Closteroviridae. We found that frequent horizontal gene transfer, gene duplication, and alteration of expression strategy have shaped the genome complexity and diversification of the family Closteroviridae. Recombination frequently occurred among distinct Closteroviridae members, thereby facilitating the evolution of Closteroviridae. Given the potential emergence of similar wild-citrus-originated novel viruses as pathogens, the need for surveillance of their pathogenic and epidemiological characteristics is of utmost priority for global citrus production.

Complete genome sequence of a novel closterovirus isolated from Dregea volubilis.

The complete genome sequence of a putative novel closterovirus, tentatively named "Dregea volubilis closterovirus 1" (DvCV1, GenBank accession no. MZ779122), infecting Dregea volubilis in China was determined using high-throughput sequencing (HTS). The complete genome sequence of DvCV1 consists of 16,165 nucleotides (nt) and contains nine ORFs. The genome structure of DvCV1 is typical of members of the genus Closterovirus. Complete genome sequence analysis showed that DvCV1 shares 41.4-48.4% nucleotide sequence identity with other known closteroviruses. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70-like protein (HSP70h), and coat protein (CP) of DvCV1 share 46.80-62.65%, 31.06-51.80%, and 28.34-37.37% amino acid sequence identity, respectively, with the RdRp, HSP70h and CP of other closteroviruses. Phylogenetic analysis based on HSP70h aa sequences placed DvCV1 alongside other members of the genus Closterovirus in the family Closteroviridae. These results suggest that DvCV1 is a new member of the genus Closterovirus. This is the first report of a closterovirus infecting D. volubilis.

Scalable Early Detection of Grapevine Viral Infection with Airborne Imaging Spectroscopy.

The U.S. wine and grape industry loses $3B annually due to viral diseases including grapevine leafroll-associated virus complex 3 (GLRaV-3). Current detection methods are labor-intensive and expensive. GLRaV-3 has a latent period in which the vines are infected but do not display visible symptoms, making it an ideal model to evaluate the scalability of imaging spectroscopy-based disease detection. The NASA Airborne Visible and Infrared Imaging Spectrometer Next Generation was deployed to detect GLRaV-3 in Cabernet Sauvignon grapevines in Lodi, CA in September 2020. Foliage was removed from the vines as part of mechanical harvest soon after image acquisition. In September of both 2020 and 2021, industry collaborators scouted 317 hectares on a vine-by-vine basis for visible viral symptoms and collected a subset for molecular confirmation testing. Symptomatic grapevines identified in 2021 were assumed to have been latently infected at the time of image acquisition. Random forest models were trained on a spectroscopic signal of noninfected and GLRaV-3 infected grapevines balanced with synthetic minority oversampling of noninfected and GLRaV-3 infected grapevines. The models were able to differentiate between noninfected and GLRaV-3 infected vines both pre- and postsymptomatically at 1 to 5 m resolution. The best-performing models had 87% accuracy distinguishing between noninfected and asymptomatic vines, and 85% accuracy distinguishing between noninfected and asymptomatic + symptomatic vines. The importance of nonvisible wavelengths suggests that this capacity is driven by disease-induced changes to plant physiology. The results lay a foundation for using the forthcoming hyperspectral satellite Surface Biology and Geology for regional disease monitoring in grapevine and other crop species. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Investigating Protein-Protein Interactions Between Grapevine Leafroll-Associated Virus 3 and Vitis vinifera.

Grapevine leafroll disease (GLD) is a globally important disease that affects the metabolic composition and biomass of grapes, leading to a reduction in grape yield and quality of wine produced. Grapevine leafroll-associated virus 3 (GLRaV-3) is the main causal agent for GLD. This study aimed to identify protein-protein interactions between GLRaV-3 and its host. A yeast two-hybrid (Y2H) library was constructed from Vitis vinifera mRNA and screened against GLRaV-3 open reading frames encoding structural proteins and those potentially involved in systemic spread and silencing of host defense mechanisms. Five interacting protein pairs were identified, three of which were demonstrated in planta. The minor coat protein of GLRaV-3 was shown to interact with 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 02, a protein involved in primary carbohydrate metabolism and the biosynthesis of aromatic amino acids. Interactions were also identified between GLRaV-3 p20A and an 18.1-kDa class I small heat shock protein, as well as MAP3K epsilon protein kinase 1. Both proteins are involved in the response of plants to various stressors, including pathogen infections. Two additional proteins, chlorophyll a-b binding protein CP26 and a SMAX1-LIKE 6 protein, were identified as interacting with p20A in yeast but these interactions could not be demonstrated in planta. The findings of this study advance our understanding of the functions of GLRaV-3-encoded proteins and how the interaction between these proteins and those of V. vinifera could lead to GLD.

Gathered from the Vine: A Survey of Seven Grapevine Viruses Within New England Vineyards.

Vineyards in the Southeastern New England American Viticultural Area were surveyed for the incidence of seven major viruses: grapevine leafroll-associated viruses (GLRaV-1, GLRaV-2, GLRaV-3, and GLRaV-4), grapevine fanleaf virus (GFLV), tomato ringspot virus (ToRSV), and tobacco ringspot virus (TRSV). Viruses were detected by DAS-ELISA and confirmed by RT-PCR and Sanger sequencing. Multiple viruses were present in 19 out of the 25 vineyards surveyed between 2018 and 2020. GLRaV-3 (27.59%) was the most prevalent virus followed by GLRaV-4 (14.90%), GLRaV-1 (13.52%), GLRaV-2 (11.03%), ToRSV (6.34%), GFLV (5.24%), and TRSV (2.62%). Furthermore, phylogenetic analyses of the viral partial genome sequences acquired in this study revealed that the grapevine viruses present in this area are diverse, indicating that they may have been introduced from different sources. Our findings stress the need for improving the sanitary status of planting materials to avoid the introduction and dissemination of viruses to vineyards in this important wine-producing region of New England.

Diagnostic and Historical Surveys of Sweet Cherry (Prunus avium) Virus and Virus-Like Diseases in Oregon.

There are over 35 known virus and virus-like diseases of sweet cherry (Prunus avium), some with potential to cause severe economic impact by reducing vegetative growth, vigor, or fruit quality. Oregon is the second-ranked state for sweet cherry production in the United States. Statewide surveys were conducted in Oregon sweet cherry orchards for virus and virus-like diversity and distribution. Orchards in key production regions with suspected virus disease symptoms were sampled. Virus-specific enzyme-linked immunosorbent assay, isothermal amplification, or quantitative real-time PCR were used to test for the presence of common or economically important sweet cherry pathogens, including cherry leaf roll virus (CLRV), little cherry virus 2 (LChV2), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), tomato ringspot virus (ToRSV), and 'Candidatus Phytoplasma pruni'. CLRV, a new virus of sweet cherry in Oregon, was found associated with enation and dieback symptoms in The Dalles. Some viruses were found in new regions, which included Hood River (PDV, PNRSV, and ToRSV) and the Umpqua Valley (PDV and PNRSV). A subsequent survey was conducted in the Mid-Columbia production region for the presence of little cherry symptoms associated with little cherry and X-Diseases. All symptomatic samples from The Dalles and Mosier, OR, or Dallesport, WA, tested positive for 'Ca. P. pruni' but not LChV2. These findings provide a foundation for the current understanding and management of virus and virus-like diseases of sweet cherry in Oregon and context for further studies into these pathogens and their vectors.

Detecting Grapevine Virus Infections in Red and White Winegrape Canopies Using Proximal Hyperspectral Sensing.

Grapevine virus-associated disease such as grapevine leafroll disease (GLD) affects grapevine health worldwide. Current diagnostic methods are either highly costly (laboratory-based diagnostics) or can be unreliable (visual assessments). Hyperspectral sensing technology is capable of measuring leaf reflectance spectra that can be used for the non-destructive and rapid detection of plant diseases. The present study used proximal hyperspectral sensing to detect virus infection in Pinot Noir (red-berried winegrape cultivar) and Chardonnay (white-berried winegrape cultivar) grapevines. Spectral data were collected throughout the grape growing season at six timepoints per cultivar. Partial least squares-discriminant analysis (PLS-DA) was used to build a predictive model of the presence or absence of GLD. The temporal change of canopy spectral reflectance showed that the harvest timepoint had the best prediction result. Prediction accuracies of 96% and 76% were achieved for Pinot Noir and Chardonnay, respectively. Our results provide valuable information on the optimal time for GLD detection. This hyperspectral method can also be deployed on mobile platforms including ground-based vehicles and unmanned aerial vehicles (UAV) for large-scale disease surveillance in vineyards.

Molecular characterization of Cordyline virus 1 isolates infecting yam (Dioscorea spp).

Cordyline virus 1 (CoV1) is a velarivirus that has so far only been reported in ornamental Ti plants (Cordyline fruticosa). Using high-throughput sequencing, we identified CoV1 infection in yam accessions from Vanuatu. Using a specific RT-PCR assay, we found that CoV1 is also present and highly prevalent in Dioscorea alata, D. cayenensis, and D. trifida in Guadeloupe. Phylogenetic analysis showed that CoV1 isolates infecting yam in Guadeloupe display a low level of molecular diversity. These data provide insights into the transmission of CoV1 in yam in Guadeloupe.

Complete genome sequence of peony leafroll-associated virus, a novel ampelovirus in subgroup I.

The complete genome sequence of peony leafroll-associated virus (PLRaV) was determined by deep sequencing of ribosomal-RNA-depleted total RNA extracted from a peony plant exhibiting leafroll symptoms. Further PCR and RACE analysis showed that the PLRaV genome consists of 15,406 nucleotides and contains 10 putative open reading frames, with an organization typical of members of the genus Ampelovirus, family Closteroviridae. Amino acid sequence comparisons showed that the viral heat shock protein 70 homolog (HSP70h) shared the highest sequence identity (41.7%) with the corresponding region of grapevine leafroll-associated virus 1, and the coat protein (CP) and RNA-dependent RNA polymerase (RdRp) shared the highest sequence identity (32.1% and 52.3%, respectively) with grapevine leafroll-associated virus 13. Phylogenetic analysis of the HSP70h, CP, and RdRp aa sequences showed that PLRaV clustered with members of subgroup I of the genus Ampelovirus.

Transmission of Areca Palm Velarivirus 1 by Mealybugs Causes Yellow Leaf Disease in Betel Palm (Areca catechu).

Yellow leaf disease (YLD) is the most destructive disease of betel palm (Areca catechu). A strong association between YLD and areca palm velarivirus 1 (APV1) has been observed. However, the causal relationship between APV1 and disease, and the transmission mode, warrant further investigation. This work showed that APV1 was transmitted by both Ferrisia virgata and Pseudococcus cryptus mealybugs and caused YLD symptoms in betel palm seedlings; therefore, we demonstrate that APV1 is a causal agent of YLD. APV1 was detected in the stylets, foreguts, midguts, and hindguts of the vectors via both immunocapture reverse transcription PCR and immunofluorescence assays. APV1 was not transmitted transovarially from viruliferous female F. virgata to their progeny. In summary, the transmission of APV1 by F. virgata may occur in a noncirculative, semipersistent manner. This study fills important gaps in our knowledge of velarivirus transmission, which is critical for developing YLD management practices.

Improved Detection of Little Cherry Virus-2 Using a Hydrolysis Probe to Manage the Pacific Northwest Little Cherry Disease Epidemic.

Little cherry virus-2 (LChV-2) is a viral pathogen that is reaching epidemic levels in Washington State. This virus is insect vectored and has significant impacts on sweet cherry production. To aid growers in making informed management decisions, we sought to develop a diagnostic assay to better detect isolates of LChV-2 currently found in Washington, allowing more accurate estimations of disease occurrence. This study showed that there were two distinct genotypes of LChV-2 present in Washington State. This information was used to develop an up-to-date reverse transcription real-time quantitative PCR assay, which was then optimized, validated, and compared with four previously published assays of a panel of field samples. This comparison demonstrated that the newly developed assay provided greater sensitivity, accurately detecting <10 copies per reaction and could detect both LChV-2 genotypes. Finally, we examined the effect of potential inhibitors in various tissue types from cherry, finding that young leaf tissue affected sensitivity of detection less than root tissues.

An Advanced One-Step RT-LAMP for Rapid Detection of Little cherry virus 2 Combined with High-Throughput Sequence-Based Phylogenomics Reveal Divergent Flowering Cherry Isolates.

Little cherry virus 2 (LChV-2, genus Ampelovirus) is considered to be the main causal agent of the economically damaging little cherry disease, which can only be controlled by removal of infected trees. The widespread viral disease of sweet cherry (Prunus avium L.) is affecting the survival of long-standing orchards in North America and Europe, hence the dire need for an early and accurate diagnosis to establish a sound disease control strategy. The endemic presence of LChV-2 is mainly confirmed using laborious time-consuming reverse-transcription (RT-PCR). A rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting a conserved region of the coat protein was developed and compared with conventional RT-PCR for the specific detection of LChV-2. This affordable assay, combined with a simple RNA extraction, deploys desirable characteristics such as higher ability for faster (<15 min), more analytically sensitive (100-fold), and robust broad-range diagnosis of LChV-2 isolates from sweet cherry, ornamental flowering cherry displaying heterogenous viral etiology and, for the first time, newly identified potential insect vectors. Moreover, use of Sanger and total RNA high-throughput sequencing as complementary metaviromics approaches confirmed the LChV-2 RT-LAMP detection of divergent LChV-2 isolates in new hosts and the relationship of their whole-genome was exhaustively inferred using maximum-likelihood phylogenomics. This entails unprecedented critical understanding of a novel evolutionary clade further expanding LChV-2 viral diversity. In conclusion, this highly effective diagnostic platform facilitates strategical support for early in-field testing to reliably prevent dissemination of new LChV-2 outbreaks from propagative plant stocks or newly postulated insect vectors. Validated results and major advantages are herein thoroughly discussed, in light of the knowledge required to increase the potential accuracy of future diagnostics and the essential epidemiological considerations to proactively safeguard cherries and Prunus horticultural crop systems from little cherry disease.

Grapevine Leafroll-Associated Virus 3 in Single and Mixed Infections Triggers Changes in the Oxidative Balance of Four Grapevine Varieties.

With the aim to characterize changes caused by grapevine leafroll-associated virus 3 (GLRaV-3) singly or in coinfection with other viruses and to potentially determine genotype-specific or common markers of viral infection, thirty-six parameters, including nutrient status, oxidative stress parameters, and primary metabolism as well as symptoms incidence were investigated in 'Cabernet Franc,' 'Merlot,' 'Pinot Noir,' and 'Tribidrag' grapevine varieties. Host responses were characterized by changes in cellular redox state rather than disturbances in nutrient status and primary metabolic processes. Superoxide dismutase, hydrogen peroxide, and proteins were drastically affected regardless of the type of isolate, the host, and the duration of the infection, so they present cellular markers of viral infection. No clear biological pattern could be ascertained for each of the GLRaV-3 genotypes. There is a need to provide a greater understanding of virus epidemiology in viticulture due to the increasing natural disasters and climate change to provide for global food production security. Finding grape varieties that will be able to cope with those changes can aid in this task. Among the studied grapevine varieties, autochthonous 'Tribidrag' seems to be more tolerant to symptoms development despite numerous physiological changes caused by viruses.

A novel RNA virus, Thesium chinense closterovirus 1, identified by high-throughput RNA-sequencing of the parasitic plant Thesium chinense.

The genome sequence of a closterovirus (genus Closterovirus, family Closteroviridae), tentatively named Thesium chinense closterovirus 1 (TcCV1), was identified by performing high-throughput RNA-sequencing of the haustoria and root tissues of Thesium chinense, a parasitic plant. The TcCV1 genome was predicted to encode nine proteins, eight of which have orthologs in previously identified closteroviruses. The TcCV1 RNA-dependent RNA polymerase (RdRp) and heat shock protein 70 homolog (Hsp70h) showed 27.8-68.2% and 23.8-55.1% amino acid identity, respectively, to orthologous proteins of known closteroviruses. The putative +1 ribosomal frameshifting site required for producing RdRp was identified as GUUUAGC with UAG stop codon and the skipped nucleotide U. Phylogenetic trees based on RdRp and Hsp70h show that TcCV1 is a novel member of the genus Closterovirus, forming a subclade with a group of known closteroviruses, including mint virus 1 and carnation necrotic fleck virus. The genome sequence of TcCV1 may be useful for studying the genome evolution of closteroviruses. Keywords: Thesium chinense closterovirus 1; Closterovirus; Closteroviridae; Thesium chinense.

Genomic diversity of Areca Palm Velarivirus 1 (APV1) in Areca palm (Areca catechu) plantations in Hainan, China.

BACKGROUND: Areca palm (Areca catechu L.) is an important commercial crop in southeast Asia, but its cultivation is threatened by yellowing leaf disease (YLD). Areca palm velarivirus 1 (APV1) was recently associated with YLD, but little is known regarding its population and genetic diversity. To assess the diversity of YLD, the APV1 genome was sequenced in YLD samples collected from different sites in Hainan. RESULTS: Twenty new and complete APV1 genomes were identified. The APV1 isolates had highly conserved sequences in seven open reading frames (ORFs; > 95% nucleotide [nt] identity) at the 3' terminal, but there was diversity (81-87% nt identity) in three ORFs at the 5' terminal. Phylogenetic analysis divided the APV1 isolates into three phylogroups, with 16 isolates (> 70%) in phylogroup A. Mixed infections with different genotypes in the same tree were identified; this was closely correlated with higher levels of genetic recombination. CONCLUSIONS: Phylogroup A is the most prevalent APV1 genotype in areca palm plantations in Hainan, China. Mixed infection with different genotypes can lead to genomic recombination of APV1. Our data provide a foundation for accurate diagnostics, characterization of etiology, and elucidation of the evolutionary relationships of APV1 populations.

Titer and distribution of little cherry virus 2 in Prunus avium.

Little cherry virus 2 (LChV-2) is a causal agent of little cherry disease, which produces small, misshapen fruit with poor color and taste. As LChV-2 symptoms are only present near harvest, molecular detection is essential for effective control. Therefore, we determined the titer and distribution of this virus in infected trees over time. While initial infections were found to be basipetal, in field trees, early-stage infection was characterized by uneven distribution and low titer, concentrated in woody stems. In contrast, established infections were systemic, and detection was consistent across tissues. These data provide improved sampling recommendations for the detection of LChV-2.

A novel ampelovirus associated with mealybug wilt of pineapple (Ananas comosus).

Mealybug wilt of pineapple (MWP) is the most important and complex viral disease affecting pineapple worldwide. High-throughput sequencing was conducted to characterize a new virus identified only in symptomatic pineapple plants and tentatively named pineapple mealybug wilt-associated virus 6 (PMWaV-6). Data analyses revealed a genome of 17,854 nucleotides with an organization resembling members of the genus Ampelovirus, family Closteroviridae. Encoded proteins shared sequence identity with the corresponding proteins of grapevine leafroll-associated virus 3, blackberry vein banding-associated virus, and PMWaV-2. The present study reports the discovery of PMWaV-6, a putative and distinct new member of the genus Ampelovirus, subgroup I, its potential involvement in MWP, and the development of PMWaV-6-specific RT-PCR assays to detect and monitor this virus in field samples.