Culinary rhubarb (Rheum spp.) comprises herbaceous perennials that are primarily clonally propagated. Like other asexually reproduced crops, rhubarb is vulnerable to virus transmission, but its virome remains poorly characterized. In April 2024, foliar virus-like symptoms including chlorotic and necrotic leaf spots, chlorosis, and stunting were observed on cultivar ‘Kerwin’ in the rhubarb collection at Cornell AgriTech in Geneva, NY. Symptomatic leaf tissue tested positive for tomato ringspot virus (ToRSV) in double antibody sandwich enzyme-linked immunosorbent assay using specific antibodies. Subsequently, the virome of 40 rhubarb entries was determined using high-throughput sequencing (HTS) and bioinformatic analyses. Sequence information suggested the presence of ToRSV (genus Nepovirus, family Secoviridae), cherry leafroll virus (CLRV, genus Nepovirus, family Secoviridae), Arabis mosaic virus (ArMV, genus Nepovirus, family Secoviridae), broad-leafed dock virus A (BLDVA, genus Macluravirus, family Potyviridae), tobacco streak virus (TSV, genus Ilarvirus, family Bromoviridae), and beet curly top virus (BCTV, genus Curtovirus, family Geminiviridae) in composite leaf samples. The occurrence of these RNA and DNA viruses was confirmed in composite and individual samples after deconvolution by RT-PCR or PCR, respectively, using one or two primer pairs, in combination with bidirectional Sanger sequencing of the amplicons. The symptoms of cultivar ‘Kerwin’ could not be definitively assigned to ToRSV due to co-infection with BLDVA. This is the first characterization of the rhubarb virome by HTS and the first report of ToRSV, BLDVA, TSV and BCTV in culinary rhubarb. These findings have implications for the propagation of clean material, distribution of germplasm, and future breeding efforts in rhubarb.

The treehopper Spissistilus festinus [Say, 1830] (Hemiptera: Membracidae) is not a direct pest of winegrapes (Vitis vinifera) but poses a threat to vineyards as a vector of grapevine red blotch virus (GRBV). While previous no-choice assays suggested a preference for Fabaceae for feeding and reproduction, recent gut content analyses revealed frequent feeding of S. festinus on Vitaceae, particularly free-living V. californica and its hybrids, within northern California vineyards. To better understand host selection and feeding behaviors of S. festinus, we conducted choice assays using excised leaves of Fabaceae (snap bean, alfalfa) and Vitaceae (winegrape, V. californica, V. californica hybrid). In two-choice assays, S. festinus more often visited snap bean, though Vitis species were also attractive when present. In four-choice contexts, snap bean remained a preferred host, followed by V. californica and winegrapes. These results confirmed the polyphagous tendencies of S. festinus and provided new insights into the feeding behaviors of this treehopper species.

Viruses of cultivated apple (Malus × domestica) are believed to be transmitted nearly exclusively via vegetative propagation, with few known biotic vectors and limited evidence of vertical transmission. To evaluate the seed transmission capabilities of six viruses and one viroid of apple, a large-scale seedling grow-out experiment was conducted using seeds harvested from 51 trees infected by several combinations of six viruses and one viroid. Virus detection via multiplex PCR-based amplicon sequencing followed by RT-qPCR validation demonstrated that citrus concave gum-associated virus and apple stem grooving virus were transmitted to seedlings at rates of 4.0% (32/792) and 0.3% (3/908), respectively. No evidence of seed transmission was obtained for apple chlorotic leaf spot virus, apple green crinkle-associated virus, apple hammerhead viroid, apple rubbery wood virus 2, or apple stem pitting virus. These findings document a previously unknown mode of transmission for two widely distributed apple viruses with direct implications for breeding programs, the selection of virus-free trees, and the exchange of germplasm.

Grapevine (Vitis spp.) propagation material is profusely exchanged across geographic and regulatory boundaries. Trading germplasm increases the diversity of cultivars and rootstocks with enhanced viticultural attributes but also risks the dissemination of pests and detrimental pathogens such as viruses in new grape production areas. Therefore, regulations are established to facilitate the safe trade of Vitis propagation material with desirable traits. Regrettably regulations are sometimes inadequate. Consequently, the accessibility of desired germplasm for growers might be unduly delayed, providing opportunities to circumvent regulations by illegally introducing germplasm of interest, amidst risking epidemics of viruses of concern, including quarantined agents, and jeopardizing the health of vineyards. To address some of the regulatory limitations, scientists from around the world recently defined phantom agents in fruit crops, including grapevines, and provided a compelling case for their exclusion from regulatory oversight. Simultaneously, a group of virologists realized the need to complement the list of phantom agents in grapevines by considering additional viruses, virus-like diseases and viroids that should not be subject to regulatory oversight. Here, we present a list of nine viruses, 14 virus-like diseases, nine viroids, and 129 presumed viruses of the grapevine, that are not phantom agents but should be excluded from regulation or should not be regulated. Our list is anticipated to assist policy makers adopt regulations that expedite the safe exchange of Vitis germplasm across regulatory boundaries while reducing incentives for illicit introductions.

Spissistilus festinus [Say, 1830] (Hemiptera: Membracidae) is a well-known pest of leguminous crops and a more recently described pest of grapevine due to its ability to transmit grapevine red blotch virus, an economic threat to grape production. Legumes (family Fabaceae), unlike grapevines (Vitis vinifera L.), are preferred feeding and reproductive hosts for S. festinus. Here, we analyzed the development and behavior of S. festinus on snap beans (Phaseolus vulgaris) in comparison to alfalfa (Medicago sativa) and grapevines, with an emphasis on colony establishment and population growth. Snap beans supported all stages of S. festinus development, promoting feeding, reproduction, and colony establishment, as well as supporting a faster life cycle, particularly on detached trifoliates in controlled environmental chambers, compared with alfalfa. Moreso, social aggregation of S. festinus adults was observed on snap bean plants with the petiole as a preferred feeding site. A preference toward grapevine petioles was also seen, though their survival and aggregation behaviors drastically declined on this nonlegume host. Dissecting the alimentary canal of S. festinus revealed more orange, refractive oil droplets, and air bubbles in specimens from grapevine compared to specimens from snap bean or alfalfa, suggesting possible disruptions in digestive processing or nutritional deficiencies with the former host. Together, our findings highlight snap bean as an ideal host for rearing populations of S. festinus to be used in grapevine red blotch virus transmission studies.

Azerbaijan is major producer of fruit crops, such as pome and stone fruits, in the Caspian Sea and Caucasus Mountains areas (FAO Stat, 2022). No information is available on the occurrence of apple chlorotic leaf spot virus (ACLSV, genus Trichovirus, family Betaflexiviridae) in the country. Therefore, the main fruit tree production areas in Azerbaijan were surveyed for ACLSV during the 2017-2019 growing seasons by DAS-ELISA using ACLSV reagents (Neogen - Scotland, UK) (Clark and Adams 1977). Of the 510 plant samples tested, 139 were positive for ACLSV. To confirm the presence of ACLSV, total RNA was isolated from leaf samples (0.5 g) exhibiting deformation, yellow veins, ringspots, and/or chlorosis symptoms as previously described (Foissac et al. 2005), and analyzed by RT-PCR using the OneStep RT-PCR kit (Qiagen) and primers designed in the coat protein gene of ACLSV: F (5′-CTACAATTTGAAGGAGGTGGTCAAC-3′), R (5′-GACGCCGGTTCATATTAGTTATGAC-3′) and primers for the NADH dehydrogenase subunit 5 fragment as a housekeeping gene (Menzel et al. 2002). Reactions were performed at 5 min at 95 °C, 34 cycles of 10 s at 95 °C, 20 s at 50 °C, 20 s at 72 °C, followed by a final extension of 2 min at 72 °C. DNA amplicons were resolved by electrophoresis on 2% agarose gels and visualized under UV illumination after staining with GelRed (Biotium, Fremont, CA). Results showed the occurrence of ACLSV in 14 apple samples (Malus domestica L.; n=38), four cherry plum sample (P. cerasifera L.; n=17), one almond samples (P. amygdalus L.; n=13), and one plum sample (P. domestica L.: n=71). All the pear (Pyrus communis L.; n=27), quince (Cydonia oblonga L.; n=2), sweet cherry (P. avium L.; n=27), sour cherry (P. cerasus L.; n=1), peach (P. persica L.; n=22), and apricot (P. armeniaca L.; n=8) samples tested negative for ACLSV in RT-PCR. Next, the 287-bp amplicons obtained by RT-PCR from two Azerbaijani ACLSV isolates from the Ganja and Kurdamir region, i.e., Ganja 43 from apple and Kurdamir 80 from plum, were cleaned with Exo SAP-IT and characterized by bidirectional Sanger sequencing at the Cornell Biotechnology Resource Center in Ithaca, NY. Sequence analyses were performed with the DNASTAR Lasergene software package (v17.3.0.57). Results revealed 87.2%-96.7% and 98.3%-100% nucleotide and amino acid identities, respectively, of ACLSV isolate Ganja 43 isolate (GenBank PP894314) with Afghan (KU749440) and Ukrainian (JQ866623) isolates, and 86.6%-95.2% and 97.9%-99.0% nucleotide and amino acid identities, respectively, of ACLSV isolate Kurdamir 80 (GenBank PP898052) with Afghan (KU749440) and Indian (FN666579) isolates. This is the first report of ACLSV in pome and stone fruit trees in Azerbaijan. Since the occurrence of mixed virus infections was not investigated in this study, the viral symptoms displayed by the infected trees cannot be ascribed to ACLSV. The occurrence of ACLSV suggests the need for certification programs in the country to reduce the presence of the virus in the propagation material of pome and stone fruit trees.

The epidemiological relationship between arthropod dispersal and plant communities is poorly understood at the landscape level. We investigated the connectivity of natural and cultivated plants in vineyard ecosystems for red blotch disease dynamics by characterizing the dietary history of Spissistilus festinus, a treehopper vector of grapevine red blotch virus (GRBV). Molecular analysis of the gut content of 205 S. festinus caught in 71 vineyard sites and the flora proximal to vineyards in Napa Valley, California, USA revealed a total of 171 genera from 61 plant families with a preeminence of Asteraceae, Fabaceae, and Vitaceae over two growing seasons, illustrating dietary profiles composed primarily of natural vegetation. An indicator species analysis identified the strength of ecological associations by showing distinct seasonal feeding trends related to weather patterns and host repertoires unique to some vineyard sites with estimated S. festinus travel distances of up to two kilometers. Potential reproductive and overwintering hosts of S. festinus were identified in natural habitats and an ecological relatedness between free-living vines in riparian corridors and vineyards for GRBV transmission was documented. Together, our findings on landscape vegetation connectivity and S. festinus dispersal transformed our understanding of red blotch disease ecology and informed disease management options.

The dynamics of cotton leafroll dwarf virus (CLRDV) symptomology in Gossypium plants was investigated in Mississippi in 2019 and 2022. High-throughput sequencing of libraries constructed from ribo-depleted total RNA or total nucleic acids and bioinformatic analyses revealed a novel virus, cotton virus A (CotVA), that clusters within the family Caulimoviridae alongside two unclassified viruses related to ruflodiviruses and caulimoviruses. Endogenous CotVA elements, referred to as endogenous caulimovirids (ECVs), were identified in multiple chromosomes of seven out of 26 Gossypium species analyzed, including two commercial species: G. hirsutum (Upland cotton) and G. barbadense (Pima cotton). The circular nature of the episomal genome of one CotVA isolate was confirmed using five overlapping primer sets. A DNase protection assay on purified virus preparations revealed evidence of CotVA virions. The presence of CotVA ECVs and/or an episomal form of CotVA in cotton fields complicates the interpretation of CLRDV symptoms, but proper studies are needed to determine if CotVA is associated with any symptomology. Possible synergistic interactions among CLRDV, CotVA, and proteins, or other molecules produced by ECVs and the impact of these interactions on any possible disease progression require further investigation.

Fanleaf degeneration disease is referred to as "ortiage" in the French literature in 1723. Nowadays it occurs in most vineyards worldwide. Grapevine fanleaf virus (GFLV) is the main causal agent of the disease. It was identified in 1960 following the identification of the ectoparasitic dagger nematode Xiphinema index as its exclusive vector. The expression of the bipartite viral genome has been characterized and functions have been assigned to most viral proteins. Remarkably, the viral RNAs are monouridylated. The virion structure was determined by crystallography and transmission determinants were mapped on the coat protein. Further, viral determinants of symptoms in leaves and roots of plant hosts, three suppressors of RNA silencing, and metabolic pathways dysregulated upon infection were identified. Despite tremendous progress, management responses are limited. Future research will undoubtfully better elucidate the different steps of the infection cycle and provide opportunities to apply transformative GFLV management solutions in the vineyard.

Grapevine red blotch disease emerged as a major threat to the North American viticulture more than 25 years ago. Prior to the discovery of its causal agent, grapevine red blotch virus (GRBV), the disease was likely mistaken for other vineyard problems. Over the last decade and a half, research on red blotch disease focused on GRBV biology; diagnostics; transmission biology; disease epidemiology; ecology of its vector, the treehopper Spissistilus festinus; and strategies for disease management. Research has also uncovered some of the physiological effects of GRBV on grapevines (inhibition of hexose translocation from leaves to fruits, transcriptional suppression of phenylpropanoid pathways), fruit (low soluble solids, poor ripening, reduced phenolic extractability, high titratable acidity), and wine (altered sensory attributes such as less fruit aromas and poor color and mouthfeel). The economic effects of the disease in different grape-producing regions of the United States are estimated to be as high as $68,548 per hectare over a 25-year vineyard lifespan. Here we reflect on major red blotch research progress and discuss future priorities. We also highlight the contribution of GRBV to the grapevine community as a major driver of enhanced cooperation among researchers, growers, nurseries, extension agents, policymakers, regulators, and service providers. We anticipate that strengthened interactions among all the members of the grapevine community and science-based disease management responses in vineyards will curtail GRBV spread and improve vineyard health.

A decade after the discovery of grapevine red blotch virus (GRBV), there is ample evidence of its detrimental impacts on grapevine physiology, grape composition, and wine production. To mitigate the spread of GRBV in vineyards, roguing is recommended as a disease management response. The imperative to identify and remove diseased vines justifies the development of autonomous scouting. In this study, nearly 700 ground-based hyperspectral images, encompassing both symptomatic and asymptomatic vine canopies, were collected in a Cabernet Franc vineyard during two growing seasons, capturing pre- and post-veraison vine development stages. Spanning 230 bands from visible (VIS) to near-infrared (NIR) domains (510 to 900 nm with 1.7 nm width), canopy spectral signals were isolated from the background through semantic segmentation using U-Net. Simultaneously, the GRBV status of each vine was established in the laboratory through polymerase chain reaction. These two intertwined datasets were used for training various machine learning algorithms and their ensembles. In addition, strategies to reduce dataset size through spectral binning and testing three different feature selection methods (Recursive Feature Elimination, Univariate Feature Selection, and taking into consideration autocorrelation) were explored. Our findings revealed that hyperspectral imagery identified GRBVinfected vines with an accuracy of 75.7 % around harvest, coinciding with the peak of disease symptom expression, utilizing only 19 bands with a 16 nm bin width. Prior to veraison when most vines are asymptomatic, an accuracy of 74.2 % was achieved, employing 5 bands with a 16 nm bin width. This study substantiates the utility of hyperspectral images in the identification of GRBV-infected vines, offering a robust foundation for the development of a streamlined sensing system that holds great promise for the grape and wine industry in effectively scouting vineyards for GRBV.

Grapevines face many threats, with viral diseases like grapevine leafroll and red blotch viruses being particularly challenging due to their elusive nature and lack of cures. Traditional containment methods involve identifying and replacing infected vines, a laborious and costly process. This highlights the need for innovative detection solutions. Advances in technology offer hope, promising more efficient methods. Since 2019, a research project at CSU Fresno has been investigating the use of hyperspectral imagery to improve the detection of leafroll and red blotch.

Grapevine red blotch virus (GRBV), the causal agent of red blotch disease of grapevines, is transmitted by Spissistilus festinus, the threecornered alfalfa hopper. Isolates of GRBV belong to two phylogenetic clades (I and II) and S. festinus is a dimorphic insect, with two genotypes found in the western (California, CA) and the southeastern (SE) regions of the United States. The transmission of GRBV by S. festinus is circulative and nonpropagative, yet some parameters of transmission remain to be characterized. Here, we compared the acquisition, transmission, and retention of GRBV isolates from phylogenetic clades I and II by S. festinus males and females of the two genotypes. Results indicated that the SE genotype acquired GRBV more efficiently (72.5%, 29/40) than the CA genotype (22.5%, 18/80), with differences in acquisition observed between males (32.5%, 26/80) and females (52.5%, 21/40) of the two S. festinus genotypes and between GRBV isolates of phylogenetic clades I (29%, 23/80) and II (60%, 24/40). Following acquisition, both S. festinus genotypes and sexes retained GRBV isolates of phylogenetic clades I and II for at least 60 days without access to an infected plant. For transmission, the GRBV isolate of phylogenetic clade II was more efficiently transmitted by the SE genotype (54%, 13/24) than the CA genotype (17%, 4/24) and SE females (75%, 12/16) were significantly more efficient transmitters of GRBV than CA females (19%, 3/16). Together, our findings revealed that S. festinus genotype, sex, and virus isolate influence GRBV acquisition and transmission but not retention. This research addressed important knowledge gaps in S. festinus-mediated transmission of GRBV that are essential for advancing red blotch disease epidemiology and developing appropriate disease management responses.

The impacts of viruses on horticultural and fruit traits of apple trees were first characterized half a century ago, yet the responses to viruses of contemporary apple cultivars and rootstocks in high-density planting systems remains largely unknown. The aim of our study was to evaluate the effects of apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), apple green crinkle-associated virus (AGCaV), and citrus concave gum-associated virus (CCGaV) on horticultural and fruit traits of young apple trees representing six commercially relevant scion-rootstock combinations in a high-density experimental orchard in New York. Analyses of horticultural traits showed statistically significant differences between infected and uninfected trees in trunk diameter (CCGaV) and terminal shoot growth (CCGaV, AGCaV); however, differences in means were small and inconsistent. Analyses of fruit traits revealed a statistically significant yield reduction of ‘Baigent’ Gala trees on ‘Geneva 935’ (G.935) rootstocks associated with ACLSV (-27

Among the three viruses of the genus Grablovirus in the plant virus family Geminiviridae, insect vector transmission is documented only for grapevine red blotch virus (GRBV), for which Spissistilus festinus (Hemiptera: Membracidae), the three-cornered alfalfa hopper, is identified as a vector. Transmission of GRBV by S. festinus is circulative, nonpropagative; it is also transstadial but neither transovarial, nor via seeds. Here, we thoroughly describe the S. festinus-mediated transmission process of GRBV with Vitis species, the natural hosts of the virus, and Phaseolus vulgaris plants, an experimental pseudo-systemic herbaceous host of the virus.

Pome fruits from the family Rosaceae are grown globally. Viruses can affect their quality, shape, and size. Apple stem grooving virus (ASGV, genus Capillovirus, family Betaflexiviridae) has a wide host range (Chofong et al., 2024), including apple and pear trees (Mustafayev, 2021). No information is available on the occurrence of ASGV in Azerbaijan. The aim of this research was to survey apple, pear and quince in commercial orchards and private gardens of various cultivars in different geographical areas of Azerbaijan for ASGV. A total 87 leaf samples was collected in 2017-2019 from 38 apple (Malus domestica L.), 27 pear (Pyrus communis L.), and 22 quince (Cydonia oblonga L.) trees exhibiting stem deformation and foliar necrotic spots for DAS-ELISA testing using ASGV serological reagents (Neogen - Scotland, UK). Positive reactions to ASGV were obtained for 27 apple, 16 pear, and 6 quince samples. To confirm the presence of ASGV, total RNA was isolated from 67 leaf samples (0.5 g), as previously described (Foissac et al., 2005), and analyzed by RT-PCR using the OneStep RT-PCR kit (Qiagen) and primers designed in the coat protein gene of ASGV (F: 5'-GCCACTTCTAGGCAGAACTCTTTGAA-3' and R: 5'-AAGCATGACTTCCTGTTTTTCCCCAA-3') and the NADH dehydrogenase subunit 5 fragment used as a housekeeping gene (Menzel et al., 2002). RT-PCR analyses were performed for 5 min at 95 °C and 34 cycles of 10 s at 95 °C, 20 s at 50 °C, 20 s at 72 °C, followed by a final extension of 2 min at 72 °C. DNA amplicons were resolved by electrophoresis on 2% agarose gels and visualized under UV illumination after staining with GelRed (Biotium, Fremont, CA) (Mustafayev et al., 2025). Results showed ASGV DNA amplicons of the expected size (273 bp) in 17 apple and 2 pear samples but not in quince samples. Discrepancies between DAS-ELISA and RT-PCR results are unclear but could be due to the primers not optimally capturing the ASGV genetic variability. DNA amplicons corresponding to one pear (PV098407) and five apple (PV098408-PV098412) samples were cleaned with Exo SAP-IT and characterized by bidirectional Sanger sequencing at the Cornell Biotechnology Resource Center in Ithaca, NY. Sequence analyses with the DNASTAR Lasergene software package (v17.3.0.57) showed a high sequence identity ranging from 98.2% to 98.9% and 97.8% to 100% at the nucleotide and amino acid, respectively, among Azerbaijani ASGV isolates and other apple and pear isolates from South Korea (JN792474, JN792477), the USA (MW322834), Pakistan (MN861413), China (OM313348), Germany (MW582790), and Brazil (MK929791). Azerbaijani ASGV isolates Aghdam 81 and Xachmaz 256 were co-infected ACLSV, isolate Sheki 27 with apple chlorotic leaf spot virus, and isolates Guba 101 and Shemkir 174 with apple stem pitting virus, as shown by RT-PCR. In consistence with other reports, stem and leaf symptoms were not associated with the occurrence of ASGV in single or mixed infections, and sequence identities of the partial ASGV coat protein gene were not distinct between apple and pear isolates (Shokri et al., 2023). This is the first report of ASGV in apple and pear trees in Azerbaijan.

Ceresini treehoppers are present in northern California vineyard ecosystems, including the closely related Spissistilus and Tortistilus (Hemiptera: Membracidae). These membracids are not direct pests of wine grapes, but S. festinus is a vector of grapevine red blotch virus (GRBV). No information is available on the ability of Tortistilus spp. to transmit GRBV. In this study, Tortistilus were collected on yellow panel cards across 102 vineyard sites and surrounding areas in Napa Valley, California, USA in 2021–2023. Specimens were morphotyped, sexed and tested for GRBV ingestion and acquisition by multiplex PCR or qPCR. Phylogenetic analysis of the partial sequence of mt-COI and ITS gene fragments of a subset of 40 Tortistilus specimens revealed clustering in a monophyletic clade with T. wickhami with the former barcode sequence. Only 6% (48/758) of the T. wickhami tested positive for GRBV, but none of the heads with salivary glands (0%, 0/50) of the dissected specimens tested positive for GRBV, indicating no virus acquisition. In contrast, half of the dissected heads with salivary glands of S. festinus (52%, 12/23), from the same collection vineyard sites, tested positive for GRBV. Together, our findings confirmed the presence of T. wickhami in northern California vineyards and suggested a dubious role of this treehopper as a vector of GRBV.