Complete Genome Sequence Resource for a Recently Isolated Potato Ring Rot Pathogen, Clavibacter sepedonicus K496.

Potato ring rot caused by Clavibacter sepedonicus has been a devastating disease in the U.S. since 1930. In this study, we isolated a recent C. sepedonicus strain, K496, from potato tubers showing discolorations of the vascular cylinder or pith tissues. We de novo assembled the genome sequence of K496 with 1,924,544,313 bp of Nanopore reads (N50 = 13,785 bp) using Flye v2.9 and polished it with 2 × 150 bp paired-end Illumina reads (855,788,703 bp in total). The resulting genome of K496 consists of a single circular chromosome 3,266,016 bp long and a linear plasmid of 135,489 bp. Using the NCBI PGAP v5.3, this genome was predicted to have 3,301 genes, encompassing 3,247 protein-coding genes, 90 pseudogenes, two 5S rRNA-coding, two 16S rRNA-coding, two 23S rRNA-coding sequences, 45 tRNAs, and three noncoding RNAs. The chromosome and plasmid sequences have been deposited at the NCBI GenBank database under the accession numbers CP088266 and CP088267, respectively.

Species of Dickeya and Pectobacterium Isolated during an Outbreak of Blackleg and Soft Rot of Potato in Northeastern and North Central United States.

An outbreak of bacterial soft rot and blackleg of potato has occurred since 2014 with the epicenter being in the northeastern region of the United States. Multiple species of Pectobacterium and Dickeya are causal agents, resulting in losses to commercial and seed potato production over the past decade in the Northeastern and North Central United States. To clarify the pathogen present at the outset of the epidemic in 2015 and 2016, a phylogenetic study was made of 121 pectolytic soft rot bacteria isolated from symptomatic potato; also included were 27 type strains of Dickeya and Pectobacterium species, and 47 historic reference strains. Phylogenetic trees constructed based on multilocus sequence alignments of concatenated dnaJ, dnaX and gyrB fragments revealed the epidemic isolates to cluster with type strains of D. chrysanthemi, D. dianthicola, D. dadantii, P. atrosepticum, P. brasiliense, P. carotovorum, P. parmentieri, P. polaris, P. punjabense, and P. versatile. Genetic diversity within D. dianthicola strains was low, with one sequence type (ST1) identified in 17 of 19 strains. Pectobacterium parmentieri was more diverse, with ten sequence types detected among 37 of the 2015-2016 strains. This study can aid in monitoring future shifts in potato soft rot pathogens within the U.S. and inform strategies for disease management.

An anomalous detection of Tomato yellow leaf curl virus in tomato in New York State.

In August 2020, a New York State vegetable grower sought assistance to identify a malady of tomato (Solanum lycopersicum). The plants were grown from saved seed that had been planted annually in NY and/or FL for over 15 years without significant disease problems, but the identity of the cultivar was not known. Submitted photos showed severely stunted plants with distorted leaves (crinkling, cupping, twisting); leaves were reduced in size and showed interveinal yellowing. Although the most likely explanation given the growing region was herbicide damage, the symptoms bore a striking resemblance to those presented by tomato yellow leaf curl (TYLCV)-infected tomato plants. TYLCV has not been reported from NY, as the whitefly vector (Bemisia tabaci) does not overwinter in the region. Stem tissue from a symptomatic plant was grafted onto a greenhouse grown rootstock of tomato breeding line 201231 (Cornell University); shoots emerging from grafted rootstocks showed symptoms consistent with those on the scion within 21 days of grafting. Total nucleic acid was extracted (Gambino et al. 2008), and a polymerase chain reaction (PCR) assay to detect TYLCV was performed using primers AV632 and AC1048 (Martínez-Culebras et al. 2001). Sanger sequencing of the expected size ~460 bp product from a representative sample showed 98% nucleotide identity with the sequence of over 52 isolates of TYLCV (blastn analysis using default parameters; Altschul et al. 1990). The total nucleic acid preparation was subjected to rolling circle ampification followed by restriction enzyme SphI digestion (Haible et al. 2006). An approximately 2.8 kb DNA fragment was resolved by agarose gel electrophoresis, gel purified, inserted into the cloning vector pUC19 and sequenced. Two clones yielded sequence of 2781 nt with only one nt mismatch (accession # MW373746, MW373747). BLAST analysis showed the sequence to be most closely related to TYLCV-IL from papaya in Texas (accession KX024647.1) with 99% identity (2752 of 2781 nt). Further inquiry revealed that the vegetable grower's plants had been seeded and grown in Florida prior to transplanting in NY; Florida is a production region where the virus and vectors are endemic. Although the virus has been shown to be associated with seed (Pérez-Padilla et al. 2020) and seed transmission has been reported (Kil et al. 2016), this subject is controversial and the epidemiology of the disease is not consistent with a seed-transmitted virus (Rojas, et al. 2018). In this reported occurrence, the most plausible explanation is that the virus was introduced into NY with transplants. All of the field grown transplants of this cultivar were infected, but no local disease spread in NY was reported, nor were there reports of the vector. The significance of this report is to highlight the importance of phytosanitation in the movement of plants and plant materials. The long-distance movement of TYLCV via infected transplants in the US and globally is well-established. The presence of a pathogen may be transient and their establishment will depend on the epidemiology of the pathogen, in this case, the presence of the vector.

Grapevine Red Blotch Virus Is Transmitted by the Three-Cornered Alfalfa Hopper in a Circulative, Nonpropagative Mode with Unique Attributes.

The transmission mode of grapevine red blotch virus (GRBV, genus Grablovirus, family Geminiviridae) by Spissistilus festinus, the three-cornered alfalfa hopper, is unknown. By analogy with other members in the family Geminiviridae, we hypothesized circulative, nonpropagative transmission. Time-course experiments revealed GRBV in dissected guts, hemolymph, and heads with salivary glands after a 5-, 8-, and 10-day exposure to infected grapevines, respectively. After a 15-day acquisition on infected grapevines and subsequent transfer on alfalfa, a nonhost of GRBV, the virus titer decreased over time in adult insects, as shown by quantitative PCR. Snap bean proved to be a feeding host of S. festinus and a pseudosystemic host of GRBV after Agrobacterium tumefaciens-mediated delivery of an infectious clone. The virus was efficiently transmitted by S. festinus from infected snap bean plants to excised snap bean trifoliates (90%) or grapevine leaves (100%) but less efficiently from infected grapevine plants to excised grapevine leaves (10%) or snap bean trifoliates (67%). Transmission of GRBV also occurred trans-stadially but not via seeds. The virus titer was significantly higher in (i) guts and hemolymph relative to heads with salivary glands, and (ii) adults emanating from third compared with first instars that emerged on infected grapevine plants and developed on snap bean trifoliates. This study demonstrated circulative, nonpropagative transmission of GRBV by S. festinus with an extended acquisition access period compared with other viruses in the family Geminiviridae and marked differences in transmission efficiency between grapevine, the natural host, and snap bean, an alternative herbaceous host.

Cultivated and Wild Grapevines in Tennessee Possess Overlapping but Distinct Virus Populations.

Viruses and viroids prevalent in a population of 42 wild grapevines (i.e., free-living, uncultivated grapevines; Vitis spp.) were compared with those in a population of 85 cultivated grapevines collected in Tennessee, United States by RNA sequencing analysis of pools of ribosomal RNA-depleted total RNA. The sequences of 10 viruses (grapevine fleck virus, grapevine leafroll-associated virus 2, grapevine rupestris stem pitting-associated virus, grapevine Syrah virus 1, grapevine vein-clearing virus, grapevine virus B, grapevine virus E, tobacco ringspot virus, tomato ringspot virus, and a novel nano-like virus) and two viroids (hop stunt viroid and grapevine yellow speckle viroid 1) were detected in both grapevine populations. Sequences of four viruses (grapevine associated tymo-like virus, grapevine leafroll-associated virus 3, grapevine red blotch virus, and grapevine virus H) were identified only from cultivated grapevines. High, moderate, and low numbers of sequence reads were identified only from wild grapevines for a novel caulimovirus, an enamovirus, and alfalfa mosaic virus, respectively. The presence of most virus sequences and both viroids was verified independently in the original samples by reverse-transcription PCR followed by Sanger sequencing. Comparison of viral sequences shared by both populations showed that cultivated and wild grapevines harbored distinct sequence variants, which suggests that there was limited virus movement between the two populations. Collectively, this study represents the first unbiased survey of viruses and viroids in both cultivated and wild grapevines within a defined geographic region.

Identification of Pectobacterium versatile Causing Blackleg of Potato in New York State.

Soft rot bacteria classified in the Pectobacteriaceae (SRP), including Pectobacterium and Dickeya spp., are responsible for soft rot and blackleg diseases of potato. Since 2014, blackleg outbreaks caused by D. dianthicola have increased in the United States and Canada. Our previous study found that the most abundant causal organisms of blackleg disease in New York State were P. parmentieri and D. dianthicola, with the latter being the only Dickeya species reported. In the present study, we identified and characterized pathogenic SRP bacteria from 19 potato samples collected in New York State during the 2017 growing season. We used genome sequence comparison to determine the pathogens' species. We found eight P. versatile, one P. atrosepticum, two P. carotovorum, two P. parmentieri, and six D. dianthicola isolates in our 2017 SRP collection. This is the first time that P. versatile has been reported to cause potato blackleg disease in New York State. We determined the phylogenetic relationships between the SRP strains by using 151 single-copy orthologous gene sequences shared among the set of bacteria in our analysis, which provided better resolution than phylogenies constructed with the dnaX gene.

Grapevine Asteroid Mosaic-Associated Virus is Resident and Prevalent in Wild, Noncultivated Grapevine of New York State.

In North America, uncultivated, free-living grapevines (Vitis spp.) frequently grow alongside their cultivated counterparts, thus increasing the potential for exchange of microbiota. For this study, we used high-throughput sequencing (HTS) of small RNAs to survey for virus populations in free-living grapevines of the Finger Lakes region of New York State. Of 32 grapevines analyzed, 23 were free-living vines, while the remaining 9 were commercially grown Vitis vinifera plants from the same region. In total, 18 (78.3%) of the free-living grapevines tested were positive for grapevine asteroid mosaic-associated virus (GAMaV) infection by HTS, with detection confirmed by seminested reverse-transcription PCR and sequencing of nine isolates. Phylogenetic analyses of an ungapped alignment of the New York GAMaV sequences (length: 2,334 nucleotides) with the five known full-length or close to full-length global sequences showed that the New York isolates were broadly grouped. Of the nine cultivated plants, eight were infected with both hop stunt viroid and grapevine yellow speckle viroid 1, three were singly infected with grapevine leafroll-associated virus 3, and one harbored GAMaV. This limited survey of free-living grapevines, one of the first to use HTS, has highlighted the high incidence of a virus associated with disease in commercial V. vinifera.

Complete Genome Sequence of a Gram-Positive Bacterium, Leifsonia sp. Strain PS1209, a Potato Endophyte.

We report the complete and annotated genome sequence of a Gram-positive bacterium, Leifsonia sp. strain PS1209, a potato endophyte that was isolated from apparently healthy tubers of potato cultivar NY166. The circular genome is 4,091,164 bp long, with a GC content of 69.08%, containing 3,926 genes.

A rapid, sensitive and inexpensive method for detection of grapevine red blotch virus without tissue extraction using loop-mediated isothermal amplification.

Grapevine red blotch virus (GRBV) is an emerging virus of significant viticultural importance throughout North America. Here, we report the development of a simple protocol for point-of-use detection of GRBV. Extraction of nucleic acids is not required; instead, the whole intact plant can simply be pricked with a sterile pipette tip, which is then incubated in sterile distilled water to provide the sample template in a loop-mediated isothermal amplification (LAMP) reaction. This method is 10,000 times more sensitive than conventional PCR, costs under a dollar per sample, and can be completed from sampling to readout in just over half an hour.

Complete Genome Sequence of Dickeya dianthicola ME23, a Pathogen Causing Blackleg and Soft Rot Diseases of Potato.

In 2014, an outbreak of potato blackleg and soft rot disease emerged in North America and continues to impact potato production. Here, we report the annotated genome sequence of Dickeya dianthicola ME23, a strain hypothesized to be representative of the bacterial population responsible for this disease outbreak.

Evidence for the splicing of grablovirus transcripts reveals a putative novel open reading frame.

Grapevine red blotch virus (GRBV) is type member of the newly identified genus Grablovirus. It possesses a single-stranded circular DNA genome of around 3200 nucleotides encoding three open reading frames (ORFs) in both the virion sense, the V1 (CP), V2 and V3, and complementary sense, C1 (RepA), C2 and C3. As shown for members of the genus Mastrevirus, the C1 and C2 ORFs are predicted to fuse through splicing to form a replication-associated protein (Rep). Data obtained using high-throughput sequencing (RNA-Seq) of three RNA-enriched populations, extracted from GRBV-infected grapevine (Vitis vinifera), confirmed the presence of the predicted C1-C2 intron (nts 2288-2450), but in addition identified a larger virion-sense intron (nts 251-589) spanning the V2 ORF. Evidence for both introns in a number of isolates was supported by bioinformatic analysis of publicly available datasets (n=20). These observations were further supported by RT-PCR analyses in both GRBV-infected grapevine and transient expression assays where GRBV genome segments were agro-inoculated onto Nicotiana benthamiana. The donor site of the virion-sense intron is located within two small ORFs, V0 and V02, while the acceptor site is two-thirds along the V2 ORF. Splicing at these positions is predicted to delete the N terminus of the encoded V2 protein. Comparative analyses of full-length GRBV sequences and the related tentative grabloviruses Prunus geminivirus A and wild Vitis virus 1 support the existence of both introns and V0. The probable regulatory role of these introns in the GRBV infection cycle is discussed.

A Small RNA-Mediated Regulatory Network in Arabidopsis thaliana Demonstrates Connectivity Between phasiRNA Regulatory Modules and Extensive Co-Regulation of Transcription by miRNAs and phasiRNAs.

Gene regulation involves the orchestrated action of multiple regulators to fine-tune the expression of genes. Hierarchical interactions and co-regulation among regulators are commonly observed in biological systems, leading to complex regulatory networks. Small RNA (sRNAs) have been shown to be important regulators of gene expression due to their involvement in multiple cellular processes. In plants, microRNA (miRNAs) and phased small interfering RNAs (phasiRNAs) correspond to two well-characterized types of sRNAs involved in the regulation of posttranscriptional gene expression, although information about their targets and interactions with other gene expression regulators is limited. We describe an extended sRNA-mediated regulatory network in Arabidopsis thaliana that provides a reference frame to understand sRNA biogenesis and activity at the genome-wide level. This regulatory network combines a comprehensive evaluation of phasiRNA production and sRNA targets supported by degradome data. The network includes ~17% of genes in the A. thaliana genome, representing ~50% annotated gene ontology (GO) functional categories. Approximately 14% of genes with GO annotations corresponding to regulation of gene expression were found to be under sRNA control. The unbiased bioinformatic approach used to produce the network was able to detect 107 PHAS loci (regions of phasiRNA production), 5,047 active phasiRNAs (~70% of which were non-canonical), and reconstruct 17 regulatory modules resulting from complex regulatory interactions between different sRNA-regulatory pathways. Known regulatory modules like miR173-TAS-PPR/TPR and miR390-TAS3-ARF/F-box were faithfully reconstructed and expanded, illustrating the accuracy and sensitivity of the methods and providing confidence for the validity of findings of previously unrecognized modules. The network presented here includes a 2X increase in the number of identified PHAS loci, a large complement (~70%) of non-canonical phasiRNAs, and the most comprehensive evaluation of sRNA cleavage activity in A. thaliana to date. Structural analysis showed similarities to networks of other biological systems and demonstrated connectivity between phasiRNA regulatory modules with extensive co-regulation of transcripts by miRNAs and phasiRNAs. The described regulatory network provides a reference that will facilitate global analyses of individual plant regulatory programs such as those that control homeostasis, development, and responses to biotic and abiotic environmental changes.

The Distribution and Detection of Grapevine red blotch virus in its Host Depend on Time of Sampling and Tissue Type.

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative polymerase chain reaction (qPCR) method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a pattern similar to that found for the greenhouse-grown plants but, most strikingly, close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.

Prevalence and Genetic Diversity of Grabloviruses in Free-Living Vitis spp.

The distribution and diversity of grapevine red blotch virus (GRBV) and wild Vitis virus 1 (WVV1) (genus Grablovirus; family Geminiviridae) were determined in free-living Vitis spp. in northern California and New York from 2013 to 2017. Grabloviruses were detected by polymerase chain reaction in 28% (57 of 203) of samples from California but in none of the 163 samples from New York. The incidence of GRBV in free-living vines was significantly higher in samples from California counties with high compared with low grape production (χ2 = 83.09; P < 0.001), and in samples near (<5 km) to compared with far (>5 km) from vineyards (χ2 = 57.58; P < 0.001). These results suggested a directional spread of GRBV inoculum predominantly from vineyards to free-living Vitis spp. WVV1 incidence was also significantly higher in areas with higher grape production acreage (χ2 = 16.02; P < 0.001). However, in contrast to GRBV, no differential distribution of WVV1 incidence was observed with regard to distance from vineyards (χ2 = 0.88; P = 0.3513). Two distinct phylogenetic clades were identified for both GRBV and WVV1 isolates from free-living Vitis spp., although the nucleotide sequence variability of the genomic diversity fragment was higher for WWV1 (94.3 to 99.8% sequence identity within clade 1 isolates and 90.1 to 100% within clade 2 isolates) than GRBV (98.3% between clade 1 isolates and 96.9 to 100% within clade 2 isolates). Additionally, evidence for intraspecific recombination events was found in WVV1 isolates and confirmed in GRBV isolates. The prevalence of grabloviruses in California free-living vines highlights the need for vigilance regarding potential grablovirus inoculum sources in order to protect new vineyard plantings and foundation stock vineyards in California.

Pectobacterium and Dickeya Responsible for Potato Blackleg Disease in New York State in 2016.

Beginning in 2014, outbreaks of blackleg disease compromised potato (Solanum tuberosum) production in the northeastern United States. Disease severity was atypical for plantings with certified seed. During 2016, 43 samples with blackleg symptoms were analyzed, originating from more than 20 farms operating in New York State. A combination of techniques was employed to identify the blackleg pathogens: isolation in vitro, diagnostic PCR assays for Pectobacterium and Dickeya sp., pathogenicity assays, and DNA sequencing. Twenty-three bacterial isolates were obtained, the majority of which were designated D. dianthicola or P. parmentieri; two of the isolates were designated P. atrosepticum. All isolates were pathogenic in stem lesion and tuber soft rot assays and exhibited pectin degrading activity (pitting) in crystal violet pectate agar medium. Phylogenetic analyses of dnaX gene sequences placed all but one of the isolates into clades corresponding to D. dianthicola, P. parmentieri, or P. atrosepticum. One atypical isolate clustered with P. carotovorum subspecies. Data are consistent with the hypothesis that D. dianthicola from New York and the northeast are part of a single clade, and at least three different soft rot bacteria were associated with blackleg during 2016 in New York.

Identification of Plant Virus Receptor Candidates in the Stylets of Their Aphid Vectors.

Plant viruses transmitted by insects cause tremendous losses in most important crops around the world. The identification of receptors of plant viruses within their insect vectors is a key challenge to understanding the mechanisms of transmission and offers an avenue for future alternative control strategies to limit viral spread. We here report the identification of two cuticular proteins within aphid mouthparts, and we provide experimental support for the role of one of them in the transmission of a noncirculative virus. These two proteins, named Stylin-01 and Stylin-02, belong to the RR-1 cuticular protein subfamily and are highly conserved among aphid species. Using an immunolabeling approach, they were localized in the maxillary stylets of the pea aphid Acyrthosiphon pisum and the green peach aphid Myzus persicae, in the acrostyle, an organ earlier shown to harbor receptors of a noncirculative virus. A peptide motif present at the C termini of both Stylin-01 and Stylin-02 is readily accessible all over the surface of the acrostyle. Competition for in vitro binding to the acrostyle was observed between an antibody targeting this peptide and the helper component protein P2 of Cauliflower mosaic virus Furthermore, silencing the stylin-01 but not stylin-02 gene through RNA interference decreased the efficiency of Cauliflower mosaic virus transmission by Myzus persicae These results identify the first cuticular proteins ever reported within arthropod mouthparts and distinguish Stylin-01 as the best candidate receptor for the aphid transmission of noncirculative plant viruses.IMPORTANCE Most noncirculative plant viruses transmitted by insect vectors bind to their mouthparts. They are acquired and inoculated within seconds when insects hop from plant to plant. The receptors involved remain totally elusive due to a long-standing technical bottleneck in working with insect cuticle. Here we characterize the role of the two first cuticular proteins ever identified in arthropod mouthparts. A domain of these proteins is directly accessible at the surface of the cuticle of the acrostyle, an organ at the tip of aphid stylets. The acrostyle has been shown to bind a plant virus, and we consistently demonstrated that one of the identified proteins is involved in viral transmission. Our findings provide an approach to identify proteins in insect mouthparts and point at an unprecedented gene candidate for a plant virus receptor.

Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements.

In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3' untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites.

A novel grablovirus from non-cultivated grapevine (Vitis sp.) in North America.

During screening of non-cultivated (wild) grapevine (Vitis sp.) from Napa County, California for the grapevine red blotch virus (GRBV; genus Glabrovirus, family Geminiviridae), an atypical polymerase chain reaction product pattern was observed. Rolling circle amplification followed by cloning and sequencing revealed the presence of a circular DNA characteristic of geminiviruses. The complete genome of nine isolates of the virus ranged from 3204 to 3278 nt in size. The genome most closely resembled that of GRBV in both sequence (57 to 59% identity) and organization. With limited sequence identity to described geminiviruses, this virus warrants designation as a new species, and the name 'Wild Vitis virus 1' is proposed.

Spatiotemporal spread of grapevine red blotch-associated virus in a California vineyard.

Grapevine red blotch-associated virus (GRBaV), the causative agent of red blotch disease, is a member of the genus Grablovirus, in the family Geminiviridae and the first known geminivirus of Vitis spp. Limited information is available on the epidemiology of red blotch disease. A 2-hectare Vitis vinifera cv. 'Cabernet franc' vineyard in Napa County, California, USA was selected for monitoring GRBaV spread over a three-year period (2014-2016) based on an initially low disease incidence and an aggregation of symptomatic vines at the edge of the vineyard proximal to a wooded riparian area. The incidence of diseased plants increased by 1-2% annually. Spatial analysis of diseased plants in each year using ordinary runs analysis within rows and Spatial Analysis by Distance IndicEs (SADIE) demonstrated aggregation. Spatiotemporal analysis between consecutive years within the association function of SADIE revealed a strong overall association among all three years (X=0.874-0.945). Analysis of epidemic spread fitting a stochastic spatiotemporal model using the Monte Carlo Markov Chain method identified strong evidence for localized (within vineyard) spread. A spatial pattern consisting of a combination of strongly aggregated and randomly isolated symptomatic vines within 8-years post-planting suggested unique epidemic attributes compared to those of other grapevine viruses vectored by mealybugs and soft scales or by dagger nematodes for which typical within-row spread and small-scale autocorrelation are well documented. These findings are consistent with the existence of a new type of vector for a grapevine virus.