Molecular characterization of a divergent genetic variant of wheat Eqlid mosaic virus from a Texas wheat field.

Investigations conducted during the spring 2020 season to diagnose the associated viral agent of a severe mosaic disease of wheat in a Texas Panhandle field revealed the presence of wheat Eqlid mosaic virus (WEqMV; genus Tritimovirus, family Potyviridae) in the analyzed samples. The complete genome sequences of two WEqMV isolates were determined, and each was found to be 9,634 nucleotides (nt) in length (excluding the polyA tail) and to contain 5' and 3' untranslated regions of 135 nt and 169 nt, respectively, based on rapid amplification of cDNA ends (RACE) assays. Both sequences contained an open reading frame (ORF) of 9,330 nt encoding a polyprotein of 3,109 amino acids (aa). The ORF sequences of the two isolates were 100% identical to each other, but only 74.7% identical to that of the exemplar WEqMV-Iran isolate, with 85.7% aa sequence identity in the encoded polyprotein. The Texas WEqMV isolates also diverged significantly from WEqMV-Iran in the individual proteins at the nt and aa levels. This is the first report of WEqMV in the United States and the first report of this virus outside of Iran, indicating an expansion of its geographical range.

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.

Incidence and Genetic Diversity of Grapevine Virus G in Croatian Vineyards.

Grapevine virus G (GVG) is a recently discovered vitivirus infecting grapevines. Historically, viruses in the genus Vitivirus have been associated with the grapevine rugose wood disease. Based on new and previously reported GVG isolates, primers and probes were developed for real-time RT-PCR. The developed assay successfully detected the virus in infected plants during dormancy and the growing season. A field study of 4327 grapevines from Croatian continental and coastal wine-growing regions confirmed the presence of GVG in 456 (~10.5%) grapevines from three collection plantations and 77 commercial vineyards, with infection rates ranging from 2% to 100%. Interestingly, the virus was confirmed only in vines considered to be Croatian autochthonous cultivars, but not in introduced cultivars. A 564-nucleotide long portion of the coat protein gene from previously known and newly characterized GVG isolates had nucleotide and amino acid identities ranging from 89% to 100% and from 96.8% to 100%, respectively. Phylogenetic analysis revealed five distinct groups, with isolates originating from the same site being close to each other, indicating possible local infection. The information presented in this manuscript sets the stage for future studies to better understand the ecology and epidemiology of GVG and the possible need for inclusion in certification schemes.

Grapevine Badnavirus 1: Detection, Genetic Diversity, and Distribution in Croatia.

Grapevine badnavirus 1 (GBV-1) was recently discovered in grapevine using high throughput sequencing. In order to carry out large-scale testing that will allow for better insights into virus distribution, conventional and real-time PCR assays were developed using sequences both from previously known, and four newly characterized isolates. Throughout the growing season and dormancy, GBV-1 can be detected by real-time PCR using available tissue, with the possibility of false-negative results early in vegetation growth. GBV-1 real-time PCR analysis of 4302 grapevine samples from the Croatian continental and coastal wine-growing regions revealed 576 (~13.4%) positive vines. In the continental wine-growing region, virus incidence was confirmed in only two collection plantations, whereas in the coastal region, infection was confirmed in 30 commercial vineyards and one collection plantation. Infection rates ranged from 1.9 to 96% at the different sites, with predominantly autochthonous grapevine cultivars infected. Conventional PCR products obtained from 50 newly discovered GBV-1 isolates, containing the 375 nucleotides long portion of the reverse transcriptase gene, showed nucleotide and amino acid identities ranging from 94.1 to 100% and from 92.8 to 100%, respectively. The reconstructed phylogenetic tree positioned the GBV-1 isolates taken from the same vineyard close to each other indicating a possible local infection event, although the tree nodes were generally not well supported.

Transmission of Grapevine Red Blotch Virus by Spissistilus festinus [Say, 1830] (Hemiptera: Membracidae) between Free-Living Vines and Vitis vinifera 'Cabernet Franc'.

Grapevine red blotch disease emerged within the past decade, disrupting North American vine stock production and vineyard profitability. Our understanding of how grapevine red blotch virus (GRBV), the causal agent of the disease, interacts with its Vitis hosts and insect vector, Spissistilus festinus, is limited. Here, we studied the capabilities of S. festinus to transmit GRBV from and to free-living vines, identified as first-generation hybrids of V. californica and V. vinifera 'Sauvignon blanc' (Vcal hybrids), and to and from V. vinifera 'Cabernet franc' (Vvin Cf) vines. The transmission rate of GRBV was high from infected Vcal hybrid vines to healthy Vcal hybrid vines (77%, 10 of 13) and from infected Vvin Cf vines to healthy Vcal hybrid vines (100%, 3 of 3). In contrast, the transmission rate of GRBV was low from infected Vcal hybrid vines to healthy Vvin Cf vines (15%, 2 of 13), and from infected Vvin Cf vines to healthy Vvin Cf vines (19%, 5 of 27). No association was found between transmission rates and GRBV titer in donor vines used in transmission assays, but the virus titer was higher in the recipient leaves of Vcal hybrid vines compared with recipient leaves of Vvin Cf vines. The transmission of GRBV from infected Vcal hybrid vines was also determined to be trans-stadial. Altogether, our findings revealed that free-living vines can be a source for the GRBV inoculum that is transmissible by S. festinus to other free-living vines and a wine grape cultivar, illustrating the interconnected roles of the two virus hosts in riparian areas and commercial vineyards, respectively, for virus spread. These new insights into red blotch disease epidemiology will inform the implementation of disease management strategies.

Thermotherapy Followed by Shoot Tip Cryotherapy Eradicates Latent Viruses and Apple Hammerhead Viroid from In Vitro Apple Rootstocks.

Virus and viroid-free apple rootstocks are necessary for large-scale nursery propagation of apple (Malus domestica) trees. Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV) are among the most serious apple viruses that are prevalent in most apple growing regions. In addition to these viruses, a new infectious agent named Apple hammerhead viroid (AHVd) has been identified. We investigated whether thermotherapy or cryotherapy alone or a combination of both could effectively eradicate ACLSV, ASGV, and AHVd from in vitro cultures of four apple rootstocks developed in the Cornell-Geneva apple rootstock breeding program (CG 2034, CG 4213, CG 5257, and CG 6006). For thermotherapy treatments, in vitro plants were treated for four weeks at 36 °C (day) and 32 °C (night). Plant vitrification solution 2 (PVS2) and cryotherapy treatments included a shoot tip preculture in 2 M glycerol + 0.8 M sucrose for one day followed by exposure to PVS2 for 60 or 75 min at 22 °C, either without or with liquid nitrogen (LN, cryotherapy) exposure. Combinations of thermotherapy and PVS2/cryotherapy treatments were also performed. Following treatments, shoot tips were warmed, recovered on growth medium, transferred to the greenhouse, grown, placed in dormancy inducing conditions, and then grown again prior to sampling leaves for the presence of viruses and viroids. Overall, thermotherapy combined with cryotherapy treatment resulted in the highest percentage of virus- and viroid-free plants, suggesting great potential for producing virus- and viroid-free planting materials for the apple industry. Furthermore, it could also be a valuable tool to support the global exchange of apple germplasm.

Virus Pathogens in Australian Vineyards with an Emphasis on Shiraz Disease.

Grapevine viruses are found throughout the viticultural world and have detrimental effects on vine productivity and grape and wine quality. This report provides a comprehensive and up-to-date review on grapevine viruses in Australia with a focus on "Shiraz Disease" (SD) and its two major associated viruses, grapevine virus A (GVA) and grapevine leafroll-associated virus 3 (GLRaV-3). Sensitive grapevine cultivars like Shiraz infected with GVA alone or with a co-infection of a leafroll virus, primarily GLRaV-3, show symptoms of SD leading to significant yield and quality reductions in Australia and in South Africa. Symptom descriptors for SD will be outlined and a phylogenetic tree will be presented indicating the SD-associated isolates of GVA in both countries belong to the same clade. Virus transmission, which occurs through infected propagation material, grafting, and naturally vectored by mealybugs and scale insects, will be discussed. Laboratory and field-based indexing will also be discussed along with management strategies including rogueing and replanting certified stock that decrease the incidence and spread of SD. Finally, we present several cases of SD incidence in South Australian vineyards and their effects on vine productivity. We conclude by offering strategies for virus detection and management that can be adopted by viticulturists. Novel technologies such as high throughput sequencing and remote sensing for virus detection will be outlined.

Identification and genomic characterization of grapevine Kizil Sapak virus, a novel grapevine-infecting member of the family Betaflexiviridae.

A novel virus with a (+) single-stranded RNA genome was detected by high-throughput sequencing (HTS) in a sample of grapevine (Vitis vinifera) cv. Kizil Sapak (sample/isolate 127) that originated from Turkmenistan. The complete genome of the virus, tentatively named "grapevine Kizil Sapak virus" (GKSV), is 7,604 nucleotides in length, excluding the poly(A) tail. The genome organization of GKSV, encoded genes, and sequence domains are typical for members of the family Betaflexiviridae, specifically those belonging to the subfamily Trivirinae. Phylogenetic analysis placed GKSV within the subfamily Trivirinae, in the same clade as fig latent virus 1 (FLV-1) but distinct from the clades formed by members of other genera. A comparative analysis of GKSV-127 with the HTS-derived sequences obtained from two additional isolates showed that they are genetic variants of the same virus species. Based on current ICTV species and genus demarcation criteria, and the results of the sequence and phylogenetic analyses, we propose that GKSV and FLV-1 represent a new genus within the subfamily Trivirinae.