Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.

Grapevine red blotch virus (GRBV) is a DNA virus in the family Geminiviridae. This pathogen is the causal agent of grapevine red blotch disease, which affects cultivated grapevines and leads to negative effects on crop quality and yield. GRBV is present in commercial vineyards across North America, indicating that spread might have been largely human mediated. That said, recent surveys have demonstrated that there appears to be secondary transmission, most likely by an insect vector. In this study, vineyard insects and plants were surveyed to identify potential candidate vectors and noncrop plants that could act as reservoirs for this pathogen. Results reconfirm that GRBV is limited to Vitis spp., including both cultivated and wild grapevines. Eleven insect genera or species, field collected in vineyards, tested positive for GRBV using quantitative polymerase chain reaction. These insect taxa include unknown Aphididae, the cicadellids Aceratagallia spp., Acinopterus angulatus, Caladonus coquilleti, Colladonus montanus reductus, Colladonus sp., Empoasca spp., Erythroneura elegantula, and Scaphytopius graneticus, along with the membracid Spissistilus festinus and an unknown delphacid. Of these organisms, S. festinus is already known to be capable of transmitting GRBV, and Scaphytopius graneticus may merit closer evaluation as a candidate vector. [ ] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Background: Climate plays an essential role in forest health, and climate change may increase forest productivity losses due to abiotic and biotic stress. Increased temperature leads to the increased formation of ozone (O₃). Ozone is formed by the interaction of sunlight, molecular oxygen and by the reactions of chemicals commonly found in industrial and automobile emissions such as nitrogen oxides and volatile organic compounds.Although it is well known that productivity of Northern red oak (Quercus rubra) (NRO), an ecologically and economically important species in the forests of eastern North America, is reduced by exposure to O₃, limited information is available on its responses to exogenous stimuli at the level of gene expression.Results: RNA sequencing yielded more than 323 million high-quality raw sequence reads. De novo assembly generated 52,662 unigenes, of which more than 42,000 sequences could be annotated through homology-based searches. A total of 4140 differential expressed genes (DEGs) were detected in response to O₃stress, as compared to their respective controls. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the O₃-response DEGs revealed perturbation of several biological pathways including energy, lipid, amino acid, carbohydrate and terpenoid metabolism as well as plant-pathogen interaction.Conclusion: This study provides the first reference transcriptome for NRO and initial insights into the genomic responses of NRO to O₃. Gene expression profiling reveals altered primary and secondary metabolism of NRO seedlings, including known defense responses such as terpenoid biosynthesis.

Despite the significance of grape production to the fruit industry in Tennessee (TN), no published information has been available on viruses affecting grapevines in the state. Hence, a survey was conducted during the 2016 and 2017 growing seasons to determine the status of nine major viruses of grapevines in TN vineyards by taking advantage of classical serological assays and confirmatory nucleic acid-based diagnostic approaches. A total of 349 samples from 23 grapevine cultivars mostly displaying viral-like symptoms were collected from 23 commercial vineyards. All samples were assayed by DAS-ELISA for arabis mosaic virus (ArMV), grapevine leafroll-associated virus (GLRaV)-1, GLRaV-2, GLRaV-3, GLRaV-4, grapevine fanleaf virus (GFLV), tobacco ringspot virus (TRSV), and tomato ringspot virus (ToRSV). Selected serologically positive samples were also tested by RT-PCR, followed by Sanger sequencing of the generated amplicons. Additionally, 19 grapevines displaying symptoms characteristic of grapevine red blotch virus (GRBV) were also assayed by PCR followed by confirmatory sequencing-based methods. Collectively, these assays verifiably detected GLRaV-1, GLRaV-2, GLRaV-3, ToRSV, and GRBV in TN vineyards. This is the first record of the presence of these viruses in TN vineyards. ArMV, GLRaV-4, GFLV, and TRSV were not detected. The majority of samples tested positive for a single virus, whereas mixed infections with more than one virus were detected in 37% of samples.

Prune dwarf virus (PDV) is one of the major positive RNA viruses which cause economical damages in stone fruit trees. The symptoms of PDV vary between different stone fruits namely sour and sweet cherry, almond, peach, apricot and plum including leaf narrowing, leaf chlorosis, vein clearing, mosaic, leaf whitening, leathery leaf, bushy branches and stunt trees. During the years 2011 and 2012, 251 leaf samples were collected for detection of PDV in stone fruit orchards of Charmahal-va-Bakhtiari province. DAS-ELISA test proved PDV presence serologically. Then, total RNA were extracted and tested by two-step RT-PCR which replicated partial and full coat protein sequence of PDV. One hundred and eighty one out of total samples (251 samples) showed PDV infection using serological and two-step RT-PCR assays, hence, incidence of PDV in Charmahal-va-Bakhtiari province was confirmed. This is the first report of PDV in stone fruit orchards of Charmahal-va-Bakhtiari province and in Iran.

Rose leaf rosette-associated virus (RLRaV) is a member of genus Closterovirus, family Closteroviridae. The virus was first discovered in China in 2015 from a mixed infected wild rose (Rosa multiflo...