Brief Summary of Minutes of Annual Meeting

For the meeitng agenda and a photo of the participants on the Zoom call, see the attachment at: https://www.nimss.org/projects/attachment/18276

The multi-state WERA-20 virtual annual meeting held during May 12 - 14, 2021, was hosted by Dr. Georgios Vidalakis, Professor & UC Extension Specialist in Plant Pathology and Director, Citrus Clonal Protection Program (CCPP), at the Department of Microbiology & Plant Pathology, University of California, Riverside, CA 92521. Dr. Timothy Paine, Divisional Dean Agricultural & Natural Resources, University of California, Riverside, welcomed the attendees followed by a brief overview of the CCPP and the role of research and extension and partnerships between institutions in agricultural sustainability. Dr. Amer Fayad, USDA-NIFA National Program Leader provided an overview of NIFA/Plant Systems Protection Programs and made a presentation on NIFA Competitive Funding Grant Programs, including opportunities for early career professionals, and Joint NSF/NIFA Program – Plant Biotic Interactions, to pursue collaborative research and extension in different areas of agriculture. Dr. Naidu Rayapati, Administrative Advisor, provided a brief account of the WERA-20 project that will be officially ending by end of September 2021. Subsequently, discussions were held about status of the new five year proposal WERA_TEMP_20 (Management of Diseases Caused by Systemic Pathogens in Temperate and Sub-Tropical Fruit Crops and Woody Ornamentals) submitted to Western Association of Agricultural Experiment Station Directors in December 2020. The Western Region’s Multistate Review Committee (MRC) provided feedback on the proposal and, in view of the importance of the project, recommended for resubmission with appropriate revisions suggested by the MRC and two peer-reviewers. Consequently, the proposal is designated as “Western Development Committee” to reflect that the proposal is “Under Review” until MRC approves the project. Based on MRC’s comments, the group discussed critical elements needed for revising the proposal. The writing committee will revise the proposal addressing MRC’s comments and proposal evaluator’s concerns and submit by January 15, 2022, for consideration by the MRC. It was agreed that Dr. Vidalakis will lead the writing committee to submit the revised proposal. Future venue for the annual meeting in 2022 will be decided when the revised proposal is approved by the MRC. As a special invitee, Dr.  Bret Hess, Executive Director, Western Association of Agricultural Experiment Station Directors, explained the process for becoming an official participant of the multi-state project using the NIMSS system (http://www.nimss.org) and encouraged participants to contact him to assist throughout the process. In addition to research and extension faculty from Land-grant universities, scientists from federal programs (USDA-ARS, USDA-APHIS, etc.), personnel from State Departments of Agriculture and Private sector and industry stakeholders can also become official participants of the multi-state projects through NIMSS system. Attendees were invited to become an official participant of the new WERA_TEMP_20 multi-state project by submitting the APPENDIX E through proper channels into the NIMSS system.

An outline of topics presented by participants:

1. State and National Reports:

Ioannis Tzanetakis (University of Arkansas)

• “What's new in the berry world”
• Introduction and elaboration on progress made on Strawberry and Blueberry viruses over the past year.

Maher Al Rwahnih (University of California, Davis)

• Overview of FPS Olive Program, Olive Virus Diseases and Certification.
• Continual research in Olive tree viruses, OQDS, virus management and sanitation.
• New control import permit is in place to allow importation of infected material from Italy.
• Olive oil production in California is increasing. Other states are producing as well.
• Due to disease, alternative Markets are needed due to demand.

Kiran Gadhave & Georgios Vidalakis (University of California, Riverside)

• Citrus yellow vein associated virus novel RNA- A 70 year old California tale
• A brief account of the problem and current understanding of the virus

Thein Ho (Driscoll’s Inc.)

• Characterization and detection of Rubus yellow net virus (RYNV) in raspberries
• RYNV strain #1 - Canada and RYNV strain #2 - UK are found.

Alexander Karasev (University of Idaho)

• Viruses of solanaceous fruit crops

Shulu Zhang (Agdia Inc.)

• “Recent developments of AmplifyRP assays for various crops”

Ramesh Pokharel (USDA-APHIS)

• Plant viruses in small berries in Maryland
• Survey, testing and documentation of multiple viruses in small fruits

Marc Fuchs (Cornell University)

• Updates on grapevine red blotch virus transmission by the three-cornered alfalfa hopper.
• Review of ongoing work and research data on this topic.

Christie Almeyda (North Carolina State University)

• Berry and grape viruses detected in North Carolina
• Review of testing capabilities for berries and new findings of viruses in 2021.
• Discussed about field survey of vineyards and testing results from 2018 to 2020

Lauri Guerra (Washington State Dept. of Agriculture)

• Washington State Fruit Trees Certification Updates

Scott Harper (Washington State University)

• Cherry Viruses, update on work over the last year.
• Impacts of little cherry disease and understanding the epidemiology of causal agents associated with the disease.
• New creation APP will be launched for growers to use as a reference in helping them identify virus symptoms in the field.

Arunabha Mitra, Sridhar Jarugula, & Naidu Rayapati (Washington State University)

• Molecular biology of Grapevine leafroll-associated viruses (GLRaVs)
• Recent efforts in building infectious cDNA clones for Grapevine leafroll-associated viruses, and current work on the molecular biology of GLRaV-1.

Adriana Larrea-Smiento & John Hu (University of Hawaii)

• Detection and characterization of plant viruses in Hawaiian pineapples.

Robert Martin (USDA-ARS)

• Presented an in-progress creation of a user-friendly Virus database for NCPN crops for end users.
• Demonstrated the NCPN database search capabilities, content for users. (multi-crop information)

Oscar Hurtado-Gonzales (USDA-APHIS)

• Status report of Fruit Trees held under Quarantine in PGOP
• Transitioning towards the use of field indicators generated in-house via tissue culture (CFIA approach).
• Review of diagnostic workflow through to Quarantine release.
• Data review of PGQP Pomes field survey in East Coast states.

Yilmaz Balci (USDA-APHIS)

• USA: Overview and updates to import requirements of pome & stone fruits
• The Controlled Import Permit (CIP) team introduced
• Review of how plants enter the United States, generally advisable plants, restricted or NAPPRA (Not approved pest plant risk assessment) plants.
• ePermits will no longer be available after the end of Fiscal year 2022.
• New system, APHIS eFile, is put in place to apply and manage your applications, registrations, permits & licenses.

1. 2021 WERA-20 Special Topics Updates:

Katlyn Staub (Washington Wine Industry Foundation)

• Update on the status of harmonization of State Certificate & Quarantine programs
• National Nursery Certification - Working Group Liaison w/ USDA APHIS on Certifications.
• Certification standards, State certification programs - funded by APHIS USDA.
• By Jan 2023, all planting materials of fruit crops exported to the EU to originate from approved certification programs.
• Start drafting documents to present for the EU.
• New focus: outreach & education; onboarding new programs
• State regulators participating: Washington, Oregon, California, Michigan, Pennsylvania. New York

Maher Al Rwahnih (UC Davis) and Ioannis Tzanetakis (University of Arkansas)

• Update on the standardization of high-throughput sequencing (HTS), diagnostics & regulatory issues
• HTS vs conventional methods for virus diagnostics in strawberry G1 plant.
• HTS vs PCR.
• Drawbacks of conventional methods for biological testing.
• Study results and ongoing work using HTS data to improve PCR test results.

Bret Hess (Executive Director, Western Association of Agricultural Experiment Station Directors)

• National Information Management & Support System (NIMSS) and Appendix E: the official process to join WERA 20
• Review of NIMSS website, and how to become a user in National Information Management & Support System (how to create new user profile)
• Creating a new Appendix E with a detailed walkthrough in the website.
• Noting Land grant University must use 25% of HATCH funds on multi-state projects.

James Stack (Kansas State University)

• A resource-conscious, near comprehensive approach to quality diagnostics for the National Plant Diagnostic Network (NPDN)
• Quality standards need to be achievable.
• Design system that is appropriate to the labs and resources.
• Quality standards should not be a burden to productivity.
• Review of Quality Proficiency, Accreditation, Core Accreditation Standards.

Patrick Sheil (USDA-APHIS)

• NPDN Project is currenting creating and implementing Professional Development courses that promote Quality Diagnostic Network (NPDN), including training materials. Funded by NPDN & supplemented by PPQ & Farm Bill

Erich Rudyj (USDA-APHIS, National Clean Plant Network)

• Understanding its quality from the perspective of National Clean Plant Network (NCPN) and it’s Strategic Plan.
• Permanent funding secured through the Farm Bill.
• Emphasis on Quality for all goals and objectives and why quality is important to NCPN on ‘National’ POV.

Irene Lavagi-Craddock and Fatima Osman (University of California, Riverside and Davis)

• Lavagi presented “Overview of the NCPN-Quality initiative”
• Restate the NCPN Creation of Quality Steering Committee and the Citrus Clonal Protection Program tasks of Therapy, diagnostics and distribution.
• Osman presented slides on Phase 3 “Building a roadmap for developing a system wide Quality plan.”
• Review of Milestones & Timeline - objectives (FY 2020 - FY 2023)
• Survey will be released soon to collect feedback from all NCPN centers.

Panel Discussion: Summary perspective of Quality- Erich Rudy (Facilitator)

• Introduction of Sarah Trujillo, USDA PDQ Office, NCPN Strategic Planner
• Emphasis on increase of question to better help the understanding of Quality across the board and on the importance of networking with other programs.
• SOP for Diagnostics is already part of the NCPN Strategic plan and surveys will assist in gathering data on how broad training should be.
• James Stack will look into allowing access to Core Quality Standards and the 360 Training Platform.
• Reminders for the importance of coordination of networks and incorporating Therapy Protocols using virus genome, multiple isolates, multiple tests.

Ioannis Tzanetakis (University of Arkansas)

In strawberry, we have fully characterized a new cytorhabdovirus and developed multiplex RT-PCR diagnostics protocols. We also completed transmission with the strawberry and small bramble aphid, but neither proved to be vectors of the virus. We also completed the characterization of another rhabdovirus in the crop. The new virus is the type member of a new genus in the family and transmission trials with the strawberry aphid were unsuccessful. As in the case with the novel cytorhabdovirus a multiplex RT-PCR test targeting two virus genes and an internal control have been developed.

In blueberry, the characterization of a novel luteovirus has been completed and revealed its close association with nectarine stem pitting associated virus. A survey of over 600 samples from the Pacific Northwest, Michigan, New Jersey and Pennsylvania indicated that the virus is very widespread as more than half of the samples tested positive. For understanding population structure of the virus, sequence analysis of the partial genome of ~ 300 isolates indicated ~ 18% diversity at the nucleotide level. The new carlavirus discovered in blueberries is most closely related to blueberry scorch virus (BlScV) and was present in ~ 2% of the aforementioned samples. The virus cross-reacts very poorly with antibodies against scorch, whereas its host range and symptomology on alternative hosts is different to that of BlScV. An infectious clone has been developed and will be used to test the reaction of major blueberry cultivars to the virus. This information is used in the development of accurate and reliable protocols for the detection of two blueberry viruses. These tests will be able to detect the vast majority of isolates of the two viruses circulating in the United States.

Ekaterina Nikolaeva (Pennsylvania Department of Agriculture)

Pennsylvania Department of Agriculture (PDA) in cooperation with Penn State University (PSU) conducted 2020 PPA 7721 funded surveys for exotic diseases in orchards and small fruits. Orchard survey targets included Plum pox virus, Asian Pear Blight (Erwinia pyrifoliae), Asiatic brown rot (Monilia polystroma), Apple brown rot (Monilinia fructigena), Apple Proliferation (Candidatus Phytoplasma mali), European stone fruit yellow (Ca. Phytoplasma prunorum), and Almond witches’ broom (Ca. Phytoplasma phoenicium). Small fruit survey targeted Asian pear blight (Erwinia pyrifolia), Nepovirus Tomato black ring virus, Australian Grapevine Yellows (Ca. Phytoplasma australiense), Flavescence Doreé Phytoplasma (Ca. Phytoplasma vitis), and Bois noir Phytoplasma (Ca. Phytoplasma solani). No exotic targets were detected.

In 2020, PDA/PSU team in collaboration with USDA-APHIS Plant Germplasm Quarantine Program (PGQP) evaluated distribution of known pome viruses and viroids on apple trees affected by Rapid Apple Decline (RAD) in PA. The samples collected from 18 orchards and 6 production sites were analyzed in PGQP via HTS and RT-PCR techniques. In result, ASGV, CCGaV,  ALV-1, ASPV, TRSV, ToRSV, and GaIV were the most common viruses detected on RAD affected trees. ACLSV, ARWaV1, ARWaV2, AHVd, AGCaV were also detected in PA apple orchards. PDA continues to operate with the Fruit Tree Improvement Program (FTIP), a specialized virus-tested fruit tree certification program. Three nurseries participated in the FTIP last year. Over 1,600 samples were tested for viruses of concern, including Prunus Necrotic Ringspot Virus (PNRSV), Prune Dwarf Virus (PDV), Tomato Ringspot Virus (ToRSV), and Plum Pox Virus (PPV). No PPV was detected in rootstock blocks or in registered source blocks. PNRSV remains most commonly found virus in Prunus in PA nurseries. The occurrence of PDV and ToRSV in registered blocks and nursery production blocks remain low. All blocks met virus-testing requirements for FTIP certification.

Maher Al Rwahnih (University of California, Davis)

Foundation Plant Services (FPS) continues to make advances in developing and refining methods using high throughput sequencing (HTS) as superior tools for the detection of existing and emerging viruses. We have used sequence information generated by HTS analysis to design new, species-specific PCR primers for use in molecular diagnostics. In addition, HTS proved to be an invaluable tool in the discovery of unknown viruses and in establishing a baseline analysis of the virome of a crop.

In 2016, FPS acquired a Controlled Import Permit (P588) to facilitate the introduction, quarantine, and release of imported Prunus for the tree fruit industry. In 2020, we were successful in obtaining USDA-APHIS and CDFA approval to revise our tree fruit protocols, eliminating four ineffective Prunus biological indicators. We conducted side-by-side studies by comparing HTS analysis to biological indexing, which revealed that the performance of the biological indicators was inferior to PCR and HTS testing. Under the new protocol, plants are subjected to greenhouse indexing and molecular testing at two different time points by PCR and HTS. Plants may be released if all test results demonstrate plants are free of viruses and virus-like agents.

FPS has now conducted three years of side-by-side studies comparing the efficacy of woody and herbaceous indexing to PCR and HTS testing in Prunus diagnostics. The results of these studies on 82 Prunus selections with 17 infected trees, indicate that GF 305 and Bing Cherry provided false negative results in every case. On the same selections, herbaceous host indexing was ineffective as well, with false negative results ranging from 33% to 100%. We have also compared biological indexing to HTS and PCR testing for detecting viruses on grapevine and roses. In 104 grapevine selections where 60 were infected with at least one virus, woody indicators (Cabernet Franc, St. George, and LN33) gave false negative results 4-6% of the time and herbaceous host indexing gave false negatives 25-100% of the time. In comparative studies on 65 rose selections with 22 infected plants, the woody indicators Rosa multiflora ‘Burr’ and Shirofugen cherry provided false negative results in 23% and 77% of the cases, respectively. The ‘Development and validation of real time quantitative PCR assays for the detection of viruses’ study evaluated the broad-range detection capacity of currently available real-time RT-PCR assays for viruses and developed new assays when current tests were inadequate or absent. assays for 15 different viruses were exhaustively evaluated in silico to determine their capacity to detect virus isolates deposited in GenBank. During this evaluation, several isolates deposited since the assay was designed exhibited nucleotide mismatches in relation to the existing assay’s primer sequences. In cases where updating an existing assay was impractical, we performed a redesign with the dual goals of assay compactness and comprehensive inclusion of genetic diversity. The efficiency of each developed assay was determined by a standard curve. To validate the assay designs, we tested them against a comprehensive set of 87 positive and negative Prunus samples independently analyzed by high throughput sequencing.  As a result, the real-time RT-PCR assays described herein successfully detected the different viruses and their corresponding isolates. To further validate the new and updated assays a Prunus germplasm collection was surveyed. The sensitive and reliable detection methods described here will be used for the large-scale pathogen testing required to maintain the highest quality nursery stock. We previously reported 15 new assays for viruses infecting Prunus were developed.

Our in-house validation studies have focused on the sensitivity, specificity, repeatability, and reproducibility of HTS as a routine diagnostic tool. We have developed the best sampling strategies by comparing two different types of tissue and we have optimized bioinformatics algorithms to efficiently separate pathogen and host sequences. In addition, we have participated in an inter-laboratory validation and standardization of HTS project with the USDA Center for Plant Health Science & Technology (CPHST) and Plant Germplasm Quarantine Program (PGQP) laboratories Maryland to coordinate the development of minimum basic requirements for the adoption of HTS technologies, including nucleic acid extractions, library preparation, depth of sequencing and bioinformatics, for the detection of viral pathogens. This cooperative study validated the HTS protocol and pipeline used by the participating laboratories for the identification of grapevine and apple viruses. All three laboratories detected 100% of the same viruses indicating the reliability of HTS as a detection method. This is  an on-going project that will include the validation of the HTS pipeline for Prunus virus detection in 2021-2022. These advances in protocol development for HTS technologies in plant virus detection are in-line with the International Plant Protection Convention Recommendation on Preparing to use high-throughput sequencing (HTS) technologies as a diagnostic tool for phytosanitary purposes. Our research and collaborative efforts have satisfied all the recommendations outlined in the document, allowing us to collaborate internationally and operate within the European framework.

Kiran Gadhave & Georgios Vidalakis (University of California, Riverside)

Our team at UC Riverside and the University of Maryland published a paper reporting the discovery of novel virus like RNA (provisionally named citrus yellow-vein associated virus or CYVaV) associated with the citrus yellow-vein disease (CYVD) which was first reported in California in 1957. The CYVaV RNA genome has 2,692 nucleotides and codes for two discernable open reading frames (ORFs). ORF1 encodes a protein of 190 amino acids (aa) whereas ORF2 is presumably generated by a −1 ribosomal frameshifting event just upstream of the ORF1 termination signal. The frameshift product (717 aa) encodes the RNA-dependent RNA polymerase (RdRp). Phylogenetic analyses suggest that CYVaV is closely related to unclassified virus-like RNAs in the family Tombusviridae. Bio-indexing and RNA-seq experiments indicate that CYVaV can induce yellow vein symptoms independently of known citrus viruses or viroids.

In an effort to develop CYVaV as a virus induced gene silencing (VIGS) vector, the first efforts involve regeneration of citrus plants from protoplasts of embryonic suspension cultures of citrus transfected with yellow vein RNA at the plant transformation facility at UCR. Over the past few months, we tested a few wild-type and recombinant CYVaV constructs in Daisy and Tango cell lines. We report the successful replication of CYVaV (via T7 RNA transcripts generated from linearized pET17b-CYVaV construct) in both ‘Daisy’ and ‘Tango’ cell lines of citrus. Transfected protoplasts appeared to regenerate cell walls, undergo cell division, and remain viable for up to 2 months post-transfection. The plant regeneration efforts via protoplast and other CYVaV delivery efforts will be continued at UC Riverside. To study the CYVaV distribution in the US, five samples from Texas A&M University and 33 samples from Puerto Rico have been analyzed using qRT-PCR. None of the 38 samples in total have been tested positive for the presence of CYVaV. Multiple researchers in Florida that have access to old citrus orchards have been contacted. Currently, efforts are being undertaken to collect samples. We studied CYVaV mobility and symptomatology (the onset of characteristic yellow vein disease symptoms) at the first time point (6 months after graft-inoculation) in all CYVaV graft-inoculated trees planted in a replicated field trial at Agricultural Operations fields at UC, Riverside. CYVaV mobility was tested via RT-qPCR analyses of root and shoot samples from all 6 graft-inoculated and 2 healthy (negative control) trees per combination (12 rootstock scion combinations in total). Of the 72 graft-inoculated trees, 32% of trees (from different rootstock scion combinations) tested positive, whereas 68% trees tested negative for CYVaV. None of the 16 older trees (various ages) with assorted rootstock scion combinations were positive for CYVaV. Only 5 of 88 trees (including additional combinations) showed characteristic yellow vein disease symptoms. The survival of grafts was checked 6 months after original graft inoculation (all original grafts had survived for at least 2 weeks) and trees with both dead grafts were re-grafted in June 2021.

Thein Ho (Driscoll’s Inc.)

Rubus yellow net virus (RYNV) belongs to genus Badnavirus. Badnaviruses are found in plants as endogenous (inactive) sequences, and/or in episomal (infectious and active) forms. To study the state of RYNV infections, we sequenced the genomes of 25 raspberry cultivars and mined eight published genome datasets. Sequence analysis revealed the presence of a diverse array of endogenous RYNV (endoRYNV) sequences that differ significantly in their structure; some lineages have nearly complete, yet non-functional genomes whereas others have rudimentary, small sequence fragments. We developed SYBR Green PCR assays to genotype the main endoRYNV lineages as well as the only known episomal lineage in commercial Rubus. This study reveals the widespread presence of endoRYNVs in commercial raspberries, likely because breeding programs have been using a limited pool of germplasm that originally harbored endoRYNVs.

Alexander Karasev (University of Idaho)

In 2018, leaf and petiole samples from five declining Chardonnay vines were collected from a single vineyard in Canyon County of Idaho. Ribodepleted total RNA prepared from these samples was subjected to a high-throughput sequencing (HTS) analysis on a MiSeq platform, yielding between 3,623,716 and 4,467,149 300-bp paired-end reads. Raw reads were adapted and quality cleaned and mapped against the Vitis vinifera L., reference genome. Unmapped paired reads were assembled, producing between 829 and 1,996 contigs over 1,000-nt in length. All five samples were found to contain GLRaV-3 and the two common viroids, hop stunt viroid and grapevine yellow speckle viroid, while four contigs ranging in size from 1,361 to 6,736 and exhibiting homology with the GRVFV were found in three out of the five Chardonnay samples analyzed. A nearly complete genome of GRVFV-ID was assembled from the HTS data of one sample, and the 3’-terminus of the genome was acquired using the RACE methodology; the 6,736-nt sequence has been deposited in the GenBank database under the accession number MZ027155. In the fall of 2020, six commercially operating vineyards in Canyon and Nez Perce Counties of Idaho, including the original one, were sampled for the total of 26 sampled plants of white and red wine grape cultivars, based on visual symptoms of leaf reddening, leaf rolling, and chlorosis, and tested by reverse transcription (RT)-PCR using newly designed GRVFV-specific primers. Four plants were found positive for GRVFV by RT-PCR; these positive samples came from three vineyards in Canyon County, from the same wine grape cultivar, Chardonnay. Amplified RT-PCR products were directly sequenced using conventional Sanger methodology and confirmed to represent 662-nt segments of the GRVFV genome exhibiting 98.6-99.1% pairwise identity to the HTS-derived full-length genome of GRVFV-ID (MZ027155). This close identity between the GRVFV sequences from three different Idaho vineyards, coming from the same cultivar Chardonnay, may suggest a common origin of the original GRVFV infection, possibly the same supplier of the original Chardonnay planting material. Presence of GRVFV might have contributed to the decline of the original Chardonnay vines, although the exact role of GRVFV in a mixed infection with GLRaV-3 is not clear at the moment.

Grapevine red blotch virus (GRBV) infection was recently identified in an Idaho commercial vineyard. Fruit quality from healthy vines (no viruses detected), and GRBV positive (infected) vines was determined for ‘Syrah’ grapes from this vineyard. GRBV infected vines produce grapes with significantly lower total sugars and lower total anthocyanins. They were also lower in a single free amino acid, yet higher in malic acid compared to grapes from healthy vines. No significant differences were seen in total organic acids, yeast assimilable nitrogen content, total free amino acids, total phenolics, or total tannins between the grapes of healthy vines versus those of GRBV infected vines. Overall, GRBV negatively influenced grape quality by reducing total sugars and total anthocyanins adversely affecting wine quality.

Shulu Zhang (Agdia Inc.)

Agdia Inc., a leading plant diagnostics company, has been utilizing advanced diagnostic technologies to help crop growers and researchers to effectively detect the presence of plant pathogens in their crops and prevent a potential crop loss from infection by pathogens. One such technology is recombinase polymerase amplification, a leading isothermal amplification technology, based on which Agdia Inc. has developed its own isothermal amplification platform – AmplifyRP^®. In recent years, Agdia has developed and commercialized 28 AmplifyRP^® kits. Among them, 26 kits are capable of specifically detecting 25 different pathogens infecting a wide range of crops and 2 kits can be used for any pathogen. Over the past year, 5 AmplifyRP^® kits were commercialized for Grapevine leafroll-associated virus 3, Potato virus Y, Potato mop-top virus, Tomato brown rugose fruit virus, and Hop latent viroid. These five kits specific to a single species of viruses or viroids can produce real-time, quantitative results and are deployable both in laboratories and in the field. They are simple to use, and no thermal cycler and DNA/RNA purification are needed as all reactions works well with plant crude extracts at a constant temperature 39-42°C. The whole assay from sample to result can be completed within 30 minutes and yet it is as sensitive as qPCR or PCR. This isothermal amplification technology AmplifyRP^® has provided a versatile detection tool for rapid detection of many important plant pathogens and helped growers to effectively manage crops and prevent significant economic losses from damages by pathogens.

Marc Fuchs (Cornell University)

Research efforts at Cornell have primarily focused on grapevine red blotch virus (GRBV) (Cieniewicz et al.,

2020; Flasco et al., 2021) and grapevine fanleaf virus (GFLV) (Osterbaan et al., 2021; Hily et al., 2021).  For GRBV, emphasis was placed on virus transmission by the three-cornered alfalfa hopper (Flasco et al., 2021) and on vector ecology (Cieniewicz et al., 2020).  For GFLV, virus-host interactions (Osterbaan et al., 2021) and virus evolution (Hily et al., 2021) were investigated.  Efforts have also focused on exploring RNA interference as a potential means to control the grape mealybug vector of grapevine leafroll viruses (Arora et al., 2020).

Christie Almeyda (North Carolina State University)

The Micropropagation and Repository Unit (MPRU) at North Carolina State University (NCSU) produces, maintains and distributes pathogen-tested G1 material of berry crops (strawberry, blackberry, raspberry and blueberry) and muscadine grapes to industry and researchers in the U.S. The MPRU presently conducts testing for targeted pathogens and therapy for pathogen elimination (heat treatment and meristem-tip culture) and maintains Fragaria, Rubus and Vaccinium G1 (foundation) blocks in vitro, in the greenhouse and the screenhouse. The same methods are applied for muscadine grapes.

In recent years, this facility has cleaned and tested mainly domestic materials from most of the berry breeding programs in the U.S. and muscadine grapes breeding programs from the Southeast. While cleaning up berry crops, the following viruses were detected on blueberries: Blueberry latent virus (BBLV) and Blueberry red ringspot virus (BRRV). Blackberry yellow vein-associated virus (BYVaV), Blackberry virus E (BlVE) and Citrus concave gum-associated virus (CCGaV-like) were detected on blackberries. Officially in 2021, the MPRU has established a partnership with the NC Plant Disease and Insect Clinic (PDIC) to test berry and grape samples from NC growers. The MPRU has a controlled imported permit (CIP) under which the unit is currently cleaning blueberries from Chile and Peru and strawberries from Korean and Japan.

In partnership with Dr. Hoffmann (NCSU strawberry and grape extension specialist), samples from NC grower vineyards were tested using protocols previously developed by Foundation Plant Services (FPS), UC-Davis in collaboration with Dr. Maher Al Rwahnih. The pathogens currently being tested are Grapevine leafroll viruses (GLRaV-2, GLRaV-3, GLRaV-4, GLRaV7), Grapevine red blotch virus (GRBV), Grapevine rupestris stem pitting associated virus (GRSPaV), vitiviruses (GVA, GVB), Tobacco RingSpot Virus (TRSV), and Xyllela fastidiosa. Eighty samples were tested from 8 vineyards in NC (7 Vitis vinifera vineyards and 1 muscadine vineyard) in order to know the incidence of viral pathogens in this area during 2018, 2019 and 2020. GLRaV-3, GRBV and Xyllela fastidiosa were detected during the second year of this survey. GLRaV-3 and GRBV were the most predominant (20/80 each). Only 6 samples were positive for Xyllela fastidiosa. GLRaV-2, GLRaV-2 and GRBV were found in the third year. We are also working into cleaning and virus testing the material we currently have at the MPRU (10 NC muscadine cultivars) and new material (5 genotypes) we obtained from the AR breeding program in 2019.

Scott Harper (Washington State University)

The Harper lab is one of the major participants in the Washington & Oregon Little Cherry Disease task force, providing the pathology component in collaboration with entomology, horticulture and extension researchers to understand the pathology and etiology of this disease.  We are studying to track the movement of X-disease phytoplasma strains between hosts in the orchard and extra-orchard environment. Preliminary results suggest that annual weeds play an incidental role in long term spread, while perennial hosts are the main reservoir for the pathogen. Cumulatively, our efforts have changed grower practices: 89% of respondents at the 2020 NW Hort Show said they had changed management practices over the last two years as a result of information from WSU researchers (N=160), 86% had scouted and sampled symptomatic trees (N=215), 77% had removed infected trees (N=197), and 70% had applied post-harvest sprays for leafhoppers (N=188).

Adriana Larrea-Smiento, Alejandro Olmedo-Velarde & John Hu (University of Hawaii)

Flat-mite transmitted plant viruses in Hawaii: update on a multi-crop study.

Hibiscus green spot virus 2 is a member of the Kitaviridae family and genus Higrevirus.  In Hawaii, hibiscus green spot virus 2 (HGSV2) infection causes leprosis-like symptoms in citrus, and green spot symptoms in Hibiscus spp., including H. arnottianus, a species native to Hawaii. A reverse genetics system for kitavirids has yet to be developed. Such a system would help us better understand basic biological mechanisms of virus-host and virus-vector interactions. HGSV2 is potentially transmitted by Brevipalpus mites (Acari: Tenuipalpidae), although adequate transmission studies are lacking. Therefore, a robust reverse genetics system of HGSV2 for agrobacterium-mediated delivery into Phaseolus vulgaris (common bean) is in development. So far, two separate biological experiments have demonstrated the infectiousness of the HGSV2 reverse genetics system. Additionally, preliminary results of transmission assays of HGSV2, using Brevipalpus mites collected from citrus, suggest that Brevipalpus mites can not only acquire HGSV2, but also potentially transmit HGSV2 from citrus to citrus.

Re-examination of mealybug wilt of pineapple

Mealybug wilt of pineapple (MWP) is the most important viral disease affecting pineapple. The disease is caused by members within the genus Ampelovirus, family Closteroviridae. The viruses are named pineapple mealybug wilt-associated virus 1 (PMWaV-1), PMWaV-2, and PMWaV-3. The identification and molecular characterization of pineapple secovirus A (PSV-A), a member within the subgenera Cholivirus, genus Sadwavirus; and the recent report of a putative new PMWaV member, tentatively named pineapple mealybug wilt-associated virus 6 (PMWaV-6) were accomplished utilizing high throughput sequencing (HTS) technologies. Complete viral genomes were obtained including the 5’ and 3’ ends. RNA-dependent RNA polymerase (RdRp), Heat shock protein 70 homolog (HSP70) and the coat protein (CP) encoded by PMWaV-6 share 30-69% identity with homologs of grapevine leafroll-associated virus 3 (GLRaV-3) and PMWaV-2, suggesting the presence of a new member belonging to the subgroup I within the genus Ampelovirus. Robust RT-PCR detection methods were implemented for both PSV-A and PMWaV-6 to assess their presence in both germplasm accessions obtained from the National Plant Germplasm Repository (NPGR) located at Hilo-Hawaii, and from commercial pineapple plants. Most of the MWP-symptomatic samples tested positive for the presence of both PSV-A and PMWaV-6 in surveys performed in Oahu-Hawaii, while healthy looking samples tested negative in RT-PCR assays for these two viruses.

Robert Martin (USDA-ARS)

The program in Corvallis works closely with the University of Arkansas on characterizing novel viruses of berry crops and developing diagnostics.  The material from the blueberry survey was used to study virus diversity of two new blueberry viruses (a luteovirus and a carlavirus), a survey of these two viruses is being carried from the archived samples in the -80C freezer from the blueberry survey done earlier. The samples were sent to University of Arkansas for testing for the novel viruses and studying virus diversity.

Other Accomplishments – Stakeholder oriented Virus database for NCPN crops.

The Corvallis program has taken the lead on developing a virus database for the NCPN crops.  The content development for the database is being done primarily by members of the WERA-20 group, based on the individual’s expertise: Berries (Martin USDA-ARS) and Tzanetakis (University of Arkansas), Citrus (Vidalakis – UC Riverside), Grapes and Roses (Al Rwahnih – UC Davis), Sweetpotato (Clark – Louisiana State University), Tree Fruit and Hops (Harper – Washington State University. The funding for the project is from NCPN.  The beta version of the database is at: http://virusdb-dev.cass.oregonstate.edu/

Arunabha Mitra, Sridhar Jarugula, & Naidu Rayapati (Washington State University)

Managing economically detrimental viral diseases in vineyards is a top priority for sustainability of the grape and wine industry in Washington State. Among the viral diseases documented in the state vineyards, grapevine leafroll disease (GLD) is widespread causing significant negative impacts to vine health and productivity and fruit quality. Of the six grapevine leafroll-associated viruses (GLRaVs) reported in grapevines, GLRaV-1, GLRaV-2, GLRaV-3, and GLRaV-4 were documented in Washington State vineyards. Currently, we are conducting fundamental research to better understand the molecular biology of GLRaV-3, the most widely prevalent among the four GLRaVs with a genome size varying between 18,433 and 18,671 nucleotides (nt) and complex genome organization.  Recently, we have built full-length genomic cDNA clones for GLRaV-3 and demonstrated that these clones can replicate in Nicotiana benthamiana leaves when introduced via Agrobacterium-mediated infiltration assays (Jarugula et al., 2018, Virology 523: 89-99). This achievement has laid a foundation to establish an in vitro reverse genetics system for elucidating virus-host interactions in a systems biology approach. Recently, we have expanded these studies to GLRaV-1 that has a genome size ranging between 18,731 and 18,946 nt with an unusually long 5ʹ non-translated region (5ʹ-NTR) ranging in length between 857 and 922 nt (Donda et al., 2017, Phytopathology 107:1069-1079). A cDNA copy of the minigenome of GLRaV-1, containing the 5ʹ-NTR, replicase gene module, Green Fluorescence Protein (GFP) gene and the 3ʹ-NTR, was built as a simplified experimental system to study the role of specific viral sequences in RNA replication. After subcloning into a modified pCAM1380 binary vector, the functionality of GLRaV-1 minigenome cDNA clone was confirmed by the expression of GFP in Nicotiana benthamiana leaves agro-coinfiltrated with a heterologous silencing suppressor. Using this minigenome cDNA clone, studies were conducted to examine the role of 5ʹ-non-translated region (5’ NTR) in RNA replication. Minigenome cDNA clones with different portions of the 5ʹ NTR deleted were tested for their functionality using agro-coinfiltration assays. The results showed that the first 32 nucleotides at the 5ʹ-terminus of the non-coding region are sufficient for replication and GFP expression in N. benthamiana leaves. The minigenome clone retained its functionality in agro-coinfiltration assays when the 5ʹ NTR was replaced with corresponding sequences from two genetic variants of GLRaV-1. These results suggest that 5’NTRs with distinct size and nucleotide sequence are exchangeable between genetic variants of GLRaV-1.

Al Rwahnih, M., Diaz-Lara, A., Arnold, K., Cooper, M.L., Smith, R.J., Zhuang, G., Battany, M.C., Bettiga, L.J., Rowhani, A. and Golino, D., 2020. Incidence and Genetic Diversity of Grapevine Pinot gris Virus in California. American Journal of Enology and Viticulture. DOI: 10.5344/ajev.2020.20044*

Al Rwahnih, M., Soltani, N., Soltero Brisbane, R., Tian, T. and Golino, D.A., 2021. First Report of Apricot vein clearing-associated virus Infecting flowering apricot (Prunus mume) in the United States. Plant Disease, (ja). DOI: 10.1094/pdis-10-20-2267-pdn*

Alabi, O.J., Appel, D. N., McBride, S., Al Rwahnih, M., and Pontasch, F. M. 2020. Complete genome sequence analysis of a genetic variant of grapevine virus L from the grapevine cultivar Blanc du Bois. Archives of Virology. 165:1905–1909. DOI: 10.1007/s00705-019-04252-7

Alejandro Olmedo-Velarde Beatriz Navarro John S. Hu Michael J. Melzer and Francesco Di Serio 2020 Novel Fig-Associated Viroid-Like RNAs Containing Hammerhead Ribozymes in Both Polarity Strands Identified by High-Throughput Sequencing Frontiers in Microbiology doi: 10.3389/fmicb.2020.01903

Arora, A.K., Clark, N., Wentworth, K.S., Hesler, S., Fuchs, M., Loeb, G. and Douglas A.E. 2020. Evaluation of RNA interference for control of the grape mealybug Pseudococcus maritimus (Hemiptera: Pseudococcidae) Insects 11:739; doi:10.3390/insects11110739

Beaver-Kanuya E, Wright AA, Szostek SA, Khuu N, Harper SJ (2021) Development of RT-qPCR assays for the detection and quantification of three Carlaviruses infecting hop. Journal of Virological Methods 292: 114124.

Bolus, S., Rwahnih, M. A., Grinstead, S. C., and Mollov, D. 2021. Rose virus R, a cytorhabdovirus infecting rose. Archives of Virology. DOI: 10.1007/s00705-020-04927-6

Chingandu, N., Jarugula, S., Adiputra, J., Bagewadi, B., Adegbola, R., Thammina, C. and Naidu, R.A. 2021. First report of grapevine rupestris vein feathering virus in grapevines from Washington State. Plant Disease 105:717.

Cieniewicz, E., Poplaski, V., Brunelli, M., Dombroswkie, J. and Fuchs, M. 2020. Two distinct Spissistilus festinus genotypes in the United States revealed by phylogenetic and morphological analyses. Insects11:80; doi:10.3390/insects11020080.

Dahan, J., Thompson, B.D., Lee, J., and Karasev, A.V. 2021. First report of grapevine rupestris vein feathering virus in wine grapes in Idaho. Plant Disease 105: published on-line May 2, 2021 (https://doi.org/10.1094/PDIS-04-21-0728-PDN).

Davis TJ, Gomez MI, Harper SJ, Twomey M. (2020) Potential economic benefits of using certified clean hop plants vs. hop stunt viroid disease. Cornell University, December 2020.

Delic, D., Radulovic, M., Vakic, M., Sunulahpašić, A., Villamor, D.E.V. and Tzanetakis, I.E. 2020. First Report of black currant reversion virus and gooseberry vein banding associated virus in currants in Bosnia and Herzegovina. Plant Disease 104:2036

Delić, D., Radulović, M., Vakić, M., Sunulahpašić, A., Villamor, D.E.V. and Tzanetakis, I.E. 2020. Raspberry leaf blotch emaravirus in Bosnia and Herzegovina: population structure and systemic movement. Molecular Biology Reports 47: 4891–4896

Diaz-Lara, A., Dangl, G., Yang, J., Golino, D. A., and Rwahnih, M. A. 2021. Identification of grapevine Pinot gris virus in free-living Vitis spp. located in riparian areas adjacent to commercial vineyards. Plant Disease. DOI: 10.1094/pdis-10-20-2121-sc*

Diaz-Lara, A., Erickson, M.T., Golino, D., and Al Rwahnih, M. Design and validation of a universal reverse transcription PCR (RT-PCR) assay for vitiviruses infecting grapevine. American-Phytopathological-Society (APS) Plant Health Annual Meeting (Plant Health) Virtual meeting Jul 31-Aug 4, 2020.*

Diaz-Lara, A., Mollov, D., Golino, D. and Al Rwahnih, M., 2020. Detection and characterization of a second carlavirus in Rosa sp. Archives of Virology 11:1-3. DOI: 10.1007/s00705-020-04864-4*

Diaz-Lara, A., Mollov, D., Golino, D., and Al Rwahnih, M. 2020. Complete genome sequence of rose virus A, the first carlavirus identified in rose. Archives of Virology. 165:241–244. DOI: 10.1007/s00705-019-04460-1*

Diaz-Lara, A., Mosier, N.J., Stevens, K., Keller, K.E. and Martin, R.R. 2020. Evidence of Rubus yellow net virus integration into the red raspberry genome. Cytogenetic and Genomic Research 160:329-334.

Diaz-Lara, A., Stevens, K., Hwang, M., Golino, D., and Al Rwahnih, M. Discovery of negative sense RNA viruses in grapevine via high throughput sequencing. American- Phytopathological-Society (APS) Plant Health Annual Meeting (Plant Health) Virtual meeting Jul 31-Aug 4 2020.*

DuPont ST, Harper SJ. (2020) Better disease detection: Scouting and sampling for X phytoplasma and little cherry virus in 2020. Goodfruit Grower.

Finn, C.E., Strik, B.C., Yorgey, B.M., Peterson, M.E., Jones, P.A., Buller, G., Lee, J., Bassil, N.V. and Martin, R.R. 2020. ‘Galaxy’ thornless semierect blackberry. Hortscience https://doi.org/10.21273/HORTSCI14985-20

Finn, C.E., Strik, B.C., Yorgey, B.M., Peterson, M.E., Jones, P.A., Buller, G., Serce, S., Lee, J., Bassil, N.V. and Martin, R.R. 2020. ‘Eclipse’ thornless semi-erect blackberry. Hortscience 55:749-754. https://doi.org/10.21273/HORTSCI14891-20

Finn, C.E., Strik, B.C., Yorgey, B.M., Peterson, M.E., Jones, P.A., Lee, J., Bassil, N.V. and Martin, R.R. 2020. ‘Twilight’ thornless semi-erect blackberry. https://doi.org/10.21273/HORTSCI14992-20

Flasco, M., Hoyle, V., Cieniewicz, E.J., Roy, B.G., McLane, H.L., Perry, K.L., Loeb, G., Nault, B., Heck M. and Fuchs, M. 2021. Grapevine red blotch virus is transmitted by the three-cornered alfalfa hopper in a circulative, nonpropagative transmission mode with unique attributes. Phytopathology https://doi/org/10/1094/phyto-02-21-0061-r

Fuchs, M., Almeyda, C. V., Rwahnih, M. A., Atallah, S. S., Cieniewicz, E. J., Farrar, K., Foote, W., Golino, D.A., Gómez, M., Harper, S. and Kelly, M. 2021. Economic Studies Reinforce Efforts to Safeguard Specialty Crops in the United States. Plant Disease. 105:14–26. DOI: 10.1094/pdis-05-20-1061-fe*

Gao, Z., Khot, L.R., Naidu, R.A. and Zhang, Q. 2020. Early detection of grapevine leafroll disease in a red-berried wine grape cultivar using hyperspectral imaging. Computers and Electronics in Agriculture 179:105807.

Green, J.C., Rwahnih, M.A., Olmedo-Velarde, A., Melzer, M.J., Hamim, I., Borth, W.B., Brower, T.M., Wall, M. and Hu, J.S., 2020. Further genomic characterization of pineapple mealybug wilt-associated viruses using high-throughput sequencing. Tropical Plant Pathology 45:64-72.

Hamilton A, Harper SJ & Critzer F. (2020) Optimization of a Method for the Concentration of Genetic Material in Bacterial and Fungal Communities on Fresh Apple Peel Surfaces. Microorganisms 8(10): 1480.

Hamim, I., Borth, W.B., Suzuki, J.Y. et al. Molecular characterization of tomato leaf curl Joydebpur virus and tomato leaf curl New Delhi virus associated with severe leaf curl symptoms of papaya in Bangladesh. Eur J Plant Pathol 158, 457–472 (2020). https://doi.org/10.1007/s10658-020-02086-7

Hernandez, R.N., Isakeit, T., Al Rwahnih, M., Hernandez, R. and Alabi, O.J., 2021. First report of Cucurbit chlorotic yellows virus infecting cantaloupe (Cucumis melo L.) in Texas. Plant Disease, (ja). DOI: 10.1094/pdis-02-21-0249-pdn

Hernandez, R.N., Isakeit, T., Al Rwahnih, M., Hernandez, R. and Alabi, O.J., 2021. First report of squash vein yellowing virus naturally infecting butternut squash (Cucurbita moschata) in Texas. Plant Disease, (ja). DOI: 10.1094/pdis-02-21-0378-pdn

Hily, J.M., Poulicard, N., Kubina, J., Reynard, J.S., Garcia, S., Spilmont, A.S., Fuchs, M., Lemaire, O. and Vigne, E. 2021. Metagenomic analysis of nepoviruses: diversity, evolution and identification of a hitherto undescribed putative amino acid motif for host range in subgroup A species. Archives of Virology, in press.

Ho, T., Broome, J. C., Buhler, J.P., O’Donovan, W., Tian, T., Diaz-Lara, A., Martin, R.R., Tzanetakis, I.E., 2021. Characterization of endogenous Rubus yellow net virus in Raspberries. bioRxiv 2021.06.17.448838; doi: https://doi.org/10.1101/2021.06.17.448838

Hoffmann M, Talton W, Nita M, Jones T, Al Rwahnih M, Sudarshana MR and Almeyda CV. 2020. First Report of Grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera in North Carolina. https://doi.org/10.1007/s42161-020-00710-3

Hoffmann M, Talton W, Nita M, Jones T, Al Rwahnih M, Sudarshana MR and Almeyda CV. 2020. First Report of Grapevine red blotch virus, the Causal Agent of Grapevine Red Blotch Disease, in Vitis vinifera in North Carolina. https://doi.org/10.1094/PDIS-07-19-1539-PDN

Jarugula, S., Chingandu, N., Adiputra, J., Bagewadi, B., Adegbola, R., Thammina, C. and Naidu, R.A. 2021. First Report of grapevine red globe virus in grapevines in Washington State. Plant Disease 105:717.

Kwon, S.J., Bodaghi, S., Dang, T., Gadhave, K.R., Ho, T., Osman, F., Al Rwahnih, M., Tzanetakis, I.E., Simon, A.E. and Vidalakis, G., 2021. Complete nucleotide sequence, genome organization and comparative genomic analyses of citrus yellow-vein associated virus (CYVaV). Frontiers in Microbiology, 12: 1371.

Larrea-Sarmiento A, Olmedo-Velarde A, Wang X, Borth W, Matsumoto TK, Suzuki JY, Wall MM, Melzer MJ, Hu JS. A novel ampelovirus associated with mealybug wilt of pineapple (Ananas comosus var. comosus). Virus genes. 2021. Accepted for publication.

Larrea-Sarmiento, A., Olmedo-Velarde, A., Green, J. C., Al Rwahnih, M., Wang, X., Li, Y.-H., et al. 2020. Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus). Archives of Virology. 165:1245–1248. DOI: 10.1007/s00705-020-04592-9

Lee, J., Rennaker, C.D., Thompson, B.D., and Karasev, A.V. 2021. Influence of Grapevine red blotch virus (GRBV) on Idaho ‘Syrah’ grape composition. Scientia Horticulturae 282: 110055 (https://doi.org/10.1016/j.scienta.2021.110055).

Mulenga, R. M., Miano, D. W., Kaimoyo, E., Akello, J., Nzuve, F. M., Rwahnih, M. A., et al. 2020. First Report of Southern Bean Mosaic Virus Infecting Common Bean in Zambia. Plant Disease. 104:1880–1880. DOI: 10.1094/pdis-11-19-2390-pdn

Naidu, R.A. 2020. Viruses of Grapevines. In: 2020 Pest Management Guide for Grapes in Washington. (pp. 53-56). EB0762 (Extension).

Olmedo-Velarde, A., Hu, J., and Melzer, M.J. 2021. A virus infecting Hibiscus rosa-sinensis represents an evolutionary link between cileviruses and higreviruses. Frontiers in Microbiology DOI: 10.3389/fmicb.2021.660237

Olmedo-Velarde, A., Waisen, P., Kong, A.T., Wang, K.-H., Hu, J.S., and Melzer, M.J. 2021. Characterization of taro reovirus and is status in taro (Colocasia esculenta) germplasm from the Pacific. Archives of Virology (in press)

Osterbaan, L.J., Hoyle V., Curtis, M., DeBlasio, S., Heck, M., Rivera, K. and Fuchs, M. 2021. Identification of protein interactions of grapevine fanleaf virus RNA-dependent RNA polymerase during infection of Nicotiana benthamiana by affinity purification and tandem mass spectrometry. Journal of General Virology, in press.

Schoelz, J., Volenberg, D., Adhab, M., Fang, Z., Klassen, V., Spinka, C., et al. 2020. A Survey of Viruses Found in Grapevine Cultivars Grown in Missouri. American Journal of Enology and Viticulture. 72:73–84. DOI: 10.5344/ajev.2020.20043

Silva, J. M. F., Fajardo, T. V. M., Rwahnih, M. A., and Nagata, T. 2020. First Report of Grapevine Associated Jivivirus 1 Infecting Grapevines in Brazil. Plant Disease. DOI: 10.1094/pdis-08-20-1689-pdn

Soltani, N., Golino, D. A., and Al Rwahnih, M., 2021. First report of Rose leaf rosette-associated virus infecting rose (Rosa spp.) in California, USA. Plant Disease. DOI: 10.1094/pdis-10-20-2268-pdn*

Spak. J., Koloniouk, I., and Tzanetakis I.E. 2021. Graft-transmissible diseases of Ribes – pathogens, impact and control. Plant Disease 105: 242-250

Tamisier L., Haegeman A., Foucart Y., Fouillien N., Al Rwahnih M., Buzkan N., …., Massart S., (2021, March 23). Semi-artificial datasets as a resource for validation of bioinformatics pipelines for plant virus detection. Zenodo. DOI: 10.5281/zenodo.4584718

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Villamor, D.E.V., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. 2021. Comparison of high throughput sequencing to standard protocols for virus detection in berry crops. Plant Disease (in Press).

Wright AA, Shires M, Beaver C, Bishop Gm DuPont ST, Naranjo R, Harper SJ (2021b) The effect of Candidatus Phytoplasma pruni infection on sweet cherry fruit. Phytopathology, Accepted 5/4/21.

Wright AA, Shires M, Harper SJ (2021a) Little cherry virus-2 titer and distribution in Prunus avium. Archives of Virology, 166: 1415–1419.

Wu, Q., Habili, N., Constable, F., Al Rwahnih, M., Goszczynski, D. E., Wang, Y., et al. 2020. Virus pathogens in Australian vineyards with an emphasis on Shiraz disease. Viruses. 128: 8. DOI: 10.3390/v12080818

Zhang S. and Vrient A. (2020): Chapter 8 - Rapid Detection of Plant Viruses and Viroids in Awasthi LP (ed) Applied Plant Virology - Advances, Detection, and Antiviral Strategies, pp. 101-109, Academic Press.