WERA20: Management of Diseases Caused by Systemic Pathogens in Fruit Crops and Woody Ornamentals

(Multistate Research Coordinating Committee and Information Exchange Group)

Status: Active

Project Menu

SAES-422 Reports

Annual/Termination Reports:

[07/08/2023] [11/25/2024] [11/10/2025]

Date of Annual Report: 07/08/2023

Report Information

Annual Meeting Dates: 05/09/2023 - 05/11/2023
Period the Report Covers: 10/01/2022 - 09/30/2023

Participants

Alan Wei Agri-Analysis LLC
Christie Almeyda North Carolina State University
John Hu University of Hawaii
Mysore R Sudarshana USDA-ARS
Robert P Jones USDA-APHIS
Victoria Hornbaker California Department of Food and Agriculture
Allison Gratz Canadian Food Inspection Agency
Dipak Poudyal Oregon Department of Agriculture
Joseph Lagner University of Maryland-College Park & USDA-APHIS
Naidu Rayapati Washington State University
Ruhui Li ARS, NGRL
Yannis Tzanetakis University of Arkansas
AMER FAYAD USDA - NIFA
Ekaterina Nikolaeva Pennsylvania Department of Agriculture
Kitty Cardwell Oklahoma State University
Oscar Hurtado Gonzales APHIS PGQP
Sage Thompson PPQ - Plants for Planting Policy
Yazmin Rivera APHIS PPQ Science & Technology
AVIJIT ROY USDA-ARS
Georgios Vidalakis University of California Riverside
Kristian Stevens UC Davis - FPS
Peter Abrahamian APHIS PPQ Science & Technology
Segun Akinbade WA State Dept of Agriculture
Yilmaz Balci PPQ - Plants for Planting Policy
Bill Howell Northwest Nursery Improvement Institute
Jarred Yasuhara-Bell USDA-APHIS-PPQ-S&T-PPCDL
Larissa Carvalho Costa APHIS PGQP
Ramesh R Pokharel USDA-APHIS
Svetlana Y. Folimonova University of Florida
Yu Yang USDA APHIS PPQ PGQP
Chellappan Padmanabhan APHIS PPQ Science & Technology
Jennifer McCallister USDA APHIS PPQ PGQP
Michael West-Ortiz Cornell University
Tami Collum USDA ARS AFRS
Yvette Tamukong University of Maryland

Brief Summary of Minutes

The multi-state WERA20 project “Management of Diseases Caused by Systemic Pathogens in Temperate and Sub-Tropical Fruit Crops and Woody Ornamentals” organized an in-person annual meeting during May 9^th through 11^th, 2023 at the National Agricultural Library in Beltsville, MD. The meeting was hosted by Drs. Yazmín Rivera, Peter Abrahamian (USDA APHIS PPQ Science and Technology), and Oscar Hurtado-Gonzales (USDA APHIS PPQ Field Operations). Dr. Wendy Jin, Associate Deputy Administrator for Science and Technology Programs at USDA APHIS PPQ, welcomed the attendees followed with an overview of the USDA APHIS partnerships with industry, academia, and government research institutions. 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 funding opportunities for collaborative research and extension in different areas of agriculture.

The business meeting started with Dr. Naidu Rayapati, Administrative Advisor from Washington State University, providing a brief account of the WERA20 project and its objectives. After discussions, the annual meeting in 2024 was selected to be hosted by Dr. Alex Karasev from University of Idaho, likely at Boise, subject to approval by Western Association of Agricultural Experiment Station Directors. Following the business meeting, the scientific program started following the agenda outlined below for May 9^th.

Special topic sessions on “Citrus Health” and “Emerging technologies” were held during May 10^th. See Meeting Agenda in Appendix 1. Presentations abstracts can be found in Appendix 2. The group photo can be found through the project's Homepage.

WERA20 Main scientific program

Getting Going on Getting it Right: Update on the activities of the Diagnostic Assay Validation Network (DAVN)

Kitty Cardwell, OSU, Institute of Biosecurity and Microbial Forensics

Phylogenetic relationships, putative vector, and possible seed transmission of Lindera severe mosaic emaravirus

John Hammond, USDA ARS, USNA, Floral and Nursery Plants Research Unit

Understanding the spread of grapevine leafroll disease in Washington state vineyards

Naidu Rayapati, WSU, Irrigated Agriculture Research and Extension Center

High-throughput sequencing detection and initial molecular characterization of two novel emaraviruses co-infecting Callicarpa (beautyberry) are associated with a mosaic disease

Ramon Jordan, USDA ARS, FNPRU, US National Arboretum

Characterizing virus populations associated with Cotton leafroll dwarf virus in the southern United States

Michael West-Ortiz, Cornell University, School of Integrative Plant Science Plant Pathology and Plant-Microbe Biology

Further Virome Analysis of Apple Decline Disease

Ruhui Li, USDA ARS, National Germplasm Resources Laboratory

Validated real-time PCR screening assay and synthetic control for detection of the apple proliferation pathogen ‘Candidatus Phytoplasma mali’

Jarred Yasuhara-Bell, USDA APHIS, PPQ, Plant Pathogen Confirmatory Diagnostics Laboratory

Updates from Hawaii Five-0

John Hu, University of Hawaii at Manoa

PGQP Status Report for Fruit Trees

Oscar Hurtado-Gonzales, USDA APHIS PPQ, Plant Germplasm Quarantine Program

Newly Discovered Virus and Virus-Like Entities – Good, Bad, or Indifferent?

Bill Howell, Northwest Nursery Improvement Institute

Developing a diagnostic standard for germplasm imports

Sage Thompson and Yilmaz Balci, USDA APHIS PPQ, Pest and Exclusion and Import Programs

The Canadian Fruit Tree Export Program and the Tree Fruit Diagnostic Testing Program

Allison Gratz, CFIA, Tree Fruit Diagnostics, Science Branch

Round Table/Discussion: Conditional Releases-States and Federal crosstalk

Dipak Poudyal, Oregon Department of Agriculture

Special Topics: Citrus Health

Updates on Citrus yellow vein clearing in California

Victoria Hornbaker, California Department of Food and Agriculture

Genetic characterization and diversity of citrus yellow vein clearing virus

Peter Abrahamian, USDA APHIS PPQ, Plant Pathogen Confirmatory Diagnostics Laboratory

MiFi®: Microbe Finder for Detection of Citrus Pathogens in Metagenomic Datasets

Georgios Vidalakis, University of California, Riverside

Geographical distribution of natural hosts of Brevipalpus transmitted viruses associated with citrus leprosis disease complex in the United States

Avijit Roy, USDA ARS, Molecular Plant Pathology Laboratory

Special Topics: Emerging Technologies in Plant Pathology

Virus-mimicking artificial positive controls in a snap and Ghost Viruses updates

Ioannis Tzanetakis, University of Arkansas

Development of machine learning models for detection of 'Ca. Liberibacter asiaticus' and its application in Citrus leprosis disease diagnosis

Jonathan Shao, USDA ARS, NEA Office Area Director, Beltsville

HiPlex for detection of fruit tree viruses and viroids

Larissa Costa, USDA APHIS PPQ, Plant Germplasm Quarantine Program

Isolation of phloem specific mRNAs using translating ribosome affinity purification (TRAP) to investigate pathogen host interactions in fruit crops

Tami Collum, USDA ARS Appalachian Fruit Research Station, Kearneysville, WV

CRISPR-Based Disease Detection Strategies for Candidatus Phytoplasma

Joseph Lagner, USDA APHIS PPQ, Plant Pathogen Confirmatory Diagnostic Laboratory

University of Maryland, College Park

Minutes Attachment:

Appendix 1_ WERA-20 meeting 2023 agenda.pdf

Accomplishments

Ioannis Tzanetakis - University of Arkansas We collaborated with colleagues from Oregon who are part of WERA-20 to develop an infectious clone for blackberry virus S, a close relative of blueberry scorch virus. We are currently evaluating its effects on popular blueberry cultivars when present as a single infection. Additionally, we completed the characterization of blueberry virus L, a new luteovirus that was present in almost 80% of the 600+ samples tested. We found that blackberry leaf mottle virus is capable of producing symptoms on blackberry 'Ouachita' when present as a single infection. Furthermore, it is very efficiently transmitted by Phyllocoptes parviflori at rates of 40% in single mite and 70% in five mite transmission experiments. Obtaining and maintaining positive controls poses a significant obstacle in the development of detection assays. It can be problematic and expensive, and without them, assays cannot be validated. To address this issue, we propose a new strategy for creating virus-mimicking positive controls (ViMAPCs). Unlike alternatives such as plasmids, gBlocks™, or RNA transcripts, which can take weeks to obtain and implement, ViMAPCs can be designed and utilized in less than five days. ViMAPCs provide a more realistic representation of natural infection than other options and make it easier to detect lab-based contamination. We evaluated the feasibility and adaptability of this strategy using several RNA and DNA viruses. ViMAPCs have the potential to be used in diagnostic labs as well as in monitoring pathogen outbreaks, where rapid response is crucial. We are in the final stages of preparing a white paper on over 100 ‘phantom’ agents of citrus, grapevine, pome and stone fruit, rose, Rubus sp. (blackberry, raspberry and their hybrids), and strawberry. Experts from around the globe collaborated on this project that aims to remove those names from regulatory lists. This is based on the cumulative experience and knowledge of the experts, and our inability to identify any isolate or sequence information available for the agents in question that would allow for their identification.   Georgios Vidalakis - University of California, Riverside In this report period, May 2022 – May 2023, we collaborated with colleagues from Oklahoma to develop and validate e-probes for the detection of 32 graft-transmissible pathogens of citrus. The in-silico-validated e-probes were uploaded to the new MiFi® platform and beta testing was initiated (<a href="https://bioinfo.okstate.edu/">https://bioinfo.okstate.edu/</a>). In collaborative efforts with experts in USA and other citrus producing countries, we developed new and updated pathogen detection assays for ‘Candidatus Liberibacter species’ and citrus viroid VII. We also developed instruments and methods for improving and streamlining sample processing for high-throughput detection of viral pathogens of citrus. In two additional studies, we identified differentially expressed citrus genes in dwarfed citrus infected with citrus dwarfing viroid and we studied the effect of huanglongbing disease on arbuscular mycorrhizal fungal communities in different citrus orchards. WERA 20 members, in collaboration with viroid experts in USA and around the world, co-edited and published a book containing 26 protocols of biological, electrophoresis, hybridization and PCR techniques for viroid detection and research. We also collaborated in the national effort to prepare a white paper on over 100 ‘phantom’ agents of citrus, grapevine, pome and stone fruit, rose, Rubus sp., and strawberry in an effort to update regulatory lists and harmonize the scientific literature. In this report period, the University of California, Riverside, National Clean Plant Network (NCPN) Citrus Center, namely the Citrus Clonal Protection Program, distributed 61,982 pathogen-tested units, of 337 citrus accessions, to nurseries, producers and public. In addition, NCPN-Citrus and WERA 20 members organized a national webinar in the principles of quality management for diagnostic laboratories with emphasis on qPCR protocols. Finally, California WERA 20 members co-organized three national/international conferences (i.e., California Citrus Nursery Society, International Society Citrus Nurserymen, and International Organization of Citrus Virologists) with participation of hundreds of scientists, regulators and growers. Christie Almeyda - Micropropagation and Repository Unit (MPRU), North Carolina State University 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. We are currently serving breeders in NC (Rubus/Fragaria/Vaccinium), AR (Rubus) and FL (Vaccinium/Rubus). We are also providing services to industry on multiple capacities: diagnostics, graft indexing and cleanup of imported material. Deliverables include the maintenance of foundation plants (100 genotypes - 3 plants per genotype in a screenhouse (Rubus & Vaccinium) and a greenhouse (Fragaria)); maintenance of in vitro genotypes (160 genotypes); cleanup of imported genotypes (Vaccinium - 18, Fragaria - 5, Rubus - 1; Total = 24) and distribution since 2018 of 42 genotypes (2-3 TC plantlets/genotype; at least 20 plugs/genotype for field trueness to type test). Nothing has been distributed under our Controlled Import Permit (CIP) yet. While cleaning up berry crops, the following viruses were detected on blueberries: Blueberry latent virus (BBLV). Blackberry yellow vein-associated virus (BYVaV), Blackberry leaf mottle associated virus (BlMav) were detected on blackberries. Since 2021, the MPRU has established a partnership with the NC Plant Disease and Insect Clinic (PDIC). Now NC growers can submit berry and grape samples to be tested for designated pathogens at the MPRU as the unit has expanded its diagnostic services. Under the CIP, the MPRU is currently cleaning berry material from Chile, Peru, Korea, Japan and Mexico. The MPRU is working closely with other berry clean centers (OR, AR and CA) as well as USDA-APHIS regulators to discuss matters related to CIP management for successful release of imported material. In partnership with Dr. Hoffmann (NCSU strawberry and grape extension specialist), the MPRU was able to collaborate with its diagnostics capacity for virus surveys on grapes. Various NC grower fields were tested to validate the establishment of molecular testing at the MPRU using protocols previously developed by Foundation Plant Services (FPS), UC-Davis in collaboration with Dr. Maher Al Rwahnih. The MPRU now has the capacity of testing for 10 pathogens affecting grapes using quantitative RT-PCR. Targeted pathogens were selected based on importance and prevalence in the region. The pathogens currently being tested are Grapevine leaf roll viruses (GLRaV-2, GLRaV-3, GLRaV-4, GLRaV-7), Grapevine red blotch virus (GRBV), Grapevine rupestris stem pitting associated virus (GRSPaV), vitiviruses (GVA, GVB), Tobacco Ring Spot Virus (TRSV), and Xyllela fastidiosa. Due to funding restrictions, not much work has been done on muscadine grapes in 2022-2023. We continue maintaining the clean material we currently have at the MPRU (10 NC muscadine cultivars) and cleaning new material (3 genotypes) we obtained from the AR breeding program in 2019.  John Hu, University of Hawaii Pineapple accessions were subjected to RNA-sequencing to study the occurrence of viral populations in pineapple. Analysis of high-throughput sequencing data obtained from 24 germplasm accessions and from public domain transcriptome shotgun assembly (TSA) data identified two novel sadwaviruses, putatively named “pineapple secovirus C” (PSV-C) and “pineapple secovirus D” (PSV-D). They shared low amino acid sequence identity (from 34.8 to 41.3%) compared with their homologs in the Pro-pol region of the previously reported PSV-A and PSV-B. The complete genome (7,485 bp) corresponding to a previously- reported partial sequence of the badnavirus, pineapple bacilliform ER virus (PBERV), was retrieved from one of the datasets. Overall, we discovered a total of 69 viral sequences representing 10 members within the Ampelovirus, Sadwavirus, and Badnavirus genera. Genetic diversity and recombination events were found in members of the pineapple mealybug wilt-associated virus (PMWaV) complex as well as PSVs. PMWaV-1, -3, and -6 presented recombination events located across the quintuple gene block while no recombination events were found for PMWaV-2. High recombination frequency of the RNA1 and RNA2 molecules from PSV-A and PSV-B were congruent with the diversity found by phylogenetic analyses. Here, we also report the development and improvement of RT-PCR diagnostic protocols for the specific identification and detection of viruses infecting pineapple based on the diverse viral populations characterized in this study. Maher Al Rwahnih – University of California, Davis At Foundation Plant Services (FPS), UC-Davis, we continue to make advances in developing and refining our methods using high throughput sequencing (HTS) as a superior diagnostic tool. We have used sequence information generated by HTS analysis to design new, species-specific polymerase chain reaction (PCR) primers for use in PCR diagnostics. In addition, HTS proves to be an invaluable tool in the discovery of unknown viruses and in establishing a baseline analysis of the virome of a crop.  FPS has been conducting work to determine if co-infections of grapevine leafroll associated virus-3 (GLRaV-3) and grapevine virus A (GVA) lead to sudden vine collapse (SVC) on Freedom rootstock and to identify rootstocks that might be more resistant. In addition, our work is aimed at determining if SVC is spreading within vineyards in a pattern consistent with the ecology of mealybugs, known vectors of GLRaV-3 and GVA. Our 2021 study of 12 vineyards found that 87% of all vines were positive for GLRaV-3, 73% were positive for both GLRaV-3 and GVA, and 26% were positive for both GLRaV-3 and GVB. All GVA and GVB infections were co-infected with GLRaV-3. Additional analysis of our research suggests that while there is a positive correlation between SVC and the presence of GLRaV-3/GVA co-infections in the blocks that we sampled, the association is not complete. While most of the SVC vines were positive for GLRaV-3 and GVA, this was also true for asymptomatic vines within the SVC cluster in most blocks. To determine if time was the missing factor in a positive correlation between SVC and co-infection by GLRaV-3 and GVA, we re-surveyed GLRaV-3/GVA asymptomatic vines in 2022. None of these vines had developed SVC symptoms but only eight of the original 18 blocks were still in place. Temporal and spatial analysis of SVC incidence in the remaining eight blocks indicated that SVC had progressed in all eight blocks via a common dispersal mechanism, but at varying rates. SVC distribution within blocks was aggregated but not to the same extent as observed for GLRaV-3 epidemics. The rootstock field trial was planted in randomized blocks in the UCD Armstrong Field Station in summer 2022 and dormant material has been collected from vines positive for GLRaV-3, GLRaV-3 and GVA, GLRaV-1 and GVA, and GLRaV-2 and GVB, in addition to virus negative controls. Vines will be graft-inoculated spring 2023 and tested by RT-qPCR in fall 2023 to verify their virus infection status. We are continuing our research on the epidemiology of grapevine red blotch virus (GRBV). The identity of a primary vector and its role in spread of the virus under field conditions remains largely unresolved. While the outbreak of GRBV in the Russell Ranch Vineyard (RRV) is a serious blow to the clean plant approach for wine grapes in California and the voluntary certification program, it also offers a unique research opportunity to characterize the statistical properties of a GRBV outbreak and gather invaluable information about mechanisms of disease spread. The objectives of the current study were to determine annual GRBV infections rates, conduct spatiotemporal analysis of GRBV infected vines, replace infected vines with healthy virus-tested (sentinel) vines, and monitor insect populations. Our work indicates that once GRBV has been introduced into vineyards, spread can be rapid with annual rates up to 18%, a 14-fold increase from the previous year. This contrasts with a Napa Valley vineyard where annual rates of natural spread were only 1-2% per year, indicating that natural spread rates within vineyards can be highly variable. We planted approximately 400 sentinel vines in the Russell Ranch Vineyard in places where previously GRBV positive vines had been removed in 2017-2019. At the time of planting, July 2020, RRV contained approximately 800 highly aggregated GRBV positive vines to serve as inoculum. We tested these sentinel vines in 2021 and 2022 at different time points to determine how early GRBV could be detected. In addition, we calculated the GRBV positive incidence in each positive vine to estimate the probability of getting false negative test results. The first new GRBV infection was detected in November 2021, 15 months after the sentinel vines were planted or five months from the time the sentinel vines had significant growth outside the planting sleeves. In 2022, the majority of new GRBV infections were detected in May and within-vine distribution was uniform in all 21 positive vines. This is noteworthy since previous work on GRBV within-vine distribution indicates that more variability exists in June compared to October. However, we are always sampling basal leaves and our May data suggests that reliable test results can be obtained in the spring when using this type of sample. Only six additional infections were detected in August and October and within-vine distribution was more variable. This data suggests that new GRBV infections cannot be detected until the year following infection and that late spring may be the optimal time to sample. At this point, we are not recommending that vineyards be sampled for GRBV in the spring. The sentinel vines are still young and do not represent the type of vines you would find in an established vineyard. In addition, policy recommendations should not be made on one or even two years of test data. However, these results have potentially important implications for early GRBV detection. Alexander V. Karasev, University of Idaho Grapevine red blotch virus (GRBV) infection was previously 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 produced grapes with significantly lower total sugars and lower total anthocyanins. They were also lower in a single free amino acid (tryptophan), yet higher in malic acid compared to grapes from healthy vines. Overall, GRBV negatively influenced grape quality by reducing total sugars and total anthocyanins, which may indicate GRBV infection delayed grape ripening and could adversely affect wine quality. To address the question of how grapevine leafroll-associated virus 3 (GLRaV-3) infected vines impact Idaho grown ‘Cabernet Sauvignon’ grape quality, a single block of plants selected based on molecular testing, with pairs of healthy and GLRaV-3 infected vines, was examined for grape quality for three consecutive growing seasons. Grapes from GLRaV-3 infected vines had significantly higher concentrations of total organic acids, and were significantly lower in total anthocyanins, total phenolics, total tannins, and total free amino acids compared to healthy vines. Cluster weights and concentrations of total sugars, and yeast assimilable nitrogen content, were not different between healthy and infected vines. We concluded that some quality elements of Idaho ‘Cabernet Sauvignon’ grapes important in wine production were negatively impacted by GLRaV-3 infection. The virome of grapevines grown in the State of Idaho was characterized in 2020-2022 for the purpose of developing diagnostic and management tools for virus and virus-like disorders in wine grapes. Over 100 leaf and petiole samples were collected from symptomatic grapevines in eight vineyards in Canyon and Nez Perce counties of Idaho and subjected to high-throughput sequencing (HTS) and RT-PCR testing. Multiple grapevine viruses were uncovered by HTS, and their presence was confirmed and validated by RT-PCR with specific primers designed based on the information obtained with HTS. Up to 18 viruses and virus strains, in addition to several genetic variants of hop stunt viroid and grapevine yellow speckle viroid 1, were identified in some declining ‘Chardonnay’ vines from Canyon County, Idaho. Three viruses were reported from Idaho for the first time, grapevine rupestris stem-pitting associated virus (GSPaV), grapevine rupestris vein feathering virus (GRVFV), and grapevine-associated tymo-like virus (GaTLV). GRSPaV was found only in one vineyard, in an asymptomatic Riesling cultivar. In the case of GRVFV and GaTLV, both were found associated with decline observed in cultivar ‘Chardonnay’. For GaTLV, this is the first confirmed report for the United States. Go to the WERA20 Homepage (Attachments Section) to see additional individual project information:  https://www.nimss.org/projects/attachment/18910

Publications

<ol> <li>Dahan, J., Orellana, G.E., Lee, J., and Karasev, A.V. Genome sequences of two grapevine rupestris stem pitting-associated virus variants from Vitis vinifera cv. Riesling in Idaho, U.S.A. Microbiology Resource Announcements 12 (4): e01366-22 (<a href="https://doi.org/10.1128/MRA.01366-22">https://doi.org/10.1128/MRA.01366-22</a>).</li> <li>Dahan, J., Orellana, G.E., Lee, J., and Karasev, A.V. Occurrence of grapevine-associated tymo-like virus in wine grapes in the United States. Plant Disease 107: 592 (<a href="https://doi.org/10.1094/PDIS-05-22-1140-PDN">https://doi.org/10.1094/PDIS-05-22-1140-PDN</a>).</li> <li>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: 3309 (<a href="https://doi.org/10.1094/PDIS-04-21-0728-PDN">https://doi.org/10.1094/PDIS-04-21-0728-PDN</a>).</li> <li>Donda, B.P., Kesoju, S.R., Arnold, K., McRoberts, N. and Naidu, R.A. 2022. Spatio-Temporal Spread of Grapevine Leafroll Disease in Washington State Vineyards. Plant Disease 107:1471-1480. <a href="https://doi.org/10.1094/PDIS-04-22-0939-RE">https://doi.org/10.1094/PDIS-04-22-0939-RE</a></li> <li>Druciarek, T., Lewandowski, M. and Tzanetakis, I.E., 2023. Identification of a second vector for rose rosette virus. Plant Disease, <a href="https://doi.org/10.1094/PDIS-11-22-2686-SC">https://doi.org/10.1094/PDIS-11-22-2686-SC</a></li> <li>Medberry, A.M. Srivastava, A., Diaz-Lara, A., Al Rwahnih, M., Villamor E.V. and Tzanetakis, I.E. 2023. A novel, divergent member of the Rhabdoviridae infects strawberry. Plant Disease 107: 620-623</li> <li>Villamor, D.V.V., Sierra Mejia, A., Martin, R.R. and Tzanetakis, I.E., 2023. Genomic analysis and development of infectious clone of a novel carlavirus infecting blueberry. Phytopathology 113: 98-103</li> <li>Medberry, A.M. and Tzanetakis, I.E. 2022. Identification, characterization, and detection of a novel strawberry cytorhabdovirus. Plant Disease 106: 2784-2787</li> <li>Vakić, M., Stainton, D., Delic, D., and Tzanetakis, I.E. 2022. Characterization of the first Rubus yellow net virus genome from blackberry. Virus Genes 58: 594-597</li> <li>Osman, F., Dang, T., Bodaghi, S., Haq, R., Lavagi-Craddock, I., Rawstern, A., Rodriguez, E., Polek, M., Wulff, N. A., Roberts, R., Pietersen, G., Englezou, A., Donovan, N., Folimonova, S. Y., & Vidalakis, G. (2022). Update and validation of the 16S rDNA qPCR assay for the detection of three ‘Candidatus’ Liberibacter species following current MIQE guidelines and workflow. PhytoFrontiers™. <a href="https://doi.org/10.1094/phytofr-04-22-0046-fi">https://doi.org/10.1094/phytofr-04-22-0046-fi</a></li> <li>Chambers, G. A., Geering, A. D. W., Holford, P., Vidalakis, G., & Donovan, N. J. (2022). Development of a one-step RT-qPCR detection assay for the newly described citrus viroid VII. Journal of Virological Methods, 299, 114330. <a href="https://doi.org/10.1016/j.jviromet.2021.114330">https://doi.org/10.1016/j.jviromet.2021.114330</a></li> <li>Lavagi-Craddock, I., Dang, T., Comstock, S., Osman, F., Bodaghi, S., & Vidalakis, G. (2022). Transcriptome Analysis of Citrus Dwarfing Viroid Induced Dwarfing Phenotype of Sweet Orange on Trifoliate Orange Rootstock. Microorganisms, 10(6), 1144. <a href="https://doi.org/10.3390/microorganisms10061144">https://doi.org/10.3390/microorganisms10061144</a></li> <li>Dang, T., Bodaghi, S., Osman, F., Wang, J., Rucker, T., Tan, S.-H., Huang, A., Pagliaccia, D., Comstock, S., Lavagi-Craddock, I., Gadhave, K. R., Quijia-Lamina, P., Mitra, A., Ramirez, B., Uribe, G., Syed, A., Hammado, S., Mimou, I., Campos, R., … Vidalakis, G. (2022). A comparative analysis of RNA isolation methods optimized for high-throughput detection of viral pathogens in California’s regulatory and disease management program for citrus propagative materials. Frontiers in Agronomy, 4. <a href="https://doi.org/10.3389/fagro.2022.911627">https://doi.org/10.3389/fagro.2022.911627</a></li> <li>Dang, T., Wang, H., Espíndola, A. S., Habiger, J., Vidalakis, G., & Cardwell, K. (2022). Development and statistical validation of e-probe diagnostic nucleic acid analysis (EDNA) detection assays for the detection of citrus pathogens from raw high throughput sequencing data. PhytoFrontiers™. <a href="https://doi.org/10.1094/phytofr-05-22-0047-fi">https://doi.org/10.1094/phytofr-05-22-0047-fi</a></li> <li>Xi, M., Deyett, E., Ginnan, N., Ashworth, V. E. T. M., Dang, T., Bodaghi, S., Vidalakis, G., Roper, M. C., Glassman, S. I., & Rolshausen, P. E. (2022). Geographic Location, Management Strategy, and Huanglongbing Disease Affect Arbuscular Mycorrhizal Fungal Communities Across U.S. Citrus Orchards. Phytobiomes Journal, 6(4), 342–353. <a href="https://doi.org/10.1094/pbiomes-03-22-0014-r">https://doi.org/10.1094/pbiomes-03-22-0014-r</a></li> <li>Pagliaccia, D., Hill, D., Dang, E., Uribe, G., De Francesco, A., Milton, R., De La Torre, A., Mounkam, A., Dang, T., Botaghi, S., Lavagi-Craddock, I., Syed, A., Grover, W., Okamba, A., & Vidalakis, G. (2023). Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering. Journal of Visualized Experiments, 194. <a href="https://doi.org/10.3791/65159">https://doi.org/10.3791/65159</a></li> <li>Rao A., Lavagi-Craddock I., and Vidalakis G. (eds.) 2022. Viroids: Methods and Protocols, Methods in Molecular Biology. Springer, Vol. 2316. <a href="https://doi.org/10.1007/978-1-0716-1464-8_4%20">https://doi.org/10.1007/978-1-0716-1464-8_4 </a></li> <li>Hoffmann, M., Volk, E., Talton, W., Al Rwahnih, M., Almeyda, C., Burrack, H., Blaauw, B., Bertone, M. 2021. Grapevine Virus Distribution, Identification, and Management in North Carolina. NC State Extension Publications. AG-911.</li> <li>Fuchs, M., Almeyda, C.V., Al Rwahnih, M., Atallah, S.S., Cieniewicz, E.J., Farrar, K., Foote, W.R., Golino, D.A., Gómez, M.I., Harper, S.J., Kelly, M.K., Martin, R.R., Martinson, T., Osman, F.M., Park, K., Scharlau, V., Smith, R., Tzanetakis, I.E., Vidalakis, G., and Welliver, R. 2021. Economic studies reinforce efforts to safeguard specialty crops in the United States. <a href="https://apsjournals.apsnet.org/doi/10.1094/PDIS-05-20-1061-FE">https://apsjournals.apsnet.org/doi/10.1094/PDIS-05-20-1061-FE</a>.</li> <li>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. <a href="https://doi.org/10.1007/s42161-020-00710-3">https://doi.org/10.1007/s42161-020-00710-3</a></li> <li>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. <a href="https://doi.org/10.1094/PDIS-07-19-1539-PDN">https://doi.org/10.1094/PDIS-07-19-1539-PDN</a></li> <li>Hamim I, Suzuki JY, Borth WB, Melzer MJ, Wall MM,Hu JS. 2022. <a href="https://pubmed.ncbi.nlm.nih.gov/36090110/">Preserving plant samples from remote locations for detection of RNA and DNA viruses.</a> Front Microbiol. 2022 Aug 25;13:930329. doi: 10.3389/fmicb.2022.930329.</li> <li>Larrea-Sarmiento A, Olmedo-Velarde A, Wang X,Borth W, Domingo R, Matsumoto TK, Suzuki JY, Wall MM, Melzer M, Hu JS 2022. Genetic Diversity of viral populations associated with ananas germplasm and improvement of virus diagnostic protocols. Pathogens 2022, 11, 1470. <a href="https://doi.org/10.3390/pathogens11121470">https://doi.org/10.3390/pathogens11121470</a></li> <li>Larrea-Sarmiento A, Geering ADW, Olmedo-Velarde A, Wang X,Borth W, Matsumoto TK, Suzuki JY, Wall MM, Melzer M, Moyle R, Sharman M, Hu JS, Thomas JE. 2022. <a href="https://pubmed.ncbi.nlm.nih.gov/36269415/">Genome sequence of pineapple secovirus B, a second sadwavirus reported infecting Ananas comosus.</a> Arch Virol. 2022 Oct 21. doi: 10.1007/s00705-022-05590-9.</li> <li>Lee, J., Rennaker, C.D., Thompson, B.D., Dahan, J., and Karasev, A.V. 2023. Idaho ‘Cabernet Sauvignon’ grape composition altered by grapevine leafroll-associated virus 3. NFS Journal 30: 1-6 (<a href="https://doi.org/10.1016/j.nfs.2023.02.002">https://doi.org/10.1016/j.nfs.2023.02.002</a>). </li> <li>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 (<a href="https://doi.org/10.1016/j.scienta.2021.110055">https://doi.org/10.1016/j.scienta.2021.110055</a>).</li> <li>Diaz-Lara A, Aguilar-Molina VH, Monjarás-Barrera JI, Vončina D, Erickson TM, Al Rwahnih M. Potential Implications and Management of Grapevine Viruses in Mexico: A Review. International Journal of Plant Biology. 2023; 14(1):177-189. DOI: <a href="https://doi.org/10.3390/ijpb14010015">3390/ijpb14010015</a></li> <li>Choi J, Osatuke AC, Erich G, Stevens K, Hwang MS, Al Rwahnih M, Fuchs M. High-Throughput Sequencing Reveals Tobacco and Tomato Ringspot Viruses in Pawpaw. Plants. 2022; 11(24):3565. <a href="https://doi.org/10.3390/plants11243565">DOI: 10.3390/plants11243565</a></li> <li>Wwang, X., Larrea-Sarmiento, A., Olmedo Velarde, A., Al Rwahnih, M., Borth, W.B., Suzuki, J., Wall, M. M., Melzer, M., and Hu, J. 2022. Survey of viruses infecting Basella alba in Hawaii. Plant Disease. DOI: <a href="https://doi.org/10.1094/PDIS-02-22-0449-SR">1094/PDIS-02-22-0449-SR</a></li> <li>Hoyle, V, Flasco, MT, Choi, J, Cieniewicz, EJ, McLane, H, Perry, K, Dangl, G, Al Rwahnih, M, Heck, M, Loeb, G, Fuchs, MF., 2022. Transmission of Grapevine Red Blotch Virus by Spissistilus festinus [Say, 1830] (Hemiptera: Membracidae) between Free-Living Vines and Vitis vinifera 'Cabernet Franc'. VIRUSES-BASEL, 14(6). <a href="https://doi.org/10.3390/v14061156">DOI: 10.3390/v14061156</a></li> <li>Voncina, D, Diaz-Lara, A, Preiner, D, Al Rwahnih, M, Stevens, K, Juri, S, Malenica, N, Simon, S, Meng, BZ, Maletic, E, Fulgosi, H, Cvjetkovic, B., 2022. Virus and Virus-like Pathogens in the Grapevine Virus Collection of Croatian Autochthonous Grapevine Cultivars. PLANTS-BASEL, 11(11). <a href="https://doi.org/10.3390/plants11111485">DOI: 10.3390/plants11111485</a></li> <li>Medberry AN, Srivastava A, Diaz-Lara A, Al Rwahnih M, Villamor DEV, Tzanetakis IE., 2023. A novel, divergent member of the Rhabdoviridae infects strawberry. Plant disease. <a href="https://doi.org/10.1094/pdis-05-22-1078-sc">DOI: 10.1094/pdis-05-22-1078-sc</a></li> <li>Kuo YW, Bednarska A, Al Rwahnih M, Falk BW., 2022. Development of Agrobacterium tumefaciens Infiltration of Infectious Clones of Grapevine Geminivirus A Directly into Greenhouse-Grown Grapevine and Nicotiana benthamiana Plants. Phytopathology, PHYTO01220015R. <a href="https://doi.org/10.1094/phyto-01-22-0015-r">DOI: 10.1094/phyto-01-22-0015-r</a></li> <li>Lebas, B., Adams, I., Al Rwahnih, M., Baeyen, S., Bilodeau, G.J., Blouin, A.G., Boonham, N., Candresse, T., Chandelier, A., De Jonghe, K. and Fox, A., 2022. Facilitating the adoption of high-throughput sequencing technologies as a plant pest diagnostic test in laboratories: A step-by-step description. EPPO Bulletin. 52(2): 394-418.<a href="https://doi.org/10.1111/epp.12863">DOI: 10.1111/epp.12863</a></li> <li>Bennypaul, H.S., Sanderson, D.S., Donaghy, P., Abdullahi, I., Green, M., Klaassen, V. and Al Rwahnih, M., 2022. Development of a one-step RT-qPCR assay for the detection of Grapevine leafroll-associated virus 7. Journal of Virological Methods, 308: 114578.<a href="https://doi.org/10.1016/j.jviromet.2022.114578">DOI: 10.1016/j.jviromet.2022.114578</a></li> <li>Jagunić, M., Diaz-Lara, A., Szőke, L., Rwahnih, M.A., Stevens, K., Zdunić, G. and Vončina, D., 2022. Incidence and Genetic Diversity of Grapevine Virus G in Croatian Vineyards. Plants. <a href="https://doi.org/10.3390/plants11182341">DOI: 10.3390/plants11182341</a></li> <li>ter Horst, A.M., Fudyma, J.D., Bak, A., Hwang, M.S., Santos-Medellin, C., Stevens, K., Rizzo, D., Al Rwahnih, M. and Emerson, J.B, 2022. RNA viral communities are structured by host plant phylogeny in oak and conifer leaves. Phytobiomes Journal. <a href="https://doi.org/10.1094/PBIOMES-12-21-0080-R">DOI: 10.1094/PBIOMES-12-21-0080-R</a></li> <li>Jagunić, M., Diaz-Lara, A., Rwahnih, M.A., Preiner, D., Stevens, K., Zdunić, G., Hwang, M. and Vončina, D., 2022. Grapevine Badnavirus 1: Detection, Genetic Diversity, and Distribution in Croatia. Plants. <a href="https://doi.org/10.3390/plants11162135">DOI: 10.3390/plants11162135</a></li> </ol>

Impact Statements

Grapevine leafroll disease continues to be an economically important viral disease affecting sustainability of vineyards in many grapevine-growing regions. Multi-season studies were conducted in Washington State on the spread of grapevine leafroll disease to young vineyards planted with certified planting stock. The data provided an improved understanding of the dynamics of leafroll disease spread to facilitate area-wide disease management strategies. Diagnostic primers were designed for the RT-PCR detection of three novel viruses found in Idaho grapevines, GSPaV, GRVFV, and GaTLV, and validated for virus detection on field-collected samples.

Date of Annual Report: 11/25/2024

Report Information

Annual Meeting Dates: 09/17/2024 - 09/19/2024
Period the Report Covers: 10/01/2023 - 09/30/2024

Participants

Akinbade, Segun - WA Department of Agriculture;
Al Rwahnih, Maher - UC-Davis;
Alvarez-Quinto, Robert - Univ. of Minnesota;
Balci, Yilmaz - USDA-APHIS-PPQ;
Hurtado-Gonzales, Oscar - USDA-APHIS;
Karasev, Alexander - Univ. of Idaho;
Melzer, Michael - Univ. of Hawaii;
Nikolaeva, Ekaterina - PA Dept. of Agriculture;
Olmedo-Velarde, Alejandro - Iowa State University;
Pandey, Binod - Oregon Dept. of Agriculture;
Pokharel, Ramesh - USDA-APHIS-PPQ;
Poudyal, Dipak - Oregon Dept. of Agriculture;
Powell, Garner - Clemson University;
Rayapati, Naidu - Washington State Univ.;
Reyes-Proano, Edison - Univ. of Idaho;
Sudarshana, Mysore - USDA-ARS;
Thompson, Sage - USDA-APHIS-PPQ;
Tzanetakis, Ioannis - Univ. of Arkansas;
Vidalakis, Georgios - UC Riverside;
Wei, Alan - Agri-Analysis, LLC

Brief Summary of Minutes

To see the group photo of Attendees, go to the WERA20 Homepage, click on the Outline module, then the Attachments selection on the left hand side.

The multi-state WERA20 project “Management of Diseases Caused by Systemic Pathogens in Temperate and Sub-Tropical Fruit Crops and Woody Ornamentals” organized an in-person annual meeting during September 17^th through 19^th, 2024, at the University of Idaho Water Center in Boise, ID. The meeting was hosted by Dr. Alexander Karasev, University of Idaho. Dr. A. Karasev welcomed the participants on behalf of the University of Idaho and Idaho Wine Commission. Dr. Naidu Rayapati, Administrative Advisor from Washington State University, provided a brief account of the WERA20 project and its objectives. After business discussions, the annual meeting in 2025 was proposed to be hosted by Dr. Christy Almeyda from North Carolina State University, likely at Raleigh in May 2025, subject to approval by Western Association of Agricultural Experiment Station Directors.

WERA20 Scientific Program

September 17, 2024 (Tuesday)

An update on grapevine viruses in Washington State (Naidu Rayapati)

WSDA fruit tree certification project report (S. Akinbade/B. Matheson)

State reports – South Carolina virus report (G. Powell)

State reports - Minnesota (R. Alvarez-Quinto)

Surveying for potential virus introduction pathways to fruit tree fields in fruit tree nursery stock certification program (D. Poudyal)

Grapevine virus research in Idaho (A. Karasev)

State reports - Pennsylvania (E. Nikolaeva)

State reports - Hawaii (M. Melzer)

Arkansas UPDATE 2024: Berry viruses infectious clones and VIGS vectors (I. Tzanetakis)

California research and operational updates – Foundation Plant Services (M. Al Rwahnih)

California citrus report (G. Vidalakis)

PGQP status report for fruit trees, APHIS report (O. Hurtado-Gonzales)

Outbreak of prune brown line in Northern California (M. Sudarshana)

September 18, 2024 (Wednesday)

NCPN discussion, led by I. Tzanetakis, G. Vidalakis, and M. Melzer

Exploring the peach virome and quantifying seasonal dynamics of PNRSV and PDV titer in peach trees (G. Powell)

An ultra-sensitive and high throughput method for screening plant viruses using grapevine leafroll associated virus 3 (GLRaV-3) as an example (A. Wei)

Regulatory updates: ACIR, eFile and Controlled Import Permits, APHIS Plants for Planting Policy (S. Thompson, R. Pokharel, Y. Balci)

Q & A session

Citrus viroids: friends or foes? (G. Vidalakis)

A new totivirus discovered in European white birch (E. Reyes-Proano)

The power of data mining: a case study with cotton leafroll dwarf virus (A. Olmedo-Velarde)

Grapevine viruses in Idaho revealed by deep sequencing (A. Karasev)

Tobacco ringspot virus in grapes and blueberries (Naidu Rayapati)

Roundtable discussion: funding sources and opportunities for collaborations - local, regional, and national projects

Recap and wrap-up (A. Karasev)

September 19, 2024 (Thursday)

Field tour, including visits to a commercial testing laboratory (Western Laboratories, Parma, ID), to research laboratories (University of Idaho Center of Plant and Soil Health, Parma, ID), and to two commercial vineyards (St. Chapelle and Bitner, Caldwell, ID).

Accomplishments

Naidu Rayapati, Washington State University Managing viral diseases in vineyards is a top priority for sustainable growth of Washington’s grape and wine industry that has an estimated $9.6 billion to the state’s economy. Vineyard surveys during the past 10 plus years and testing samples using molecular diagnostic assays and high-throughput sequencing technology revealed the presence of fifteen viruses in Washington vineyards. Among them, Grapevine leafroll-associated virus 3 (GLRaV-3) causing leafroll disease (GLD) was found to be more widespread than Grapevine red blotch virus (GRBV) causing red blotch disease (GRBV) in Washinton vineyards. Since GLRaV-3 and GRBV produce similar symptoms in red-fruited cultivars and mild symptoms or no obvious symptoms in white-fruited cultivars, accurate diagnosis of these two viruses is critical for managing GLD and GRBD. Multi-season studies were conducted in commercial vineyards to better understand spatial and temporal spread of GLD and GRBD. The results showed that apparently healthy young vineyards are subject to the constant risks of GLD pressure from neighboring infected older vineyards. The results, based on the temporal and spatial increase of GLD, suggested random patterns of symptomatic vines within the block during initial years indicating primary spread of GLRaV-3. Clustering of symptomatic vines during subsequent years suggested vine-to-vine secondary spread within the block. In addition, the data showed a disease gradient in which more number of symptomatic vines in newly infected blocks were in rows proximal to infected old blocks suggesting ‘edge effect.’ Overall, our observations on the spatial and temporal dynamics of GLD provided a baseline dataset to conduct further research for a better understanding of confounding factors contributing to the spread of the disease across vineyards. In contrast, studies on spatial and temporal spread of GRBD showed no vine-to-vine spread in vineyards. Based on these results, it can be concluded that the field spread of GRBD is less likely to occur in Washington vineyards and roguing can be implemented as an effective strategy for controlling the disease. Tobacco ring spot virus (TRSV) and Grapevine fanleaf virus (GFLV) were detected in vineyards showing fanleaf degeneration/decline symptoms. However, these two viruses were found sporadically in Washington vineyards. Field studies have shown that TRSV significantly affects vineyard lifespan and fruit yield, and the dagger nematode (Xiphinema rivesi), identified based on morphological features and genome sequence analysis, present in vineyard soil can spread the virus from infected to healthy vines. Preliminary studies indicated that X. rivesi is unlikely to spread GFLV. Thus, GFLV can be eliminated by using virus-tested ‘clean’ plants due to the absence of its nematode vector, X. index, in Washington vineyards, whereas management of TRSV needs a combination of ‘clean’ plants and post-planting management of dagger nematodes.  Due to the heightened importance of viral diseases to blueberry production in Washington State, experiments were conducted to analyze symptomatic highbush blueberry (Vaccinium corymbosum L.) plants that led to detection of TRSV in plants showing severe defoliation and stunting symptoms. We completed further studies on impact of TRSV on fruit quality attributes of blueberries and phylogenetic relationships of the RNA1 and RNA2 genome segments of TRSV with corresponding sequences of viruses in the genus Nepovirus. We also demonstrated in-field transmission of TRSV using cucumber bait plant assays and identified the dagger nematode, X. rivesi, as a putative vector transmitting the virus. In collaboration with grapevine nurseries and Plant Services Program of the Washington State Department of Agriculture (WSDA), we employed robust sampling protocols and high-throughput molecular diagnostic methods to improve the sanitary status of grapevines in registered Mother Blocks in grapevine nurseries. Multi-year diagnostic research revealed that grapevines in registered Mother Blocks of Washington nurseries are free of GRBV that plagued grapevine nurseries in other regions. Together with nurseries and WSDA, we implemented certification standards to safeguard vines in registered Mother Blocks leading to national and global recognition of Washington nurseries as the best source for virus-tested planting stock. Outreach and educational activities were conducted during the project period to strengthen partnerships with regulatory agencies, nurseries, growers, and researchers for a unified approach to advance clean plant campaigns for healthy vineyards in Washington State. Segun Akinbade, Washington State Department of Agriculture The Fruit Tree Certification Program was established to facilitate national and international export of clean planting stock for the tree fruit industry. The program works with fruit tree nurseries to ensure that over 90,000 mother trees maintained across 13 certified fruit tree nurseries and are as healthy as the Generation 1 plant material sourced from the Clean Plant Center Northwest (CPCNW). Various forms of testing are routinely conducted throughout the growing season to achieve this objective. For instance, all Prunus mother trees are tested annually for the presence of pollen-transmitted viruses such as Prunus necrotic ringspot virus (PNRSV) and Prune dwarf virus (PDV). Additionally, all cherry mother trees are tested for Cherry leaf roll virus (CLRV), while non-cherry Prunus mother trees are tested for Plum pox virus (PPV). Testing for pathogens in Pyrus and Malus trees is primarily done through visual inspections, with molecular analyses conducted when necessary. In addition to routine testing, the program seeks funding to address emerging diseases. In 2023, WSDA was awarded funding through the Plant Protection Act (PPA) 7721 to conduct a survey for Little Cherry Disease (LCD) in Prunus mother blocks in nurseries participating in the certification program.   Approximately 1,900 mother trees were sampled and tested for Western X phytoplasma and Little Cherry Virus 2 (LChV2). WSDA tested 9% of the samples, while the Washington State University (WSU) Plant Pest Diagnostic Clinic in Pullman, WA, tested the remaining 91%. The survey results indicated that 10.5% of the samples tested positive for Western X phytoplasma, and 3.7% were positive for LChV2. Most of the infected trees were concentrated in two nurseries. One of the nurseries decided to remove all Prunus trees and implement rigorous insect control measures, with plans to restart Prunus mother tree propagation in 2025. WSDA deregistered the block in the second nursery where most positive samples originated. The 2024 LCD survey is still ongoing. WSDA also secured funding from a separate PPA 7721 award to update Chapter 16-350 of the Washington Administrative Code (WAC), which governs fruit tree planting stock registration and certification. From June 2023 to March 2024, the project established a working group composed of fruit tree certification participants, Washington State orchardists, the Oregon State Department of Agriculture (ODA), Washington State University (WSU), and WSDA. Led by a facilitator, the working group was tasked with updating the existing rules to align with current industry practices and emerging challenges since the last revision was done in 2005. The second objective of this project was to survey for vectors of LCD in registered blocks. Leafhoppers, known to transmit Western X phytoplasma, were found in 5 out of 10 nurseries surveyed. Leafhopper management strategies were proposed, and the findings were shared with all nurseries participating in the program. Dipak Poudyal, Oregon Department of Agriculture and Segun Akinbade, Washington State Department of Agriculture The Oregon Department of Agriculture (ODA) and Washington State Department of Agriculture (WSDA) administer nursery certification programs for Cydonia, Malus, Prunus, and Pyrus spp. Together, nurseries that participate in the ODA and WSDA programs produce over 60 million certified planting stocks annually. ODA and WSDA jointly implemented a survey project to investigate potential introduction of viruses or virus-like agents (VLAs) (Apple mosaic virus (ApMV), Citrus concave gum-associated virus (CCGaV), Cherry leaf roll virus (CLRV), Cherry rasp leaf virus (CRLV), Tobacco ring spot virus (TRSV), Tomato ringspot virus (ToRSV) and Western X phytoplasma) through weeds and cover crops into registered fields producing certified nursery stocks in Oregon and Washington. Weed and cover crop host samples were collected from nurseries participating in the certification programs in each state. Samples were collected in and around registered fields and were tested for these pathogens. In Oregon, 21 nurseries participate in the certification program. In late spring/early summer, 181 samples representing 23 different plant species were collected from 16 Oregon nurseries. The most sampled weed species were dandelion (42), common thistle (21), and Himalayan blackberry (18). The most sampled cover crop was clover (22). No samples tested positive for any of the seven target pathogens. In Washington, 108 samples representing 18 different plant species were collected from 11 Washington nurseries. The most sampled weed species were dandelion (13), common knotweed (11), goosefoot (11), clover (11), common mallow and common thistle (8 each). The remaining samples were puncture vine, flixweed, sheep sorrel, tall tumble mustard, chickweed, wild carrot, common mullein, broadleaf plantain, pennycress, fireweed, amaranth, and alfalfa. One clover sample tested positive for ApMV. This was determined to be a weedy clover and not a cover crop and was removed from the nursery. In early fall, 39 samples representing nine different plant species were collected from three Oregon nurseries. The most sampled weeds were dandelion (17) and broadleaf plantain (6). The most sampled cover crop was clover (7). Fewer samples were collected compared to the spring because nurseries had either mowed or sprayed as part of their management practices and material was not available. No samples tested positive for any of the seven target pathogens. In Washington, 110 samples representing 35 different plant species were collected from 11 Washington nurseries. No samples tested positive for any of the seven target pathogens.  In total, 438 weed and cover crop samples were tested for seven target pathogens. Only one clover sample from Washington collected in spring 2023 tested positive. Maher Al Rwahnih, University of California, Davis Foundation Plant Services (FPS) continued to advance the development and refinement of high throughput sequencing (HTS) as a superior diagnostic tool. Sequence information generated by HTS analysis was used to design new, species-specific primers for use in PCR diagnostics. Currently, the use of HiPlex PCR is being tested for use in strawberry and grape virus detection. The epidemiology of grapevine red blotch virus (GRBV) is under investigation now, although the identity of a primary vector and its role in spread of the virus under field conditions remains largely unresolved. The work indicates that once GRBV has been introduced into vineyards, spread can be rapid with annual rates up to 18%, a 14-fold increase from the previous year. This contrasts with a Napa Valley vineyard where annual rates of natural spread were found only 1-2% per year, indicating that natural spread rates within vineyards can be highly variable. Approximately 400 sentinel vines in the Russell Ranch Vineyard (RRV) were planted in places where previously GRBV positive vines had been removed in 2017-2019. At the time of planting in July 2020, RRV contained approximately 800 highly aggregated GRBV positive vines able to serve as inoculum sources. These sentinel vines were tested in 2021 and 2022 at different time points to determine how soon GRBV could be detected. In addition, we calculated the GRBV positive incidence in each positive vine to estimate the probability of getting false negative test results. The first new GRBV infection was detected in November 2021, 15 months after the sentinel vines were planted or five months from the time the sentinel vines had significant growth outside the planting sleeves. In 2022, the majority of new GRBV infections were detected in May and within-vine distribution was uniform in all 21 positive vines. This is noteworthy since previous work on GRBV within-vine distribution indicates that more variability exists in June compared to October. However, we are always sampling basal leaves and our May data suggests that reliable test results can be obtained in the spring when using this type of sample. Only six additional infections were detected in August and October and within-vine distribution was more variable. This data suggests that new GRBV infections cannot be detected until the year following infection and that late spring may be the optimal time to sample. At this point, we are not recommending that vineyards be sampled for GRBV in the spring. The sentinel vines are still young and do not represent the type of vines one would find in an established vineyard. In addition, policy recommendations should not be made on one or even two years of test data. However, these results have potentially important implications for early GRBV detection. FPS is party to a collaborative research project with other WERA-20 participants to identify ‘high risk’ strawberry viruses by region in the continental U.S. with the intent of developing evidence-based information on how to best monitor for and develop Best Management Practices (BMPs) to exclude viruses and other targeted pathogens during plant production in nurseries. This complements the projects to develop harmonized strawberry certification standards of CA, OR and WA to establish pilot projects to implement the harmonized strawberry certification standards. The information obtained in this project will be used by nursery managers to develop and implement BMPs for detecting, controlling and mitigating the occurrence of targeted pathogens. The information will also be used by the State Departments of Agriculture to focus their inspections with an emphasis towards ‘high risk’ pathogens in their state or region. Samples were collected from nurseries in 2023 and 2024 and testing for 25 different pathogens is underway. We will continue analyzing the assays and look at improving the PCRs, sequencing virus positive samples to identify viral genetic diversity and improve the PCR assays as needed. We are considering development of Hiplex (multiplex PCR) to screen for multiple viral variants at once, to further reduce the risk of false negatives due to virus variability. These sequencing efforts to evaluate virus diversity and screen negative samples using HTS to eliminate the risk of false negative are key to completing the survey of high-risk viruses circulating in US strawberry nurseries. FPS has a parallel strawberry project to design, adapt, and update molecular diagnostic techniques for detection and identification of strawberry viruses. CDFA Strawberry Registration & Certification (R&C) program currently relies on biological indexing to determine pathogen status of strawberries. Annually, CDFA tests approximately 10,000 strawberry samples, each of which requires grafting to at least two indicator plants for evaluation of pathogen symptom expression.  Indexing has been proven inadequate compared to molecular methods in other crops, such as grapes, Prunus, and rose, and research evaluating its performance in strawberries is underway. If indexing is proven ineffective in strawberry, CDFA and others relying on virus test results must be prepared to implement molecular testing. We are evaluating two molecular diagnostic methods for use in place of indexing: qPCR, and HiPlex qPCR. The qPCR sets the baseline for assays that are developed for pathogen detection. Individual qPCR is an effective technique for detection, but it allows each sample to be tested for just one pathogen. HiPlex PCR is an advanced multiplex that allows for a great number of pathogens to be screened for in just one sample. These two methods are being evaluated for reliability, sensitivity, and effectiveness, as well as implementation practicality. Assay and primer development and deployment are the current focus of the research. Side-by-side comparison of the results of each testing method will take place in year two, (anticipated for 2025) followed by adoption recommendation and training, if needed. Adoption of molecular testing method(s) is expected to reduce the amount of time and space required for testing materials for inclusion in R&C program and for phytosanitary export documents, and perhaps more importantly, provide a reliable diagnosis of plant pathogen status. In addition to evaluating HiPlex for use in strawberry, FPS is also evaluating its use for detection of grapevine leafroll-associated viruses (GLRaVs) and grapevine red blotch virus (GRBV). Previous studies clearly showed an increase in the diversity of grapevine-infecting viruses, especially GLRaVs and GRBV. With this proposal, we aim to develop Hiplex PCR (a multiplex PCR coupled with HTS), a new method able to employ multiple primers simultaneously in a single reaction that can detect genetic variants and report them in formats that work with complementary annotation tools. In this way, variants can be broadly categorized according to their likely clinical significance. In this context, Hiplex PCR will allow us to (i) efficiently differentiate and characterize various variants of GLRaV and GRBV, (ii) reduce processing time and costs, (iii) continue the successful employment of HTS in the production and distribution of pathogen tested propagative material, and (iv) identify GLRaV-3 variants to help with epidemiological studies understanding spatial-temporal progress of GLRaV-3 epidemic. The application of Hiplex PCR at our clean plant center is essential due to the heightened risk of emerging variants of grapevine viruses especially GLRaV-3 in California. Routine qPCR assays may fail to detect these variants leading to false negatives requiring the innovative Hiplex PCR technique. This research is just begun, and assays are being constructed. FPS continues research to determine if co-infections of grapevine leafroll associated virus-3 (GLRaV-3) and grapevine virus A (GVA) lead to sudden vine collapse (SVC) on Freedom rootstock and to identify rootstocks that might be more resistant. Past years of the current research included surveying vineyards afflicted with SVC and monitoring spread pattern. In 2024, FPS continues to maintain a rootstock field trial (randomized blocks established at UCD Armstrong Field Station in summer 2022) inoculated with GLRaV-3, GLRaV-3 and GVA, GLRaV-1 and GVA, and GLRaV-2 and GVB, in addition to virus negative controls. Georgios Vidalakis, University of California, Riverside In this report period, June 2023 – September 2024, we continued to support the suppression and eradication efforts against Huanglongbing (HLB) in California, where the number of positive trees has reached 8,679 and the HLB quarantine zones are expanding in the coastal and southern regions of the state. The University of California, Riverside, National Clean Plant Network (NCPN) Citrus Center, namely the Citrus Clonal Protection Program (CCPP) collaborated with the California Department of Food and Agriculture's (CDFA) Citrus Nursery Stock Pest Cleanliness Program and distributed 79,742 clean citrus propagation units (buds), tested for HLB as well as virus and viroid diseases, from 356 different citrus accessions, to 827 nurseries, producers, scientists, and the public, sourced from 1,819 pathogen-tested citrus budwood trees. The CCPP also tested 81 citrus introductions from 7 countries with 2,258 diagnostic tests intercepting 10 different types of pathogens in 21 introductions (25.9%), including HLB, viruses and viroids and performed pathogen elimination/therapy on 96 citrus accessions with 826 tissue cultures. The CCPP maintained 615 inquiries under quarantine and performed 1,941 laboratory and 3,760 biological pathogen detection tests for HLB, virus and viroid diseases resulting in the release from quarantine of 80 citrus accessions. In collaborative efforts with WERA 20 members and experts in USA, and other citrus producing countries, we continued the development and validation of e-probes for the detection of graft-transmissible pathogens of citrus; we characterized the citrus yellow vein clearing virus, an emerging pathogen in California; we identified two distinct viral suppressors of RNA silencing encoded by the citrus variant of the apple stem grooving virus, i.e., citrus tatter leaf virus; we continued the field trials on the impact on commercial citrus of the newly discovered umbra-like viral RNA of citrus yellow vein associated virus and the citrus dwarfing viroid; and we developed a pathogen detection assay for citrus exocortis viroid. We developed a micro-homogenizer for citrus tissue processing and continued improving on the instruments and methods for streamlining sample processing for high-throughput detection of viral pathogens of citrus. We also performed research on principals of circular economy for the production of pathogen tested citrus propagative materials using citrus fruit waste and control environment agriculture including studying the effects of various light wave lengths in citrus plants growth and disease bioindexing. Ioannis Tzanetakis, University of Arkansas Through collaborations with more than 10 members of this group, we have completed a comprehensive white paper addressing over 120 'phantom' agents affecting citrus, grapevine, pome and stone fruit, rose, Rubus species (blackberry, raspberry, and their hybrids), and strawberry. This project involved contributions from more than 180 experts across 40 countries, aiming to remove these agents from regulatory lists. The initiative is based on the collective experience of the experts and the absence of any isolate or sequence data to definitively identify the agents in question. In parallel, we successfully developed infectious clones for both Blackberry chlorotic ringspot virus (BCRV) and Blackberry yellow vein associated virus (BYVaV). The biological properties of these clones were evaluated and found to be consistent with those of the wild-type viruses. Additionally, virus-induced gene silencing (VIGS) vectors were created for both viruses, allowing us to monitor gene silencing throughout the infection process. Regarding Rose rosette virus (RRV) and emaraviruses more broadly, a long-standing question has been whether these viruses replicate within their mite vectors. Our study employed quantitative real-time RT-PCR to assess RRV genome copy numbers in two mite species, Phyllocoptes fructiphilus and P. adalius. The results provide new insights into viral dynamics and vector competence, revealing active virus replication in P. fructiphilus—a confirmed vector—while no replication was observed in P. adalius, confirming its non-vector status. Furthermore, the research highlights fluctuations in virus concentration in mites over time, suggesting developmental stage-specific responses and the influence of mite lifestyle on RRV retention and replication. This work marks an important step towards understanding virus-mite interaction mechanisms, which is critical for developing effective management strategies for rose rosette and other emaravirus-related diseases. Alexander V. Karasev, University of Idaho The virome of grapevines grown in the State of Idaho was continued to be characterized in 2020-2024, with the overall goal of developing diagnostic tools for virus and virus-like disorders in wine grapes. More than 200 leaf and petiole samples were collected from symptomatic grapevines in 10 vineyards in Canyon and Nez Perce counties of Idaho and subjected to high-throughput sequencing (HTS) and RT-PCR testing. Multiple grapevine viruses and their genetic variants were uncovered by HTS, and their presence was confirmed and validated by RT-PCR with specific primers designed based on the sequencing information obtained by HTS. In 2023-2024, three endornaviruses were reported from Idaho for the first time, grapevine endophyte endornavirus (GEEV), grapevine endornavirus 1 (GEV1), and grapevine endornavirus 2 (GEV2). In addition, two grapevine viroids, grapevine yellow speckle viroid 2 (GYSVd-2) and Australian grapevine viroid AGVd) were found in an unknown table grape cultivar. In January 2023, the virome of one-year-old birch plants that were kept at the greenhouse and displayed virus-like symptoms such as leaf yellowing, stunting, leaf rolling in lower leaves, mottling, and mosaic, was subjected to analysis using HTS. One contig resembling the classic genomic structure of totiviruses was found, which was named birch toti-like virus (BTLV). The genome of BTLV is 4,967 nucleotides long and contains two overlapping open reading frames (ORFs) coding for the capsid protein (CP) and an RNA-dependent RNA-polymerase (RdRP). Phylogenetic inferences based on the CP and RdRP amino acid sequences placed this virus within a clade of plant-associated totiviruses in the family Orthototiviridae. Alan Wei, Agri-Analysis LLC, Davis, CA We have developed a convenient and sensitive test for diagnosis of Grapevine leafroll associated virus type 3 (GLRaV-3).  Agri-Analysis has a proprietary patent pending technology for direct antigen direction without nucleic acid amplification. Specifically, our testing reagent is composed of llama derived nanobodies against the capsid protein of GLRaV-3 linked with fragments of nanoluciferase. When no GLRaV-3 is present, the reagent has no bioluminescence because the enzyme fragments are inactive. When the GLRaV-3 is present, the nanobodies bind to it, bringing the luciferase fragments together to form an active luciferase, producing a bright signal.  We coined the term “next generation ELISA (ngElisa)” for this method because it overcomes the disadvantages of poor sensitivity and specificity inherent in conventional ELISA. Specifically, the high sensitivity is attributable to the extremely low background noise because the enzyme fragments are inactive if they are non-specifically bound to the surface substrate, where the enzyme-linked detection antibody is always active regardless it is bound specifically to the target or nonspecifically to substrate surface. The high specificity is derived from the fact that two nanobodies are required to bind simultaneously to the target to create a signal. If the enzyme linked antibody is nonspecifically bound to a structurally similar interferent molecule, a signal will be produced in conventional ELISA but not in ngELISA, hence improving specificity.  We have demonstrated a signal-to-noise ratio of over 2200 while conventional ELISA typically has an S/N ratio of 15. This method was shown to be more sensitive than PCR and qPCR when compared side-by-side to test GLRaV-3 in serially diluted field samples.  It is envisioned that reagents can be freeze-dried in the wells of 96-well plates, signals can be read out upon sample addition without the need for additional wash steps. This “mix-and-read” format, when combined with portable luminescence readers, enables high throughput screening of multiple samples in vineyards and/or nurseries.  It can also be adapted to handheld luminescence readers for single sample testing.

Publications

Abrahamian, P., Tian, T., Posis, K., Guo, Y. Y., Yu, D., Blomquist, C. L., Wei, G., Adducci, B. A., Vidalakis, G., Bodaghi, S., Osman, F., Roy, A., Nunziata, S., Nakhla, M. K., Mavrodieva, V., & Rivera, Y. 2023. Genetic analysis of the emerging citrus yellow vein clearing virus reveals a divergent virus population in American isolates. Plant Disease. <a href="https://doi.org/10.1094/pdis-09-23-1963-re">https://doi.org/10.1094/pdis-09-23-1963-re</a> Aknadibossian, V., Freitas-Astúa, J., Vidalakis, G., & Folimonova, S. Y. 2023. Citrus Phantom Disorders of Presumed Virus and Virus-like Origin: What Have We Learned in the Past Twenty Years? Journal of Citrus Pathology, 10(1). <a href="https://doi.org/10.5070/c410161176">https://doi.org/10.5070/c410161176</a> Aknadibossian, V., Freitas-Astúa, J., Vidalakis, G., Thermoz, J.-P., Licciardello, G., Catara, A., Batista, L., Pérez, J. M., Peña, I., Zamora, V., & Folimonova, S. Y. 2024. Further investigation on citrus phantom disorders of unconfirmed viral etiology. Journal of Citrus Pathology, 11(2). <a href="https://doi.org/10.5070/c411263792">https://doi.org/10.5070/c411263792</a> Alabi, O.J., Stevens, K., Oladokun, J.O., Villegas, C., Hwang, M.S., Al Rwahnih, M., Tian, T., Hernandez, I., Ouro-Djobo, A., Sétamou, M. and Jifon, J.L. 2024. Discovery and characterization of two highly divergent variants of a novel potyvirus species infecting Madagascar periwinkle (Catharanthus roseus L.). Plant Disease 108: 2494-2502. Bodaghi, S., Tyler Dang, Huizi Wang, Andres Espindola, Irene Lavagi-Craddock, Fatima Osman, Marcos Ribeiro, Danielle Do Nascimento, Arunabha Mitra, Josh Habiger, Kitty Cardwell and Georgios Vidalakis. 2024. E-probes targeting citrus pathogens as a new diagnostic standard. Citrograph. Vol. 15:2, Spring 2024 p.44-47. <a href="https://citrusresearch.org/citrograph/archive">https://citrusresearch.org/citrograph/archive</a> Chambers, G. A., Geering, A. D., Holford, P., Kehoe, M. A., Vidalakis, G., & Donovan, N. J. 2023. A reverse transcription loop-mediated isothermal amplification assay for the detection of citrus exocortis viroid in Australian citrus. Australasian Plant Pathology. <a href="https://doi.org/10.1007/s13313-023-00903-1">https://doi.org/10.1007/s13313-023-00903-1</a> Dahan, J., Orellana, G.E., Lee, J., and Karasev, A.V. 2023. Grapevine endophyte endornavirus and two new endornaviruses found associated with grapevines (Vitis vinifera L.) in Idaho, USA. Viruses 15 (6): 1347 (<a href="https://doi.org/10.3390/v15061347">https://doi.org/10.3390/v15061347</a>). Dahan, J., Orellana, G.E., Lee, J., and Karasev, A.V. 2024. Occurrence of grapevine yellow speckle viroid 2 and Australian grapevine viroid in Idaho grapevines. Plant Disease 108: 1121 (<a href="https://doi.org/10.1094/PDIS-01-24-0034-PDN">https://doi.org/10.1094/PDIS-01-24-0034-PDN</a>).   Douhan, G., Vidalakis, G., 2023. A new citrus virus to North America, citrus yellow vein clearing virus, was recently detected in residential properties in the San Joaquin Valley of California. International Organization of Citrus Virologists (IOCV). Newsletter. Riverside, CA. <a href="https://iocv.ucr.edu/sites/default/files/2024-01/iocv_news_letter_202307.pdf">https://iocv.ucr.edu/sites/default/files/2024-01/iocv_news_letter_202307.pdf</a> Druciarek, T., Sierra Mejia, A., Zagrodzki, S.K., Singh, S., Ho, T., Lewandowski, M. and Tzanetakis, I.E. 2024. Phyllocoptes parviflori is a distinct species and a vector of the pervasive blackberry leaf mottle associated virus.  Infection, Genetics and Evolution: 105538   <a href="https://doi.org/10.1016/j.meegid.2023.105538">https://doi.org/10.1016/j.meegid.2023.105538</a> Hajizadeh, M., Amirnia, F., Srivastava, A. and Tzanetakis I.E. 2024. First Report of Strawberry Virus 3 Infecting Strawberry in Iran. Plant Disease 108: 539. <a href="https://doi.org/10.1094/PDIS-06-23-1072-SR">https://doi.org/10.1094/PDIS-06-23-1072-SR</a> Ho, T., Broome, J.C., Buhler, J.P., O’Donovan, W., Tian, T., Diaz-Lara, A., Martin, R.R. and Tzanetakis, I.E. 2024. Integration of Rubus yellow net virus in the raspberry genome: A story centuries in the making. Virology 591: 109991 <a href="https://doi.org/10.1016/j.virol.2024.109991">https://doi.org/10.1016/j.virol.2024.109991</a> Lavagi, V., Kaplan, J., Vidalakis, G., Ortiz, M., Rodriguez, M. V., Amador, M., Hopkins, F., Ying, S., & Pagliaccia, D. 2024. Recycling Agricultural Waste to Enhance Sustainable Greenhouse Agriculture: Analyzing the Cost-Effectiveness and Agronomic Benefits of Bokashi and Biochar Byproducts as Soil Amendments in Citrus Nursery Production. Sustainability, 16(14), 6070. <a href="https://doi.org/10.3390/su16146070">https://doi.org/10.3390/su16146070</a> Lavagi-Craddock, I., Harper, S., Krueger, R., Quijia-Lamiña, P., & Vidalakis, G. 2024. Policies, regulations, and production of viroid-free propagative plant materials for sustainable agriculture. Fundamentals of Viroid Biology, 337–361. <a href="https://doi.org/10.1016/b978-0-323-99688-4.00023-7">https://doi.org/10.1016/b978-0-323-99688-4.00023-7</a> Liu, C.-W., Bodaghi, S., Vidalakis, G., & Tsutsui, H. 2024. Quick Plant Sample Preparation Methods Using a Micro-Homogenizer for the Detection of Multiple Citrus Pathogens. Chemosensors, 12(6), 105. <a href="https://doi.org/10.3390/chemosensors12060105">https://doi.org/10.3390/chemosensors12060105</a> Mejia, A.S., Villamor, D.E.V. and Tzanetakis, I.E. 2024. A step closer in dissecting individual virus attributes in the blackberry yellow vein disease complex. Acta Horticulturae 1388: 373-376<a href="https://doi.org/10.17660/ActaHortic.2024.1388.54"> DOI10.17660/ActaHortic.2024.1388.54</a> Mitra, A., Sohrab Bodaghi, Kiran R. Gadhave, Sydney Helm, Rodriguez, Raymond K. Yokomi, Abigail M. Frolli, Ashraf El-Kereamy, and Georgios Vidalakis. 2024. Biology and transmissibility of CYVaV-like RNA. Citrograph. Vol. 15:2, Spring 2024 p.64-70. <a href="https://citrusresearch.org/citrograph/archive">https://citrusresearch.org/citrograph/archive</a> Pagliaccia, D., Ortiz, M., Rodriguez, M. V., Abbott, S., De Francesco, A., Amador, M., Lavagi, V., Maki, B., Hopkins, F., Kaplan, J., Ying, S., & Vidalakis, G. 2024. Enhancing soil health and nutrient availability for Carrizo citrange (X Citroncirus sp.) through bokashi and biochar amendments: An exploration into indoor sustainable soil ecosystem management. Scientia Horticulturae, 326, 112661. <a href="https://doi.org/10.1016/j.scienta.2023.112661">https://doi.org/10.1016/j.scienta.2023.112661</a> Reyes-Proano, E., Knerr, A.J., and Karasev, A.V. 2024. Molecular characterization of birch toti-like virus, a plant-associated member in the new family Orthototiviridae. Archives in Virology 169: 140 (<a href="https://doi.org/10.1007/s00705-024-06067-7">https://doi.org/10.1007/s00705-024-06067-7</a>). Singh, S., Stainton, D. and Tzanetakis, I.E. 2024. Development of rapid and affordable virus-mimicking artificial positive controls. Plant Disease 108: 30-34 <a href="https://doi.org/10.1094/PDIS-06-23-1072-SR">https://doi.org/10.1094/PDIS-06-23-1072-SR</a> Singh, S., Stainton, D. and Tzanetakis, I.E. 2024. No controls? No problem. A novel approach to develop controls that mimic natural virus infection. Acta Horticulturae 1388: 213-216 <a href="https://doi.org/10.17660/ActaHortic.2024.1388.54"> DOI 10.17660/ActaHortic.2024.1388.32</a> Tan, S., Bodaghi, S., Mitra, A., Comstock, S., Huang, A., Hammado, S., Liu, J., Abu-Hajar, S., Quijia-Lamina, P., Villalba-Salazar, G. R., Douhan, G. W., Lavagi-Craddock, I., Frolli, A. M., El-Kereamy, A., & Vidalakis, G. 2024. Two distinct viral suppressors of RNA silencing encoded by citrus tatter leaf virus. Journal of Citrus Pathology, 11(2). <a href="https://doi.org/10.5070/c411262591">https://doi.org/10.5070/c411262591</a> Vidalakis, G. 2024. The Citrus Clonal Protection Program. Ten stages of budwood production and distribution and performance metrics. Citrograph. Vol. 15:2, Spring 2024 p.38-42. <a href="https://citrusresearch.org/citrograph/archive">https://citrusresearch.org/citrograph/archive</a>. Voncina, D., Jagunic, M., De Stradis, A., Diaz-Lara, A., Al Rwahnih, M., Scepanovic, M., Almeida, R.P.P. 2024. New host plant species of grapevine virus A identified with vector-mediated infections. Plant Disease 108: 125-130.

Impact Statements

The WERA-20 multistate project members (Land-grant University researchers and USDA ARS & USDA APHIS) conducted team-based collaborative research to advance fundamental and applied knowledge on emerging and remerging viruses infecting specialty crops, including fruit crops and woody ornamentals. Members participating in the annual meeting shared latest research-based knowledge on molecular characterization of viruses, advances in high-throughput detection of viruses and current strategies for the management of viral diseases. Members of the state departments of Agriculture (OR, PA and WA) and federal regulatory agencies (USDA APHIS-PPQ) shared advances in fruit tree planting stock registration and certification programs
Attendees discussed funding available from the USDA APHIS to support pest detection and surveillance, identification, threat mitigation, and safeguard the nursery production systems under the Plant Protection Act’s Section 7721 (PPA 7721). Collaborations between researchers and state regulatory agencies are strengthening nursery certification programs to maintain virus-tested planting materials for end users.
WERA 20 members, in collaboration with hundreds of experts in the USA and around the world prepared three review articles on ‘phantom’ disorders and agents of eight fruit crops and woody ornamentals to facilitate global germplasm exchange and reducing regulatory burdens, while maintaining rigorous pathogen exclusion standards. WERA 20 members published peer-reviewed articles in scientific journals and delivered talks at grower meetings for broader dissemination of research outcomes. Members also authored a book chapter on policies, regulations, and production of viroid-free propagative plant materials and co-organized. WERA20 members participated in a workshop for controlled environment agriculture for nursery production as well as the 7th International Research Conference on Huanglongbing (HLB) with participation of hundreds of scientists, regulators and growers from around the world.

Date of Annual Report: 11/10/2025

Report Information

Annual Meeting Dates: 09/08/2025 - 09/10/2025
Period the Report Covers: 10/01/2024 - 09/30/2025

Participants

In-person attendance
Nº Name Affiliation
1. Akinbade, Segun WA Department of Agriculture
2. Allen, Zachary North Carolina State University
3. Almeyda, Christie North Carolina State University
4. Al Rwahnih, Maher University of California, Davis
5. Alvarez-Quinto, Robert University of Minnesota
6. Auman, Dustin North Carolina Crop Improvement Association Inc.
7. Cardwell, Kitty Oklahoma State University
8. Chingandu, Nomatter Canadian Food Inspection Agency
9. Groth-Helms, Deborah Agdia Inc.
10. Guzman, Tania North Carolina State University
11. Herrera, Rafael North Carolina State University
12. Karasev, Alexander University of Idaho
13. Li, Chunying North Carolina State University
14. Lialiuk, Olga North Carolina State University
15. Nicholson, Jennifer USDA APHIS-PPQ
16. Olaya, Cristian Oregon State University
17. Osman, Fatima University of California, Davis
18. Park, Kristen Cornell University
19. Rayapati, Naidu Washington State University
20. Reiland, Danny North Carolina State University
21. Rentzel, Kay US Sweetpotato Council
22. Salazar, Jhoselin North Carolina State University
23. Santos, Carlos North Carolina State University
24. Serrano, Joana University of Minnesota
25. Tzanetakis, Ioannis University of Arkansas
26. Vidalakis, Georgios University of California, Riverside
27. Villamor, Dan University of Arkansas
28. Whitfield, Anna North Carolina State University
29. Young, Carolyn North Carolina State University
30. Zhou, Jing University of Hawaii
Virtual attendance
Nº Name Affiliation
1. Abrahamian, Peter USDA ARS
2. Burkhardt, Alyssa Driscoll’s Inc.
3. Cieniewicz, Elizabeth Clemson University
4. Fayad, Amer USDA-NIFA
5. French, Ronald USDA APHIS PPQ PGQP
6. Gratz, Allison Canadian Food Inspection Agency
7. Habecker, Nicole Driscoll’s Inc.
8. Hammond, John USDA-ARS, US National Arboretum
9. Hara, Kenji Canadian Food Inspection Agency
10. Hurtado-Gonzales, Oscar USDA-APHIS PPQ PGQP
11. Jones, Robert USDA-APHIS PPQ PGQP
12. Jordan, Ramon USDA-ARS, US National Arboretum
13. Kambic, Lukas University of Hawaii
14. Lavagi-Craddock, Irene University of California, Riverside
15. Ling, Kai-Shu USDA-ARS, US Vegetable Laboratory
16. Krueger, Robert USDA-ARS-NCGRCD
17. Melzer, Michael University of Hawaii
18. Meyer-Jertberg, Melody Driscoll's Inc.
19. Mitra, Arunabha University of California, Riverside
20. Nikolaeva, Ekaterina PA Department of Agriculture
21. Ponniah, Sathish University of Arkansas at Pine Bluff
22. Port, Lauren University of California, Davis
23. Poudyal, Dipak Oregon Department of Agriculture
24. Power, Imana Louisiana State University
25. Sutton, Mary University of Georgia
26. Suzuki, Jon USDA ARS DKI US PBARC
27. Topham, Katherine University of Minnesota
28. Zhai, Ying USDA ARS

Brief Summary of Minutes

The multi-state WERA20 project “Management of Diseases Caused by Systemic Pathogens in Temperate and Sub-Tropical Fruit Crops and Woody Ornamentals” organized an in-person annual meeting during September 8^th through 10^th, 2025, at the Embassy Suites in Cary, NC. The meeting also had a virtual option for participants with travel restrictions. The meeting was hosted by Dr. Christie Almeyda from North Carolina State University. Dr. Almeyda welcomed the participants on behalf of NC State University. Dr. Naidu Rayapati, Administrative Advisor from Washington State University, provided a brief account of the WERA20 project and its objectives. Dr. Amer Fayed, National Program Leader, provided a USDA-NIFA update (remote). After business discussions, the annual meeting in 2026 was proposed to be hosted by Dr. Jing Zhang from the University of Hawaii, likely at Honolulu, time to be determined in 2026 and subject to approval by Western Association of Agricultural Experiment Station Directors.

The group photo for the meeting can be found at: https://nimss.org/projects/photos/18910

WERA20 Scientific Program

September 8, 2025 (Monday)

Field tour, included visits to the NC State Clean Plant Center and the Strawberry production program at the NC State Plant Science Initiative Building followed by a visit to Pairwise Inc. at Research Triangle Park (RTP) and Union Grove Farms in Chapel Hill, NC.

September 9, 2025 (Tuesday)

An update on grapevine viruses in Washington State (N. Rayapati).

Grapevine virus research in Idaho (A. Karasev).

Grapevine virus research in Minnesota (J. Serrano).

HTS Validation (K. Cardwell).

PGQP status report for fruit trees, APHIS report (O. Hurtado-Gonzales) – remote.

WSDA fruit tree certification project report (S. Akinbade).

CFIA Center for Plant Health Report (N. Chingandu).

Virus and viroids research in South Carolina (E. Cieniewicz) – remote.

Arkansas update (I. Tzanetakis).

California Citrus Report (G. Vidalakis).

Foundation Plant Services Report (M. Al Rwahnih).

Minnesota Report (R. Alvarez-Quinto)

Hawaii Update (J. Zhou)

Woody Ornamental virus research in Agdia Inc. (D. Groth-Helms).

Diagnostic Assay Validation Network Report (K. Cardwell).

September 10, 2025 (Wednesday)

NCPN Cooperators Session led by Dr. Jennifer Nicholson, NCPN Program Director, USDA APHIS.

WERA-20 participants include several National Clean Plant Network members, and this year the meeting featured a special full day session focused on the NCPN. The goal of this NCPN Cooperators Session was to feature the progress and technical advances of the NCPN over the program history, and to discuss cross-network initiatives and future strategies to sustain into the future. The session included a highlights of advances over the last several years in clean plant center operations, diagnostic technologies, and therapeutics as well as accomplishments in cross-network initiatives for outreach, quality, economic analysis, and data management. The final session focused on a review of national and crop group strategic planning efforts, and a group discussion of potential areas of focus for the next few years. As the current national NCPN 2021-2025 Strategic Plan is reaching the end of its original planning period, this was an opportunity to review how the network has made progress in its goals, and set the stage to review and refresh the plan over the coming year. Session presentations were as follow:

Advances in the National Clean Plant Network

Progress in the National Clean Plant Network Program (J. Nicholson).

NCPN Advances at Foundation Plant Services (M. Al Rwahnih).

Advances in Clean Citrus (G. Vidalakis).

Three years in: Building a fully functional Arkansas Center (I. Tzanetakis).

Update from the Oregon Clean Center (C. Olaya).

Update from the North Carolina Clean Center (C. Almeyda).

Cross-Network Initiatives

NCPN Economic Working Group Studies (K. Park).

NCPN Education/Outreach Initiative (J. Nicholson).

NCPN Qualitative: A Forum for Exchange of Best Practices (I. Lavaggi-Craddock (remote) and F. Osman).

NCPN ADAPT Core (K. Krist).

Group Discussion

Future Strategies

Overview of NCPN Strategic Planning efforts, challenges and opportunities (J. Nicholson).

Strategies for advancing NCPN – Group discussion.

Recap and wrap-up (J. Nicholson).

Accomplishments

Naidu Rayapati, Washington State University Managing viral diseases in vineyards is a top priority for sustainable growth of Washington’s grape and wine industry that had an estimated $9.5 billion impact on the state’s economy in 2022. Vineyard surveys and testing samples using molecular diagnostic assays and high-throughput sequencing revealed to date the presence of fifteen viruses in Washington vineyards. They are: Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, and GLRaV-4 and two strains of GLRaV-4 (GLRaV-5, GLRaV-9), Grapevine red blotch virus (GRBV), Grapevine fanleaf virus, Tobacco ring spot virus, Grapevine Rupestris stem pitting-associated virus, Grapevine virus A, Grapevine virus B, Grapevine virus E, Grapevine fleck virus, Grapevine Syrah virus 1, Grapevine rupestris vein feathering virus and Grapevine red globe virus. We have conducted molecular characterization and genetic diversity of a few these viruses. This information was used in grapevine certification and quarantine programs to implement measures for protecting Washington’s young wine industry from ‘alien’ viruses reported in grapevines worldwide.  In Washington vineyards, GLRaV-3, the main etiological agent of grapevine leafroll disease (GLD), was found to be widespread than GRBV that causes red blotch disease and other viruses listed above. Since GLRaV-3 and GRBV produce similar symptoms in red-fruited wine grape cultivars and mild symptoms or no obvious symptoms in white-fruited cultivars, accurate diagnosis was found to be critical for managing these two distinct diseases. Studies in commercial vineyards have shown that GLRaV-3 and GRBV can cause significant impacts on fruit yield and grape quality in both own-rooted and top-grafted, red-fruited wine grape cultivars. Field studies have indicated the absence of vine-to-vine spread of GRBV and roguing of infected vines followed by replanting with virus-tested cuttings can be used as a low-cost strategy to manage red blotch disease in vineyards. In contrast, GLRaV-3 can be spread by insect vectors, such as grape mealybugs (Pseudococcus maritimus) and scale insects (Parthenolecanium corni), with mealybugs playing a dominant role in virus spread because of their higher mobility and dispersal. Studies on the field spread of GLRaV-3 showed a gradual increase in GLD incidence in otherwise healthy vineyard blocks over successive years due to new infections from external sources, mostly from neighboring infected blocks. Thus, a combination of preventive and sanitary tactics and mealybug control via insecticides and mating disruption strategies must be used for synergistic effects in reducing the spread of GLD in vineyards.  Collaborations with Plant Services Program of the Washington State Department of Agriculture and certified nurseries to ensure grapevines in registered mother blocks, the main source of certified planting stock for current and future vineyards, are protected under state certification and quarantine programs.  Annual inspections for visual symptoms and testing regularly for harmful viruses of concern to the industry ensured the availability of virus-tested planting stock for growers to plant. Outreach and educational activities were conducted to increase awareness of viruses and grower adoption of certified planting stock for healthy vineyards. Research outcomes were presented at professional scientific meetings and published in peer-reviewed scientific journals for broader dissemination of knowledge and basic science impacts.  Segun Akinbade, Washington State Department of Agriculture The Fruit Tree Certification Program of the Washington State Department of Agriculture (WSDA) comprises approximately 90,000 mother fruit trees, including cultivars from the genera Malus, Pyrus, and Prunus, and is housed in 12 certified nurseries across the state. The program's key responsibility is to support the national and international distribution of clean planting cultivars to Washington State growers. Nurseries in the program are required to source planting materials from Generation One (G1) foundation blocks kept at the Clean Plant Centers (CPCs) in the United States or, with written permission, from the Canadian Food Inspection Agency. Participating nurseries must keep a dedicated mother block (G2) in thrifty condition, with access restricted to authorized personnel. The G2 block can be expanded to make G3 for commercial purposes. Finished trees (G4) are made from materials from G2 or G3. The G1 materials are tested on a routine basis by CPCs, while the WSDA Fruit Tree Certification Program carries out inspections and testing of G2, G3, and G4 trees. Testing for pathogens in the mother trees varies by genus. Prunus mother trees are tested using biological, serological, and molecular methods because many vectors are known to transmit pathogens to this genus. Visual inspection is used for Malus and Pyrus because most of their pathogens are graft-transmitted; however, these tree cohorts are subjected to a full panel molecular assay if symptoms are observed during inspection. The annual testing of cherries in the certification program for pathogens has been expanded to include mandatory testing of all cherry trees for Little cherry virus 1 (LChV 1), Little cherry virus 2 (LChV 2), and Western x Phytoplasma. This was made possible through funding received from the Animal and Plant Health Inspection Service (APHIS) through the Plant Protection Act Section 7721 (PPA 7721). Each of the cherry trees must be tested at least once every 36 months for Little Cherry Disease (LCD) pathogens. To accomplish this, nurseries in the program were divided into three, and cherry trees in each nursery will be tested in the year assigned to them. This year, around 3,000 cherry trees were screened for LChV 1, LChV 2, Western x phytoplasma, Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry leaf roll virus (CLRV) using quantitative Polymerase chain reaction (qPCR). Trees that were positive for LCD in the lab were sniffed by canine dogs trained to smell and identify infected LCD trees. The dogs were able to confirm lab results as well as other trees with low titters that had previously not been picked up in the lab. Remaining Prunus trees in the program were tested using the Enzyme-linked immunosorbent assay (ELISA). Results obtained through ELISA were confirmed using qPCR.  Infected trees are promptly removed after treatment with an herbicide. A follow-up visit to each of the nurseries with infected trees will be conducted in the Fall of 2025 to ensure that the nurseries follow the protocol for tree removal. Lastly, a greenhouse experiment was set up to determine if LCD can be transferred from seed to seedlings. The experiment began in 2023 and is scheduled to conclude in 2026. This work will help determine the registration status of nurseries producing certified seeds for rootstock production.  Maher Al Rwahnih, University of California, Davis Sudden Vine Collapse (SVC) research initiated in 2021, continues. In 2022 a field trial to evaluate virus effects on Pinot gris grown on nine rootstocks was planted. The vines have been inoculated, with treatments: GLRaV-3, GLRaV-3 + GVA, GLRaV-1 + GVA, GLRaV-2 + GVB, no virus, no graft + no virus. PCR testing in 2024 was used to confirm successful virus inoculation. 2025 scoring for symptoms is yet to come. The research vineyard will continue to be maintained, and after the virus effect is determined, the research plan calls for inoculating with fungal pathogens to determine their potential role in SVC.  FPS has been collaborating with other berry researchers on two USDA-APHIS PPA 7721 projects that are now in their third years. The first is an evaluation of HTS in place of biological indexing for Fragaria and Rubus. USDA-ARS Corvallis (Walt Mahaffee with OSU PhD Student Dan Fager) is responsible for grafting and index evaluation. FPS conducts the HTS sequencing, bioinformatic analysis, and curation of data. The preliminary results are showing HTS is superior to biological indexing. The second collaborative project is survey of strawberry viruses. In the first two year of the project, samples to survey viruses in nursery production were collected and tested by Oregon, California, and Arkansas. Year 3 of the project began September 1, and will focus on HTS of native Fragaria to determine if they may be a green bridge or reservoir for viruses infecting nursery fields.  FPS has two research projects underway for development and testing of HiPlex PCR for virus detection. With funding from CDFA Pierce’s Disease Control Board, FPS is developing HiPlex assays for detection of multiple clades of grapevine red blotch virus and multiple variants of GLRaV-3. In the year 1 work, sensitivity and specificity of the assays are good. Further testing in year 2 will help clarify the read threshold. The second HiPlex project is funded by the California Strawberry Commission, and work has focused on detecting 16 strawberry viruses. This work is in year 2, and the final project months will be focused on refining the assays and finalizing the protocols so they can be shared with CDFA, to encourage their adoption of HiPlex for more efficient virus testing. HiPlex PCR shows a lot of promise, and FPS has applied for FY27 funding via the PPA7721 program to develop HiPlex assays for Vaccinium and Rubus.  Agave is an up-and-coming crop in California, with much interest in production due to its drought tolerance. Viruses have not been well-studied in agave, and it is important to build an awareness of the pathogen transmission possibilities of vegetative propagation. As part of a CDFA-funded project looking at agave production systems, FPS is surveying agave plantings and using HTS to screen for viruses. From 10 samples tested to date, six known viral agents were detected and two new viruses were identified.  The new federal funding year began on September 1, 2025, and FPS has three new projects starting. The first project is to optimize a new Qiagen +Kapa kit for HTS library preparation, using the Hamilton STAR liquidhandler. Most of us use Illumina for ribodepletion, but Illumina is not best for eliminating rRNA. QIAseq FastSelect-rRNA plant and KAPA Hyper-Prep kits (Qiagen + KAPA) may produce a greater percentage of virus reads than using the Illumina TruSeq stranded total RNA with Ribo-zero plant kit (Illumina). Before we switch, and request update of our SOPs, we need to do our homework.  USDA-APHIS-PPCDL is coordinating an interlaboratory validation of HTS protocol for detection of plant viruses in berry crops. FPS is a participating lab, in addition to PPCDL and Arkansas Clean Plant Center. The samples for the first year of this project (with a second year planned for FY27) have been received. Libraries will be prepared, the samples will be sequenced, and bioinformatic analysis conducted.  For sweetpotato, FPS is running a comparison of indexing to HTS+PCR. Typically, annual virus testing of sweetpotato is done by indexing to Ipomoea setosa. A test population will be selected from the virus positive and clean plant collections at FPS. The test population will be tested using the typical grafting to I. setosa, which are scored for virus symptoms and tested with PCR. Storage roots and leaf tissue from the test mothers will be sampled directly and tested using HTS and PCR. We will compare the test results generated from each testing method. This is anticipated to be a two-year project, requiring updating of RT-qPCR assays.  Georgios Vidalakis, University of California, Riverside In this report period, October 2024 – September 2025, we continued to support the suppression and eradication efforts against Huanglongbing (HLB) in California, where the number of positive trees has reached 10,183 and the HLB quarantine zones are expanding in the coastal and southern regions of the state. The University of California, Riverside, National Clean Plant Network (NCPN) Citrus Center, namely the Citrus Clonal Protection Program (CCPP) collaborated with the California Department of Food and Agriculture's (CDFA) Citrus Nursery Stock Pest Cleanliness Program and distributed 83,763 clean citrus propagation units (buds), tested for HLB as well as virus and viroid diseases, from 384 different citrus accessions, to 804 nurseries, producers, scientists, and the public, sourced from 1,192 pathogen-tested citrus budwood trees. The CCPP also tested 78 citrus introductions from 7 countries with 2,424 diagnostic tests intercepting 11 different types of pathogens in 30 introductions (38.5%), including HLB, viruses and viroids and performed pathogen elimination/therapy on 83 citrus accessions with 727 tissue cultures. The CCPP maintained 574 inquiries under quarantine and performed 1, 846 laboratory and 5,284 biological pathogen detection tests for HLB, virus and viroid diseases resulting in the release from quarantine of 50 citrus accessions.  In collaborative efforts with WERA 20 members and experts in USA, and other citrus producing countries, we continued the development and validation of e-probes for the detection of graft-transmissible pathogens of citrus; evaluated duplex qPCR interference from common citrus endogenous standards (COX/MDH) and designed a synthetic internal standard to mitigate competition during reactions; sequenced and analyzed citrus viroid VII populations from multiple hosts/locations to assess genetic diversity and variant structure for the development of detection assays; designed and 3D-printed a palm-sized, field-deployable device for rapid citrus tissue lysis and nucleic-acid extraction; and identified stress-specific signaling patterns and candidate regulators linked to herbivory vs. pathogen pressure.  WERA 20 members, presented their work (presentations, keynote and invited speakers) at the 21st Panhellenic Phytopathological Conference, 23rd Conference of the International Organization of Citrus Virologists, 2025 International Citrus Congress, World Ag Expo, Conference of the Mediterranean Phytopathological Union, Plant Health, Conference of the International Society of Citrus Nurseries, and the 2025 International Conference on Viroids with participation of hundreds of scientists, regulators and growers from around the world.  Ioannis Tzanetakis, University of Arkansas The Arkansas program continued to focus on advancing plant virus diagnostics, infectious clone development, and vector–virus interaction studies across berry crops, rose, and other specialty crops. Highlights include: Work on strawberry pseudo mild yellow edge virus (SPMYEV) led to the complete genome characterization of the U.S. isolate using HTS. An infectious clone was developed, and Koch’s postulates were validated through agroinfiltration and dodder transmission assays in Fragaria vesca. In addition, PCR-based diagnostics were established to address a regulatory gap for this long-suspected “phantom” agent.  The blackberry chlorotic ringspot virus (BCRV), infectious clone was employed to study virus–host dynamics in Rubus. Comparative studies between ‘Munger’ black raspberry and ‘Natchez’ blackberry revealed strong host-dependent differences in viral accumulation patterns. Quantitative assays demonstrated consistently high levels of virus in ‘Munger’, while detection in ‘Natchez’ was more variable and significantly lower, providing insights into the reliability of indicator plants.  Studies on blueberry scorch virus (BlScV) and blueberry virus S (BluVS) involved comparative analyses using ELISA, PCR, and HTS. The results highlighted population diversity in BlScV and confirmed BluVS as a distinct and potentially significant pathogen of blueberry. To strengthen diagnostic capacity, a triplex endpoint PCR and duplex quantitative PCR assay targeting BlScV and BluVS was developed incorporating internal controls for reliable detection. Also we developed a ViMAPC to assist other laboratories that may not have the positive controls needed to run the tests.  In the continued clean plant initiatives and in collaboration with many WERA-20 members, we have continued to streamline virus diagnostics and germplasm exchange. The phantom agent paper based on national and international collaborations addressing phantom agents and advancing clean plant programs was published in April 2025.  Alexander V. Karasev, University of Idaho The virome of grapevines grown in the State of Idaho was continued to be characterized in 2020-2024, with the overall goal of developing diagnostic tools for virus and virus-like disorders in wine grapes. More than 360 leaf and petiole samples were collected from symptomatic grapevines in 10 vineyards in Canyon and Nez Perce counties of Idaho and in Malheur County of Oregon and subjected to high-throughput sequencing (HTS) and RT-PCR testing. A new tri-partite, negative-sense RNA cogu-like virus was uncovered in grapevines from a 38-year-old ‘Chardonnay’ block in Idaho through HTS of total RNA, its presence was confirmed and validated by RT-PCR with specific primers designed based on the sequencing information obtained by HTS. The virus was named grapevine-associated cogu-like Idaho virus (GaCLIdV). In phylogenetic analysis based on the RdRP, GaCLIdV grouped within the family Phenuiviridae and was placed in a lineage of plant-infecting phenuiviruses as a sister clade of the genus Laulavirus, clustering most closely with switchgrass phenui-like virus 1 (SgPLV-1) and more distantly related to grapevine-associated cogu-like viruses from the Laulavirus and Coguvirus clades. The presence of GaCLIdV in the original ‘Chardonnay’ samples was confirmed by RT-PCR amplification and Sanger sequencing. This new virus was found in five wine grape cultivars and in six vineyards sampled in Idaho and in Oregon during the 2020–2024 seasons.  Noma Chingandu, Canadian Food Inspection Agency (Centre for Plant Health) The CFIA Centre for Plant Health (CPH) in North Saanich, BC, serves as Canada’s only post-entry quarantine facility for plant viruses and virus-like diseases. Recently modernized in 2024, the facility now includes advanced diagnostics and research laboratories, growth chambers, screenhouses, and bioassay field blocks. It supports virus testing for both certified and non-certified plant material, including imports, exports, and domestic breeding programs. The Centre also maintains and distributes Generation 1 virus-tested propagative stock for Canada’s Fruit Tree and Grapevine Export Program, with over 780 accessions shared globally. A key advancement in 2024 was the full integration of High Throughput Sequencing (HTS) into virus indexing workflows, significantly improving diagnostic speed and accuracy. HTS is expected to replace bioassays starting in 2028.  Research at CPH focuses on developing and validating new diagnostic assays and supporting regulatory decisions. The Virus/Viroid Collection Bank (VVCB) project, led by Dr. Yahya Gaafar, aims to sequence and profile over 460 accessions infected with more than 80 viruses and viroids. Preliminary findings reveal high genetic variability and mixed infections, which are instrumental in refining HTS protocols and developing PCR-based confirmatory tests. Automation of HTS processes using robotics has drastically reduced sample preparation time and minimized contamination risks, enabling broader research into aspects like virus seasonality and genetic diversity.  Cristian Olaya, Oregon Clean Plant Center, Oregon State University Pathogen-free germplasm is vital to economical and sustainable production since a single clean mother plant can be propagated to 15,000 and 20 million Blackberry, Raspberry, Blueberry, or Strawberry plants over a five-year period. To ensure plants are free of pathogens, breeding material is tested and maintained by centers within the National Clean Plant Network (NCPN). One such center, the Oregon Clean Plant Center (OCPC) at USDA-ARS in Corvallis, OR, has supported the international berry industry by providing clean plants throughout the world for more than 20 years. Recently, the OCPC began a restructuring initiative in collaboration with Oregon State University’s Plant Clinic to enhance and expand its testing capacity and ability to further serve the industry. The center serves commercial and public stakeholders by offering pathogen testing for approximately 37 viruses in Rubus, 22 in Vaccinium, and around 20 in Fragaria, as well as for Phytoplasma and Xylella fastidiosa, using PCR, qPCR, ELISA, and grafting techniques. OCPC maintains and distributes over 200 different genotypes, including 99 patented cultivars (79 Rubus, 15 Fragaria, and 5 Vaccinium), 101 advanced selections (56 Rubus, 12 Fragaria, and 33 Vaccinium), and a collection of more than 150 virus-positive control plants. This partnership has allowed OCPC to serve more than 5 private stakeholders, four public breeders, updates standard operation procedures and testing methods, and curate the genotype collections.  Christie Almeyda, North Carolina State University The North Carolina Clean Center continues to clean and test mainly domestic materials from berry and muscadine grapes breeding programs in the Southeast. We currently served breeders in NC (Rubus/Fragaria/Vaccinium), AR (Rubus and muscadine grapes) and FL (Vaccinium/Rubus). We continue to provide services to industry on multiple capacities: diagnostics, graft indexing and cleanup of imported material. Deliverables include the maintenance of foundation plants (100 genotypes, Rubus, Vaccinium and Fragaria); maintenance of in vitro genotypes (200 genotypes); cleanup of imported genotypes and distribution since 2018 of 60 genotypes as clean stock.  While cleaning up berry crops, Blueberry latent virus (BBLV) is the main virus fond in blueberries. Blackberry yellow vein-associated virus (BYVaV) and Blackberry leaf mottle associated virus (BlMaV) were detected on blackberries. Since 2021, the MPRU has established a partnership with the NC Plant Disease and Insect Clinic (PDIC). Now NC growers can submit berry and grape samples to be tested for designated pathogens at the MPRU as the unit has expanded its diagnostic services. The NC Clean Center continues to work closely with other berry clean centers (OR, AR and CA) as well as USDA-APHIS regulators to discuss matters related to CIP management for successful release of imported material.  A muscadine survey was conducted in 2024 aiming to identify viruses infecting the crop and to assess their incidence in NC. Samples were collected from 210 asymptomatic vines across 8 locations and virus tested at the NC Clean Plant Center. Vines were sampled in summer, June through August, and again after harvest, October and November. Due to the impact of Hurricane Helene, three vineyards (70 vines) could not be resampled in the fall. The cultivars sampled were Noble, Carlos, Regale, Supreme, Late Fry, Triumph, and Paulk. One to three blocks were sampled at each vineyard location. A block was considered a group of 10 to 40 rows consisting of the same cultivar. Ten vines per block were selected for sampling following a zig-zag schematic. All vines sampled were asymptomatic for viral diseases. Samples were delivered on the same day to the NC Clean Plant Center. Samples were stored at -80 C until total RNA was extracted. RT qPCR assays were performed on each sample to evaluate for the presence/absence for pathogens/viruses. Protocols for virus detection were adopted from Foundation Plant Services (FPS), UC-Davis. Muscadine vines were tested for grapevine leafroll associated virus complex (GLRaV-2, 3, 4, 7), grapevine red blotch virus (GRBV), grapevine virus A and B (GVA and GVB), grapevine rupestris stem pitting-associated virus (GRSPaV), tobacco ringspot virus (TRSV) and grapevine Syrah virus-1 (GSyV-1). Amplicons were generated by conventional RT-PCR and send to sequence. Preliminary results revealed 39% incidence of GLRaV-2 and 23% incidence of GVB. Sequenced amplicons showed 91% identity to the GVB replicase and 97% to 99% identify to the GLRaV-2 coat protein. This is the first time viruses were reported to be present on NC muscadine vineyards. As a next step, an additional round of muscadine virus testing is ongoing in 2025 and samples from 2024 and 2025 will be send to FPS for HTS analysis.  Jing Zhou, University of Hawaii Jing Zhou just started the plant virologist position at University of Hawaii in August 2025, a month prior to the 2025 WERA20 meeting. Since the previous plant virologist Dr. John Hu retired in 2023, continuous work related to WERA20 has barely been conducted, and there is a gap in the reporting period as a result. As the new plant virology laboratory is setting up and research focuses are established, we expect to start implementing work regarding viruses infecting fruits, woody ornamentals, and sweet potatoes very soon. We hope to share the new findings in the coming 2026 WERA20 meetings, which will be held in Hawaii presumably during the late spring to early summer. As the meeting host, we’ve started preparing for this annual meeting and we look forward to having our group next year in Hawaii!  Mary Sutton, University of Georgia Citrus is a new industry for the state of Georgia. As a result, many of the quarantine diseases have not yet found a foot hole in Georgia citrus. The current goal of the UGA citrus extension program is to increase grower awareness of such quarantine disease before they become established. In 2025, seven trainings were provided to provide citrus growers, county agents, landscapers, homeowners, and state plant protection inspectors, with the knowledge to identify symptoms of Huanglongbing (HLB), or citrus greening, and its vector, the Asian citrus psyllid (ACP). These training courses reached over 150 people. On average, attendees reported a 46% increase in knowledge following such meetings. On a scale of 1 to 10, attendees reported their concern regarding quarantine diseases rated an average of 6.8.  Together, this suggests that conducted meetings and trainings have been successful in increasing awareness of this disease and in increasing attendee knowledge in how to recognize the associated diseases and pests.  Ekaterina Nikolaeva, Pennsylvania Department of Agriculture Pennsylvania Department of Agriculture (PDA) safeguards PA agriculture and natural resources against the entry, establishment, and spread of economically and environmentally significant pests, and facilitates the safe trade of agricultural products. In 2024-2025, PDA in cooperation with Pennsylvania State University conducted state-wide exotic disease surveys of fruit trees (Malus and Prunus) and small fruits (strawberry and grapes) commodities. The targets of the Orchard survey were Potyvirus Plum pox virus, Apple Proliferation (Candidatus Phytoplasma mali), European stone fruit yellow (Ca. Phytoplasma prunorum), Jujube witches' broom (Ca. Phytoplasma ziziphi), Little cherry disease (Little cherry viruses 1 and 2), and Almond witches’ broom (Ca. Phytoplasma phoenicium). Small fruit survey included surveillance activities for Nepovirus Tomato black ring virus on strawberry, Australian Grapevine Yellows (Ca. Phytoplasma australiense), Flavescence Doreé Phytoplasma (Ca. Phytoplasma vitis), and Bois noir Phytoplasma (Ca. Phytoplasma solani) on grapes. A total of 2,919 Prunus samples were collected and tested for PPV. Additionally, 66 Malus, 78 Prunus, 52 Strawberry and 99 Grape samples were collected and tested according to USDA PPQ approved protocols. No exotic targets were detected.  PDA continues to operate the Fruit Tree Improvement Program (FTIP), specialized inspection and virus testing program for participating PA fruit tree nurseries. In 2024, 4,459 total samples were tested, representing 15,611 trees. A total of 3,481 Prunus samples were processed through the FTIP laboratory this year for Plum pox virus (PPV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Tomato ringspot virus (ToRSV).  In result, PNRSV and PDV were the most commonly found viruses in the FTIP. Some samples were positive for multiple viruses. No PPV was detected.  Apple samples were collected from budwood source blocks. In total, 522 samples were tested for apple mosaic virus (ApMV), apple chlorotic leafspot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), tobacco ringspot virus (TRSV), and tomato ringspot virus (ToRSV). Sixteen samples were determined positive: eight samples for ACLSV, four samples for ASGV, and four samples for ToRSV.  Two cherry budwood blocks were inspected for cherry leaf roll virus (CLRV) with 19 samples collected and tested. All samples tested negative for CLRV. Phytoplasma inspections were conducted at twelve blocks consisting of registered source, common source budwood, and nursery blocks. Eighty-five samples were collected and tested. All samples were found negative. A total of 288 broadleaf weed samples were collected and tested for ToRSV. Twenty-eight soil samples were collected from current or future source blocks and proposed sites for nursery production to determine the presence of Xiphinema sp.  nematodes. ToRSV was detected in two broadleaf weed samples. Xiphinema sp. nematodes were present at low but detectable levels in budwood source locations and proposed sites for nursery production. Presence of dagger nematodes makes broadleaf weed control imperative to prevent transmission from virus-positive weeds to fruit trees.  In 2024, we implemented confirmatory testing for FTIP ELISA positive and inconclusive results. Total RNA was extracted using Qiagen kits and amplified in qRT-PCR protocols with primers specific to ASPV, ASGV, ToRSV, PNRSV and PDV (Li et al., 2018; Osman et al., 2014; Beaver-Kanya et al., 2019). Results confirmed by PCR were reported to growers to avoid using positive trees as grafting material.  In 2024, PDA in collaboration with PSU (Dr. K. Peter) and USDA APHIS PPQ Plant Germplasm Quarantine Program (PGQP) (Dr. O. Hurtado Gonzales) has started work on implementation of HiPlex technology in FTIP program. Our current virus detection workflow relies on DAS ELISA for 5 viruses and requires the use of two incompatible commercial kits (Agdia and Bioreba), resulting in duplication of the sample grinding step without possibilities to streamline the entire process. In addition, these protocols require significant time and labor to process the volume of samples necessary to adequately screen nursery plant material, not to mention that ELISA detection protocols are known to produce false positive and false negative results. In 2024-2025, we started work on validating a novel approach (HiPlex) that combines a single-step PCR and HTS for the simultaneous detection of 22 viruses and five viroids known to infect apple trees across hundreds of samples. This approach offers several advantages over traditional methods such as low amounts of cDNA input, streamline workflow from PCR to sequencing, simple data analysis and simultaneous detection of multiple viruses in multiple samples in a single sequencing run, which significantly reduces turnaround time for diagnostic results. Evaluation of this technique will help prepare the PA certification program to offer a more rapid and early detection for fruit growers and spearhead the potential transformation of diagnostics across other states by implementing simultaneous virus and viroid diagnostics and moving away from time-consuming DAS ELISA-based methods.

Publications

Abou Kubaa R, Ouro-Djobo A, Stevens KA, Alabi OJ, Al Rwahnih M. 2025. Genome characterization of prunus maculavirus 1 (PrMcV-1), a novel member of the genus maculavirus identified in prunus spp. Archives of virology, 170(8): 168.  Ault, N., Ren, S., Payne, D.  Li, Y., Srinivasan, A., Zheng, Y., Sunkar, R. and Naidu, R. A. 2025. Dynamics of small RNAs in a red-fruited wine grape cultivar infected with Grapevine red blotch virus. BMC Genomics 26: 417.  Al Rwahnih M, Klaassen V, Erickson T, Alabi OJ, Stevens K, Hwang MS, Port L. 2025. A New Era in Federal Quarantine and State Certification Diagnostics at Clean Plant Centers in the United States. Plant disease, 109(7): 1392-1403.  Chambers, G. A., Geering, A. D. W., Holford, P., Kehoe, M. A., Vidalakis, G., & Donovan, N. J. 2025. Genetic diversity of citrus viroid VII (CVd-VII). Archives of Virology, 170:12. <a href="https://doi.org/10.1007/s00705-024-06191-4">https://doi.org/10.1007/s00705-024-06191-4</a>  Cifuentes R., Brito M.L., Cornejo-Franco J.F., Alvarez-Quinto R.A., Mollov D., Martínez A., Ochoa J., Villamor D.E., Tzanetakis I.E., Quito-Avila D.F. 2025. Insights into the virome of the Andean blackberry (Rubus glaucus). Eur. J. Plant Pathol. 173:197–208. <a href="https://doi.org/10.1007/s10658-025-03058-5">https://doi.org/10.1007/s10658-025-03058-5</a>  Costanzo, S., Jones, T., Peter, K., Nikolaeva, E. 2025. First Report of ‘Candidatus Phytoplasma fraxini’-Related Strain Associated with Peach Yellows in Pennsylvania. Plant Disease, <a href="https://doi.org/10.1094/PDIS-06-25-1240-PDN">https://doi.org/10.1094/PDIS-06-25-1240-PDN</a>  Dahan, J., Orellana, G.E., Reyes-Proano, E., Lee, J., and Karasev, A.V. 2025. A novel cogu-like virus identified in wine grapes. Viruses 17 (9): 1175 (<a href="https://doi.org/10.3390/v17091175">https://doi.org/10.3390/v17091175</a>).  de Souza JO, Klaassen V, Stevens K, Erickson TM, Heinitz C, Al Rwahnih M. 2024. Characterization of Genetic Diversity in the Capsid Protein Gene of Grapevine Fleck Virus and Development of a New Real-Time RT-PCR Assay. Viruses, 16(9).  Druciarek T., Tzanetakis I.E. 2025. Invisible vectors, visible impact: The role of eriophyoid mites in emaravirus disease dynamics. Virology 606:110478. https://doi.org/10.1016/j.virol.2025.110478  Fuchs, M., Rwahnih, M. A., Blouin, A. G., Burger, J., Chooi, K.M., Constable, F., Ertunc, F., Fiore, N., Habili, N., Hily, J.-M., Katis, N., Lemaire, O., Maliogka, V. I., Maree, H. J., Minafra, A., Naidu, R.A, Pietersen, G., Saldarelli, P., Schmidt, A.-M., Music. M. &Scaron;., Várallyay, É. 2025. A list of eclectic viruses, virus-like diseases and viroids of grapevines that should not be considered for regulatory oversight: a global plea from virologists. Journal of Plant Pathology, 107(2): 847-858.  Fust C, Lameront P, Shabanian M, Song Y, Abou Kubaa R, Bester R, Maree HJ, Al Rwahnih M, Meng B. 2025. Grapevine leafroll-associated virus 3: a global threat to grapevine and wine industries but a gold mine for scientific discovery. Journal of experimental botany, 76(11): 2985-3000.  Haegeman A, Foucart Y, De Jonghe K, Goedefroit T, Al Rwahnih M, Boonham N, Candresse T, Gaafar YZA, Hurtado-Gonzales OP, Kogej Zwitter Z, Kutnjak D, Lamov&scaron;ek J, Lefebvre M, Malapi M, Mavri# Ple&scaron;ko I, Önder S, Reynard JS, Salavert Pamblanco F, Schumpp O, Stevens K, Pal C, Tamisier L, Uluba# Serçe Ç, van Duivenbode I, Waite DW, Hu X, Ziebell H, Massart S. 2024. Correction: Haegeman et al. Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests. Plants 2023, 12, 2139. Plants (Basel, Switzerland), 13(5).  Hajizadeh M., Ghaderi Zandan N., Koloniuk I., Sierra-Mejia A., Tzanetakis I.E. 2025. Characterization, detection, and prevalence of a novel strawberry crinivirus. Plant Dis. 109:988–991.  Hardigan, M. A., Finn, C. E., Jones, P. A., Strik, B. C., Peterson, M. E., Bassil, N. V., King, R. M., Wiegand, Z. J., Olaya, C., Martin, R. R., Lee, J., & Lukas, S. B. (2025). ‘Thunderhead’ Erect Primocane Fruiting Blackberry. HortScience, 60(8), 1366–1371. <a href="https://doi.org/10.21273/HORTSCI18617-25">https://doi.org/10.21273/HORTSCI18617-25</a>  Harper, S, Molnar, C, Nikolaeva, E, Jones, T, and Peter, K. 2024. Draft Genome Sequence of North American Grapevine Yellows phytoplasma strain PDA15. Microbiology. <a href="https://mra.msubmit.net/cgi-bin/main.plex?el=A6Nv7CVdN1A3GPKl2F2A9ftdN1dYz2Xow9S7CzQtabJGgZ">https://mra.msubmit.net/cgi-bin/main.plex?el=A6Nv7CVdN1A3GPKl2F2A9ftdN1dYz2Xow9S7CzQtabJGgZ</a>  Larrea-Sarmiento AE, Galanti R, Olmedo-Velarde A, Wang X, Al Rwahnih M, Borth W, Lutgen H, Fitch MM, Sugano J, Sewake K, Suzuki J, Wall MM, Melzer M, Hu J. 2024. Characterization of Two Novel Viruses Within a Complex Virome from Flowering Ginger in Hawaii. Plant disease, 108(10): 3001-3009.  Lavagi-Craddock, I., El-Kereamy, A., Hajeri, S., Lovatt, C. and Vidalakis, G. 2025. Dwarfing of commercial citrus varieties using TsnRNAs: Evaluation of yield, size and tree care from the 1990s commercial and university field trials. Citrograph. Vol. 16:4, Fall 2025 p.32–36. <a href="https://citrusresearch.org/citrograph/archive">https://citrusresearch.org/citrograph/archive</a>  Liu, C.-W., Kalish, B., Bodaghi, S., Vidalakis, G., & Tsutsui, H. 2025. A 3D-printed handheld device for quick citrus tissue lysis and nucleic-acid extraction. Advances in Sample Preparation. <a href="https://doi.org/10.1016/j.sampre.2025.100199">https://doi.org/10.1016/j.sampre.2025.100199</a>  Manoharan, B., Qi, S.-S., Vidalakis, G., El-Kereamy, A., Satheesh, V., Elango, D., Dhandapani, V., Dai, Z.-C., & Du, D.-L. (2025). Roles of hormone signaling on defense responses of invasive Sphagneticola trilobata to pathogen and insect herbivore. Physiological and Molecular Plant Pathology. (2025): 102722. <a href="https://doi.org/10.1016/j.pmpp.2025.102722">https://doi.org/10.1016/j.pmpp.2025.102722</a>  Mitra, A., Jarugula, S. and Naidu, R.A. 2025. Development of a minireplicon for Grapevine leafroll-associated virus 1 and genetic analyses of sequences in the 5ʹ non-translated region required for replication. Phytopathology 115: 1065-1075.  Mohammed, Mohammed S., Lahuf, Adnan Abdaljeleel, Jeddoa, Zuhair M., de Souza, Juliana Osse, Al-Rwahnih, Maher. 2025. Survey and high throughput sequencing revealed mixed infections of cucurbit-infecting viruses in zucchini fields in Iraq. Tropical Plant Pathology, 50(1): 67.  Molnar C., M.K. Shires, A.A. Wright, M.C. Hoskins, S.J. Cowell, E.V. Nikolaeva, R. Knier, M.T. Nouri, B. Black, S.J. Harper. 2024. Putting ‘X’ into context: the diversity of ‘Candidatus Phytoplasma pruni’ strains associated with the induction of X-disease. Plant Dis. 108 (9).  Nascimento, D. M., Bodaghi, S., Wang, H., Ribeiro-Junior, M. R., Campos, R., Dang, T., Osman, F., Habiger, J., Espindola, A. S., Vidalakis, G., & Cardwell, K. F. 2025. Development and validation of a suite of e-probes for Electronic Diagnostic Nucleic Acid Analysis (EDNA) for 20 graft-transmissible pathogens of citrus using MiFi and blind ring testing among novice users. PhytoFrontiers, 5, 243–253. <a href="https://doi.org/10.1094/PHYTOFR-12-24-0140-FI">https://doi.org/10.1094/PHYTOFR-12-24-0140-FI</a>   Neugebauer, Kerri A., Gillett, Jerri M., Klaassen, Vicki, Miles, Laura A., Rwahnih, Maher Al, Miles, Timothy D. 2025. Occurrence of Grapevine Viruses in Different Cultivars and Regions Within Michigan. Plant Health Progress, 26(2): 155-160.  Olaya, C., Ohkura, M., Lake A, and Mahaffee, W. 2025. Oregon Clean Plant Center: supporting the berries industry in partnership with the OSU Plant Clinic. Annual Meeting of the American Phytopathological Society, Honolulu, Hawaii. August 2-5, 2025.  Olaya, C., Reinhold, L., Platt, McK., Peetz, A., Donahue, K., Zasada, I. 2024. Assessment of the distribution of Xiphinema spp and associated nepoviruses in the Pacific Northwest small fruits. https://doi.org/10.1094/PHP-04-24-0034-RS  Ouro-Djobo A, Obasa K, Oladokun JO, Sétamou M, Al Rwahnih M, Alabi OJ. 2025. Relative occurrence and seasonal variations of wheat-infecting viruses in Texas. Plant disease.  Osse de Souza J, Erickson TM, Stoddard CS, Almeyda CV, Al Rwahnih M. 2025. Evidence of Rapid Infection of Four Sweetpotato Potyviruses in a Commercial Field in California. Plant disease, 109(2): 308-312.  Reyes-Proano, E., Knerr, J., Karasev, A.V. 2024. Characterization of birch toti-like virus infecting ornamental European birch. (Abstr.) Phytopathology 114: S1.82. <a href="https://doi.org/10.1094/PHYTO-114-11-S1.1">https://doi.org/10.1094/PHYTO-114-11-S1.1</a>.  Phillips, J., Bodaghi, S., Vidalakis, G., & Blaha, G. 2025. Optimizing qPCR detection of ‘Candidatus Liberibacter asiaticus’: Introducing a new type of internal standard. Plant Disease. <a href="https://doi.org/10.1094/PDIS-12-24-2714-RE">https://doi.org/10.1094/PDIS-12-24-2714-RE</a>   Shires M. K., Molnar, C. Cowell, S. J. Bishop, G. Voelker, J. Thompson, A. A. Nikolaeva, E. Copp, C. Flandermeyer, L. Melton, T. Northfield, T. D. Marshall, A. T. Cooper W. R., Harper S. J. 2025. Alternative Hosts of ‘Candidatus Phytoplasma pruni’ Identified Through Surveys and Vector Gut Content Analysis. Plant Health Progress, 26 (201).  Sierra-Mejia A., Hajizadeh M., Atanda H.Y., Tzanetakis I.E. 2025. Overcoming the woody barrier: Dodder enables efficient transfer of infectious clones to woody plants. J. Virol. Methods 334:115114. https://doi.org/10.1016/j.jviromet.2025.115114  Sierra-Mejia A., Villamor D.V.V., Rocha A., Wintermantel W.M., Tzanetakis I.E. 2024. Engineering a robust infectious clone and gene silencing vector from blackberry yellow vein associated virus. Virus Res. 350:199488. <a href="https://doi.org/10.1016/j.virusres.2024.199488">https://doi.org/10.1016/j.virusres.2024.199488</a>  Sierra-Mejia A., Villamor D.V.V., Tzanetakis I.E. 2024. Development and application of an infectious clone and gene silencing vector derived from blackberry chlorotic ringspot virus. Virus Res. 350:199460. <a href="https://doi.org/10.1016/j.virusres.2024.199460">https://doi.org/10.1016/j.virusres.2024.199460</a>  Singh S., Villamor D.V.V., Sharma Poudyal D., Sierra-Mejia A., Tzanetakis I.E. 2025. A systems-based approach to ensure berry crops health status: from the breeder to the field. Eur. J. Plant Pathol. 172:55–73. https://doi.org/10.1007/s10658-024-02985-z  Stevens KA, Al Rwahnih M. 2024. High-Throughput Sequencing for the Detection of Viruses in Grapevine: Performance Analysis and Best Practices. Viruses, 16(12).  Tzanetakis I.E., Aknadibossian V., &Scaron;pak J., Constable F., Harper S.J., Hammond J., Candresse T., Folimonova S.Y., Freitas-Astúa J., Fuchs M., Jelkmann W., Maliogka V.I., Marais A., Martin R.R., Mollov D., Vidalakis G. and another 170 authors. 2025. Streamlining global germplasm exchange: Integrating scientific rigor and common sense to exclude phantom agents from regulation. Plant Dis. 109:736–755. https://doi.org/10.1094/PDIS-04-24-0745-FE  Vidalakis, G. 2025. The Citrus Clonal Protection Program: Protecting California’s citriculture. Citrograph. Vol. 16:2, Spring 2025 p.50–54. <a href="https://citrusresearch.org/citrograph/archive">https://citrusresearch.org/citrograph/archive</a>  Whitfield, A.E., Karasev, A.V., Alabi, O.J., Batuman, O., Cieniewicz, E.J., Fall, M.L., Jacobson, A.L., Pelz-Stelinski, K., Qiu, W., Rayapati, N.A., Reitz, S.R., Turpen, T.H. 2025. Advancing Vineyard Health: Insights and Innovations for Combating Grapevine Red Blotch and Leafroll Diseases. Consensus Study Report, 269 pp. National Academies of Sciences, Engineering, and Medicine. Washington, DC: The National Academies Press. <a href="https://doi.org/10.17226/27472">https://doi.org/10.17226/27472</a>.

Impact Statements

The WERA-20 multistate project members from Land-grant Universities, USDA ARS & USDA APHIS and State Departments of Agriculture conducted team-based collaborative research to advance fundamental and applied knowledge on emerging and remerging viruses infecting specialty crops, including fruit crops and woody ornamentals. Members participating in the 2025 annual meeting shared latest research-based knowledge on molecular characterization of viruses, advances in high-throughput detection of viruses and current strategies for the management of viral diseases. Members of the State Departments of Agriculture (WA, PA) and federal regulatory agencies (USDA APHIS-PPQ) shared advances in fruit tree planting stock registration and certification programs. Attendees discussed funding available from the USDA APHIS to support pest detection and surveillance, identification, threat mitigation, and safeguard the nursery production systems under the Plant Protection Act’s Section 7721 (PPA 7721). Collaborations between researchers and state regulatory agencies have strengthened nursery certification programs to maintain virus-tested planting materials for end users. These efforts have increased growers' confidence in the value of using certified stock for planting new orchards and vineyards.
WERA 20 members, in collaboration with subject-matter experts in the USA and around the world, published review articles on ‘phantom’ disorders and agents of eight fruit crops. These efforts have advanced harmonization of policies towards seamless exchange of global germplasm by reducing regulatory burdens that affects cross-border movement of germplasm, while maintaining rigorous pathogen exclusion standards. WERA 20 members published peer-reviewed articles in scientific journals and delivered talks at grower meetings for broader dissemination of research outcomes. These outputs are empowering plant health certification, regulatory policy, and stakeholder-driven clean plant programs.
Since WERA-20 participants include several members of the National Clean Plant Network (NCPN), the 2025 annual meeting facilitated collaborative efforts to develop, validate, and deliver virus diagnostics that are specific, sensitive, scalable, and affordable benefiting researchers, regulatory agencies and specialty crop industries (Program Progress and Strategic Planning Discussed in NCPN Cooperators Meeting). Collectively, these collaborative activities supported sustainable production of specialty crops, safeguarding the exchange of planting materials in international trade, and reinforcing the role of science-driven regulatory frameworks benefiting specialty crop industries.