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

(Multistate Research Coordinating Committee and Information Exchange Group)

Status: Active

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SAES-422 Reports

Annual/Termination Reports:

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

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. 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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.