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]

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. 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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.