Brief Summary of Minutes of Annual Meeting

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

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

WERA20 Scientific Program 

September 8, 2025 (Monday) 

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

September 9, 2025 (Tuesday) 

• An update on grapevine viruses in Washington State (N. Rayapati).
• Grapevine virus research in Idaho (A. Karasev).
• Grapevine virus research in Minnesota (J. Serrano).
• HTS Validation (K. Cardwell).
• PGQP status report for fruit trees, APHIS report (O. Hurtado-Gonzales) – remote.
• WSDA fruit tree certification project report (S. Akinbade).
• CFIA Center for Plant Health Report (N. Chingandu).
• Virus and viroids research in South Carolina (E. Cieniewicz) – remote.
• Arkansas update (I. Tzanetakis).
• California Citrus Report (G. Vidalakis).
• Foundation Plant Services Report (M. Al Rwahnih).
• Minnesota Report (R. Alvarez-Quinto)
• Hawaii Update (J. Zhou)
• Woody Ornamental virus research in Agdia Inc. (D. Groth-Helms).
• Diagnostic Assay Validation Network Report (K. Cardwell). 

September 10, 2025 (Wednesday) 

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

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

Advances in the National Clean Plant Network

• Progress in the National Clean Plant Network Program (J. Nicholson).
• NCPN Advances at Foundation Plant Services (M. Al Rwahnih).
• Advances in Clean Citrus (G. Vidalakis).
• Three years in: Building a fully functional Arkansas Center (I. Tzanetakis).
• Update from the Oregon Clean Center (C. Olaya).
• Update from the North Carolina Clean Center (C. Almeyda).

Cross-Network Initiatives

• NCPN Economic Working Group Studies (K. Park).
• NCPN Education/Outreach Initiative (J. Nicholson).
• NCPN Qualitative: A Forum for Exchange of Best Practices (I. Lavaggi-Craddock (remote) and F. Osman).
• NCPN ADAPT Core (K. Krist).
• Group Discussion

Future Strategies

• Overview of NCPN Strategic Planning efforts, challenges and opportunities (J. Nicholson).
• Strategies for advancing NCPN – Group discussion.
• Recap and wrap-up (J. Nicholson).

Naidu Rayapati, Washington State University

Managing viral diseases in vineyards is a top priority for sustainable growth of Washington’s grape and wine industry that had an estimated $9.5 billion impact on the state’s economy in 2022. Vineyard surveys and testing samples using molecular diagnostic assays and high-throughput sequencing revealed to date the presence of fifteen viruses in Washington vineyards. They are: Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3, and GLRaV-4 and two strains of GLRaV-4 (GLRaV-5, GLRaV-9), Grapevine red blotch virus (GRBV), Grapevine fanleaf virus, Tobacco ring spot virus, Grapevine Rupestris stem pitting-associated virus, Grapevine virus A, Grapevine virus B, Grapevine virus E, Grapevine fleck virus, Grapevine Syrah virus 1, Grapevine rupestris vein feathering virus and Grapevine red globe virus. We have conducted molecular characterization and genetic diversity of a few these viruses. This information was used in grapevine certification and quarantine programs to implement measures for protecting Washington’s young wine industry from ‘alien’ viruses reported in grapevines worldwide. 

In Washington vineyards, GLRaV-3, the main etiological agent of grapevine leafroll disease (GLD), was found to be widespread than GRBV that causes red blotch disease and other viruses listed above. Since GLRaV-3 and GRBV produce similar symptoms in red-fruited wine grape cultivars and mild symptoms or no obvious symptoms in white-fruited cultivars, accurate diagnosis was found to be critical for managing these two distinct diseases. Studies in commercial vineyards have shown that GLRaV-3 and GRBV can cause significant impacts on fruit yield and grape quality in both own-rooted and top-grafted, red-fruited wine grape cultivars. Field studies have indicated the absence of vine-to-vine spread of GRBV and roguing of infected vines followed by replanting with virus-tested cuttings can be used as a low-cost strategy to manage red blotch disease in vineyards. In contrast, GLRaV-3 can be spread by insect vectors, such as grape mealybugs (Pseudococcus maritimus) and scale insects (Parthenolecanium corni), with mealybugs playing a dominant role in virus spread because of their higher mobility and dispersal. Studies on the field spread of GLRaV-3 showed a gradual increase in GLD incidence in otherwise healthy vineyard blocks over successive years due to new infections from external sources, mostly from neighboring infected blocks. Thus, a combination of preventive and sanitary tactics and mealybug control via insecticides and mating disruption strategies must be used for synergistic effects in reducing the spread of GLD in vineyards. 

Collaborations with Plant Services Program of the Washington State Department of Agriculture and certified nurseries to ensure grapevines in registered mother blocks, the main source of certified planting stock for current and future vineyards, are protected under state certification and quarantine programs.  Annual inspections for visual symptoms and testing regularly for harmful viruses of concern to the industry ensured the availability of virus-tested planting stock for growers to plant. Outreach and educational activities were conducted to increase awareness of viruses and grower adoption of certified planting stock for healthy vineyards. Research outcomes were presented at professional scientific meetings and published in peer-reviewed scientific journals for broader dissemination of knowledge and basic science impacts. 

Segun Akinbade, Washington State Department of Agriculture

The Fruit Tree Certification Program of the Washington State Department of Agriculture (WSDA) comprises approximately 90,000 mother fruit trees, including cultivars from the genera Malus, Pyrus, and Prunus, and is housed in 12 certified nurseries across the state. The program's key responsibility is to support the national and international distribution of clean planting cultivars to Washington State growers. Nurseries in the program are required to source planting materials from Generation One (G1) foundation blocks kept at the Clean Plant Centers (CPCs) in the United States or, with written permission, from the Canadian Food Inspection Agency. Participating nurseries must keep a dedicated mother block (G2) in thrifty condition, with access restricted to authorized personnel. The G2 block can be expanded to make G3 for commercial purposes. Finished trees (G4) are made from materials from G2 or G3. The G1 materials are tested on a routine basis by CPCs, while the WSDA Fruit Tree Certification Program carries out inspections and testing of G2, G3, and G4 trees. Testing for pathogens in the mother trees varies by genus. Prunus mother trees are tested using biological, serological, and molecular methods because many vectors are known to transmit pathogens to this genus. Visual inspection is used for Malus and Pyrus because most of their pathogens are graft-transmitted; however, these tree cohorts are subjected to a full panel molecular assay if symptoms are observed during inspection.

The annual testing of cherries in the certification program for pathogens has been expanded to include mandatory testing of all cherry trees for Little cherry virus 1 (LChV 1), Little cherry virus 2 (LChV 2), and Western x Phytoplasma. This was made possible through funding received from the Animal and Plant Health Inspection Service (APHIS) through the Plant Protection Act Section 7721 (PPA 7721). Each of the cherry trees must be tested at least once every 36 months for Little Cherry Disease (LCD) pathogens. To accomplish this, nurseries in the program were divided into three, and cherry trees in each nursery will be tested in the year assigned to them. This year, around 3,000 cherry trees were screened for LChV 1, LChV 2, Western x phytoplasma, Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry leaf roll virus (CLRV) using quantitative Polymerase chain reaction (qPCR). Trees that were positive for LCD in the lab were sniffed by canine dogs trained to smell and identify infected LCD trees. The dogs were able to confirm lab results as well as other trees with low titters that had previously not been picked up in the lab. Remaining Prunus trees in the program were tested using the Enzyme-linked immunosorbent assay (ELISA). Results obtained through ELISA were confirmed using qPCR. 

Infected trees are promptly removed after treatment with an herbicide. A follow-up visit to each of the nurseries with infected trees will be conducted in the Fall of 2025 to ensure that the nurseries follow the protocol for tree removal. Lastly, a greenhouse experiment was set up to determine if LCD can be transferred from seed to seedlings. The experiment began in 2023 and is scheduled to conclude in 2026. This work will help determine the registration status of nurseries producing certified seeds for rootstock production. 

Maher Al Rwahnih, University of California, Davis

Sudden Vine Collapse (SVC) research initiated in 2021, continues. In 2022 a field trial to evaluate virus effects on Pinot gris grown on nine rootstocks was planted. The vines have been inoculated, with treatments: GLRaV-3, GLRaV-3 + GVA, GLRaV-1 + GVA, GLRaV-2 + GVB, no virus, no graft + no virus. PCR testing in 2024 was used to confirm successful virus inoculation. 2025 scoring for symptoms is yet to come. The research vineyard will continue to be maintained, and after the virus effect is determined, the research plan calls for inoculating with fungal pathogens to determine their potential role in SVC. 

FPS has been collaborating with other berry researchers on two USDA-APHIS PPA 7721 projects that are now in their third years. The first is an evaluation of HTS in place of biological indexing for Fragaria and Rubus. USDA-ARS Corvallis (Walt Mahaffee with OSU PhD Student Dan Fager) is responsible for grafting and index evaluation. FPS conducts the HTS sequencing, bioinformatic analysis, and curation of data. The preliminary results are showing HTS is superior to biological indexing. The second collaborative project is survey of strawberry viruses. In the first two year of the project, samples to survey viruses in nursery production were collected and tested by Oregon, California, and Arkansas. Year 3 of the project began September 1, and will focus on HTS of native Fragaria to determine if they may be a green bridge or reservoir for viruses infecting nursery fields. 

FPS has two research projects underway for development and testing of HiPlex PCR for virus detection. With funding from CDFA Pierce’s Disease Control Board, FPS is developing HiPlex assays for detection of multiple clades of grapevine red blotch virus and multiple variants of GLRaV-3. In the year 1 work, sensitivity and specificity of the assays are good. Further testing in year 2 will help clarify the read threshold. The second HiPlex project is funded by the California Strawberry Commission, and work has focused on detecting 16 strawberry viruses. This work is in year 2, and the final project months will be focused on refining the assays and finalizing the protocols so they can be shared with CDFA, to encourage their adoption of HiPlex for more efficient virus testing. HiPlex PCR shows a lot of promise, and FPS has applied for FY27 funding via the PPA7721 program to develop HiPlex assays for Vaccinium and Rubus. 

Agave is an up-and-coming crop in California, with much interest in production due to its drought tolerance. Viruses have not been well-studied in agave, and it is important to build an awareness of the pathogen transmission possibilities of vegetative propagation. As part of a CDFA-funded project looking at agave production systems, FPS is surveying agave plantings and using HTS to screen for viruses. From 10 samples tested to date, six known viral agents were detected and two new viruses were identified. 

The new federal funding year began on September 1, 2025, and FPS has three new projects starting. The first project is to optimize a new Qiagen +Kapa kit for HTS library preparation, using the Hamilton STAR liquidhandler. Most of us use Illumina for ribodepletion, but Illumina is not best for eliminating rRNA. QIAseq FastSelect-rRNA plant and KAPA Hyper-Prep kits (Qiagen + KAPA) may produce a greater percentage of virus reads than using the Illumina TruSeq stranded total RNA with Ribo-zero plant kit (Illumina). Before we switch, and request update of our SOPs, we need to do our homework. 

USDA-APHIS-PPCDL is coordinating an interlaboratory validation of HTS protocol for detection of plant viruses in berry crops. FPS is a participating lab, in addition to PPCDL and Arkansas Clean Plant Center. The samples for the first year of this project (with a second year planned for FY27) have been received. Libraries will be prepared, the samples will be sequenced, and bioinformatic analysis conducted. 

For sweetpotato, FPS is running a comparison of indexing to HTS+PCR. Typically, annual virus testing of sweetpotato is done by indexing to Ipomoea setosa. A test population will be selected from the virus positive and clean plant collections at FPS. The test population will be tested using the typical grafting to I. setosa, which are scored for virus symptoms and tested with PCR. Storage roots and leaf tissue from the test mothers will be sampled directly and tested using HTS and PCR. We will compare the test results generated from each testing method. This is anticipated to be a two-year project, requiring updating of RT-qPCR assays. 

Georgios Vidalakis, University of California, Riverside

In this report period, October 2024 – September 2025, we continued to support the suppression and eradication efforts against Huanglongbing (HLB) in California, where the number of positive trees has reached 10,183 and the HLB quarantine zones are expanding in the coastal and southern regions of the state. The University of California, Riverside, National Clean Plant Network (NCPN) Citrus Center, namely the Citrus Clonal Protection Program (CCPP) collaborated with the California Department of Food and Agriculture's (CDFA) Citrus Nursery Stock Pest Cleanliness Program and distributed 83,763 clean citrus propagation units (buds), tested for HLB as well as virus and viroid diseases, from 384 different citrus accessions, to 804 nurseries, producers, scientists, and the public, sourced from 1,192 pathogen-tested citrus budwood trees. The CCPP also tested 78 citrus introductions from 7 countries with 2,424 diagnostic tests intercepting 11 different types of pathogens in 30 introductions (38.5%), including HLB, viruses and viroids and performed pathogen elimination/therapy on 83 citrus accessions with 727 tissue cultures. The CCPP maintained 574 inquiries under quarantine and performed 1, 846 laboratory and 5,284 biological pathogen detection tests for HLB, virus and viroid diseases resulting in the release from quarantine of 50 citrus accessions. 

In collaborative efforts with WERA 20 members and experts in USA, and other citrus producing countries, we continued the development and validation of e-probes for the detection of graft-transmissible pathogens of citrus; evaluated duplex qPCR interference from common citrus endogenous standards (COX/MDH) and designed a synthetic internal standard to mitigate competition during reactions; sequenced and analyzed citrus viroid VII populations from multiple hosts/locations to assess genetic diversity and variant structure for the development of detection assays; designed and 3D-printed a palm-sized, field-deployable device for rapid citrus tissue lysis and nucleic-acid extraction; and identified stress-specific signaling patterns and candidate regulators linked to herbivory vs. pathogen pressure. 

WERA 20 members, presented their work (presentations, keynote and invited speakers) at the 21st Panhellenic Phytopathological Conference, 23rd Conference of the International Organization of Citrus Virologists, 2025 International Citrus Congress, World Ag Expo, Conference of the Mediterranean Phytopathological Union, Plant Health, Conference of the International Society of Citrus Nurseries, and the 2025 International Conference on Viroids with participation of hundreds of scientists, regulators and growers from around the world. 

Ioannis Tzanetakis, University of Arkansas

The Arkansas program continued to focus on advancing plant virus diagnostics, infectious clone development, and vector–virus interaction studies across berry crops, rose, and other specialty crops. Highlights include:

Work on strawberry pseudo mild yellow edge virus (SPMYEV) led to the complete genome characterization of the U.S. isolate using HTS. An infectious clone was developed, and Koch’s postulates were validated through agroinfiltration and dodder transmission assays in Fragaria vesca. In addition, PCR-based diagnostics were established to address a regulatory gap for this long-suspected “phantom” agent. 

The blackberry chlorotic ringspot virus (BCRV), infectious clone was employed to study virus–host dynamics in Rubus. Comparative studies between ‘Munger’ black raspberry and ‘Natchez’ blackberry revealed strong host-dependent differences in viral accumulation patterns. Quantitative assays demonstrated consistently high levels of virus in ‘Munger’, while detection in ‘Natchez’ was more variable and significantly lower, providing insights into the reliability of indicator plants. 

Studies on blueberry scorch virus (BlScV) and blueberry virus S (BluVS) involved comparative analyses using ELISA, PCR, and HTS. The results highlighted population diversity in BlScV and confirmed BluVS as a distinct and potentially significant pathogen of blueberry. To strengthen diagnostic capacity, a triplex endpoint PCR and duplex quantitative PCR assay targeting BlScV and BluVS was developed incorporating internal controls for reliable detection. Also we developed a ViMAPC to assist other laboratories that may not have the positive controls needed to run the tests. 

In the continued clean plant initiatives and in collaboration with many WERA-20 members, we have continued to streamline virus diagnostics and germplasm exchange. The phantom agent paper based on national and international collaborations addressing phantom agents and advancing clean plant programs was published in April 2025. 

Alexander V. Karasev, University of Idaho

The virome of grapevines grown in the State of Idaho was continued to be characterized in 2020-2024, with the overall goal of developing diagnostic tools for virus and virus-like disorders in wine grapes. More than 360 leaf and petiole samples were collected from symptomatic grapevines in 10 vineyards in Canyon and Nez Perce counties of Idaho and in Malheur County of Oregon and subjected to high-throughput sequencing (HTS) and RT-PCR testing. A new tri-partite, negative-sense RNA cogu-like virus was uncovered in grapevines from a 38-year-old ‘Chardonnay’ block in Idaho through HTS of total RNA, its presence was confirmed and validated by RT-PCR with specific primers designed based on the sequencing information obtained by HTS. The virus was named grapevine-associated cogu-like Idaho virus (GaCLIdV). In phylogenetic analysis based on the RdRP, GaCLIdV grouped within the family Phenuiviridae and was placed in a lineage of plant-infecting phenuiviruses as a sister clade of the genus Laulavirus, clustering most closely with switchgrass phenui-like virus 1 (SgPLV-1) and more distantly related to grapevine-associated cogu-like viruses from the Laulavirus and Coguvirus clades. The presence of GaCLIdV in the original ‘Chardonnay’ samples was confirmed by RT-PCR amplification and Sanger sequencing. This new virus was found in five wine grape cultivars and in six vineyards sampled in Idaho and in Oregon during the 2020–2024 seasons. 

Noma Chingandu, Canadian Food Inspection Agency (Centre for Plant Health)

The CFIA Centre for Plant Health (CPH) in North Saanich, BC, serves as Canada’s only post-entry quarantine facility for plant viruses and virus-like diseases. Recently modernized in 2024, the facility now includes advanced diagnostics and research laboratories, growth chambers, screenhouses, and bioassay field blocks. It supports virus testing for both certified and non-certified plant material, including imports, exports, and domestic breeding programs. The Centre also maintains and distributes Generation 1 virus-tested propagative stock for Canada’s Fruit Tree and Grapevine Export Program, with over 780 accessions shared globally. A key advancement in 2024 was the full integration of High Throughput Sequencing (HTS) into virus indexing workflows, significantly improving diagnostic speed and accuracy. HTS is expected to replace bioassays starting in 2028. 

Research at CPH focuses on developing and validating new diagnostic assays and supporting regulatory decisions. The Virus/Viroid Collection Bank (VVCB) project, led by Dr. Yahya Gaafar, aims to sequence and profile over 460 accessions infected with more than 80 viruses and viroids. Preliminary findings reveal high genetic variability and mixed infections, which are instrumental in refining HTS protocols and developing PCR-based confirmatory tests. Automation of HTS processes using robotics has drastically reduced sample preparation time and minimized contamination risks, enabling broader research into aspects like virus seasonality and genetic diversity. 

Cristian Olaya, Oregon Clean Plant Center, Oregon State University

Pathogen-free germplasm is vital to economical and sustainable production since a single clean mother plant can be propagated to 15,000 and 20 million Blackberry, Raspberry, Blueberry, or Strawberry plants over a five-year period. To ensure plants are free of pathogens, breeding material is tested and maintained by centers within the National Clean Plant Network (NCPN). One such center, the Oregon Clean Plant Center (OCPC) at USDA-ARS in Corvallis, OR, has supported the international berry industry by providing clean plants throughout the world for more than 20 years. Recently, the OCPC began a restructuring initiative in collaboration with Oregon State University’s Plant Clinic to enhance and expand its testing capacity and ability to further serve the industry. The center serves commercial and public stakeholders by offering pathogen testing for approximately 37 viruses in Rubus, 22 in Vaccinium, and around 20 in Fragaria, as well as for Phytoplasma and Xylella fastidiosa, using PCR, qPCR, ELISA, and grafting techniques. OCPC maintains and distributes over 200 different genotypes, including 99 patented cultivars (79 Rubus, 15 Fragaria, and 5 Vaccinium), 101 advanced selections (56 Rubus, 12 Fragaria, and 33 Vaccinium), and a collection of more than 150 virus-positive control plants. This partnership has allowed OCPC to serve more than 5 private stakeholders, four public breeders, updates standard operation procedures and testing methods, and curate the genotype collections. 

Christie Almeyda, North Carolina State University

The North Carolina Clean Center continues to clean and test mainly domestic materials from berry and muscadine grapes breeding programs in the Southeast. We currently served breeders in NC (Rubus/Fragaria/Vaccinium), AR (Rubus and muscadine grapes) and FL (Vaccinium/Rubus). We continue to provide services to industry on multiple capacities: diagnostics, graft indexing and cleanup of imported material. Deliverables include the maintenance of foundation plants (100 genotypes, Rubus, Vaccinium and Fragaria); maintenance of in vitro genotypes (200 genotypes); cleanup of imported genotypes and distribution since 2018 of 60 genotypes as clean stock.  While cleaning up berry crops, Blueberry latent virus (BBLV) is the main virus fond in blueberries. Blackberry yellow vein-associated virus (BYVaV) and Blackberry leaf mottle associated virus (BlMaV) were detected on blackberries. Since 2021, the MPRU has established a partnership with the NC Plant Disease and Insect Clinic (PDIC). Now NC growers can submit berry and grape samples to be tested for designated pathogens at the MPRU as the unit has expanded its diagnostic services. The NC Clean Center continues to work closely with other berry clean centers (OR, AR and CA) as well as USDA-APHIS regulators to discuss matters related to CIP management for successful release of imported material. 

A muscadine survey was conducted in 2024 aiming to identify viruses infecting the crop and to assess their incidence in NC. Samples were collected from 210 asymptomatic vines across 8 locations and virus tested at the NC Clean Plant Center. Vines were sampled in summer, June through August, and again after harvest, October and November. Due to the impact of Hurricane Helene, three vineyards (70 vines) could not be resampled in the fall. The cultivars sampled were Noble, Carlos, Regale, Supreme, Late Fry, Triumph, and Paulk. One to three blocks were sampled at each vineyard location. A block was considered a group of 10 to 40 rows consisting of the same cultivar. Ten vines per block were selected for sampling following a zig-zag schematic. All vines sampled were asymptomatic for viral diseases. Samples were delivered on the same day to the NC Clean Plant Center. Samples were stored at -80 C until total RNA was extracted. RT qPCR assays were performed on each sample to evaluate for the presence/absence for pathogens/viruses. Protocols for virus detection were adopted from Foundation Plant Services (FPS), UC-Davis. Muscadine vines were tested for grapevine leafroll associated virus complex (GLRaV-2, 3, 4, 7), grapevine red blotch virus (GRBV), grapevine virus A and B (GVA and GVB), grapevine rupestris stem pitting-associated virus (GRSPaV), tobacco ringspot virus (TRSV) and grapevine Syrah virus-1 (GSyV-1). Amplicons were generated by conventional RT-PCR and send to sequence. Preliminary results revealed 39% incidence of GLRaV-2 and 23% incidence of GVB. Sequenced amplicons showed 91% identity to the GVB replicase and 97% to 99% identify to the GLRaV-2 coat protein. This is the first time viruses were reported to be present on NC muscadine vineyards. As a next step, an additional round of muscadine virus testing is ongoing in 2025 and samples from 2024 and 2025 will be send to FPS for HTS analysis. 

Jing Zhou, University of Hawaii

Jing Zhou just started the plant virologist position at University of Hawaii in August 2025, a month prior to the 2025 WERA20 meeting. Since the previous plant virologist Dr. John Hu retired in 2023, continuous work related to WERA20 has barely been conducted, and there is a gap in the reporting period as a result. As the new plant virology laboratory is setting up and research focuses are established, we expect to start implementing work regarding viruses infecting fruits, woody ornamentals, and sweet potatoes very soon. We hope to share the new findings in the coming 2026 WERA20 meetings, which will be held in Hawaii presumably during the late spring to early summer. As the meeting host, we’ve started preparing for this annual meeting and we look forward to having our group next year in Hawaii! 

Mary Sutton, University of Georgia

Citrus is a new industry for the state of Georgia. As a result, many of the quarantine diseases have not yet found a foot hole in Georgia citrus. The current goal of the UGA citrus extension program is to increase grower awareness of such quarantine disease before they become established. In 2025, seven trainings were provided to provide citrus growers, county agents, landscapers, homeowners, and state plant protection inspectors, with the knowledge to identify symptoms of Huanglongbing (HLB), or citrus greening, and its vector, the Asian citrus psyllid (ACP). These training courses reached over 150 people. On average, attendees reported a 46% increase in knowledge following such meetings. On a scale of 1 to 10, attendees reported their concern regarding quarantine diseases rated an average of 6.8.  Together, this suggests that conducted meetings and trainings have been successful in increasing awareness of this disease and in increasing attendee knowledge in how to recognize the associated diseases and pests. 

Ekaterina Nikolaeva, Pennsylvania Department of Agriculture

Pennsylvania Department of Agriculture (PDA) safeguards PA agriculture and natural resources against the entry, establishment, and spread of economically and environmentally significant pests, and facilitates the safe trade of agricultural products. In 2024-2025, PDA in cooperation with Pennsylvania State University conducted state-wide exotic disease surveys of fruit trees (Malus and Prunus) and small fruits (strawberry and grapes) commodities. The targets of the Orchard survey were Potyvirus Plum pox virus, Apple Proliferation (Candidatus Phytoplasma mali), European stone fruit yellow (Ca. Phytoplasma prunorum), Jujube witches' broom (Ca. Phytoplasma ziziphi), Little cherry disease (Little cherry viruses 1 and 2), and Almond witches’ broom (Ca. Phytoplasma phoenicium). Small fruit survey included surveillance activities for Nepovirus Tomato black ring virus on strawberry, Australian Grapevine Yellows (Ca. Phytoplasma australiense), Flavescence Doreé Phytoplasma (Ca. Phytoplasma vitis), and Bois noir Phytoplasma (Ca. Phytoplasma solani) on grapes. A total of 2,919 Prunus samples were collected and tested for PPV. Additionally, 66 Malus, 78 Prunus, 52 Strawberry and 99 Grape samples were collected and tested according to USDA PPQ approved protocols. No exotic targets were detected. 

PDA continues to operate the Fruit Tree Improvement Program (FTIP), specialized inspection and virus testing program for participating PA fruit tree nurseries. In 2024, 4,459 total samples were tested, representing 15,611 trees. A total of 3,481 Prunus samples were processed through the FTIP laboratory this year for Plum pox virus (PPV), Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), and Tomato ringspot virus (ToRSV).  In result, PNRSV and PDV were the most commonly found viruses in the FTIP. Some samples were positive for multiple viruses. No PPV was detected. 

Apple samples were collected from budwood source blocks. In total, 522 samples were tested for apple mosaic virus (ApMV), apple chlorotic leafspot virus (ACLSV), apple stem pitting virus (ASPV), apple stem grooving virus (ASGV), tobacco ringspot virus (TRSV), and tomato ringspot virus (ToRSV). Sixteen samples were determined positive: eight samples for ACLSV, four samples for ASGV, and four samples for ToRSV. 

Two cherry budwood blocks were inspected for cherry leaf roll virus (CLRV) with 19 samples collected and tested. All samples tested negative for CLRV. Phytoplasma inspections were conducted at twelve blocks consisting of registered source, common source budwood, and nursery blocks. Eighty-five samples were collected and tested. All samples were found negative.

A total of 288 broadleaf weed samples were collected and tested for ToRSV. Twenty-eight soil samples were collected from current or future source blocks and proposed sites for nursery production to determine the presence of Xiphinema sp.  nematodes. ToRSV was detected in two broadleaf weed samples. Xiphinema sp. nematodes were present at low but detectable levels in budwood source locations and proposed sites for nursery production. Presence of dagger nematodes makes broadleaf weed control imperative to prevent transmission from virus-positive weeds to fruit trees. 

In 2024, we implemented confirmatory testing for FTIP ELISA positive and inconclusive results. Total RNA was extracted using Qiagen kits and amplified in qRT-PCR protocols with primers specific to ASPV, ASGV, ToRSV, PNRSV and PDV (Li et al., 2018; Osman et al., 2014; Beaver-Kanya et al., 2019). Results confirmed by PCR were reported to growers to avoid using positive trees as grafting material. 

In 2024, PDA in collaboration with PSU (Dr. K. Peter) and USDA APHIS PPQ Plant Germplasm Quarantine Program (PGQP) (Dr. O. Hurtado Gonzales) has started work on implementation of HiPlex technology in FTIP program. Our current virus detection workflow relies on DAS ELISA for 5 viruses and requires the use of two incompatible commercial kits (Agdia and Bioreba), resulting in duplication of the sample grinding step without possibilities to streamline the entire process. In addition, these protocols require significant time and labor to process the volume of samples necessary to adequately screen nursery plant material, not to mention that ELISA detection protocols are known to produce false positive and false negative results. In 2024-2025, we started work on validating a novel approach (HiPlex) that combines a single-step PCR and HTS for the simultaneous detection of 22 viruses and five viroids known to infect apple trees across hundreds of samples. This approach offers several advantages over traditional methods such as low amounts of cDNA input, streamline workflow from PCR to sequencing, simple data analysis and simultaneous detection of multiple viruses in multiple samples in a single sequencing run, which significantly reduces turnaround time for diagnostic results. Evaluation of this technique will help prepare the PA certification program to offer a more rapid and early detection for fruit growers and spearhead the potential transformation of diagnostics across other states by implementing simultaneous virus and viroid diagnostics and moving away from time-consuming DAS ELISA-based methods.

Abou Kubaa R, Ouro-Djobo A, Stevens KA, Alabi OJ, Al Rwahnih M. 2025. Genome characterization of prunus maculavirus 1 (PrMcV-1), a novel member of the genus maculavirus identified in prunus spp. Archives of virology, 170(8): 168. 

Ault, N., Ren, S., Payne, D.  Li, Y., Srinivasan, A., Zheng, Y., Sunkar, R. and Naidu, R. A. 2025. Dynamics of small RNAs in a red-fruited wine grape cultivar infected with Grapevine red blotch virus. BMC Genomics 26: 417. 

Al Rwahnih M, Klaassen V, Erickson T, Alabi OJ, Stevens K, Hwang MS, Port L. 2025. A New Era in Federal Quarantine and State Certification Diagnostics at Clean Plant Centers in the United States. Plant disease, 109(7): 1392-1403. 

Chambers, G. A., Geering, A. D. W., Holford, P., Kehoe, M. A., Vidalakis, G., & Donovan, N. J. 2025. Genetic diversity of citrus viroid VII (CVd-VII). Archives of Virology, 170:12. https://doi.org/10.1007/s00705-024-06191-4 

Cifuentes R., Brito M.L., Cornejo-Franco J.F., Alvarez-Quinto R.A., Mollov D., Martínez A., Ochoa J., Villamor D.E., Tzanetakis I.E., Quito-Avila D.F. 2025. Insights into the virome of the Andean blackberry (Rubus glaucus). Eur. J. Plant Pathol. 173:197–208. https://doi.org/10.1007/s10658-025-03058-5 

Costanzo, S., Jones, T., Peter, K., Nikolaeva^, E. 2025. First Report of ‘Candidatus Phytoplasma fraxini’-Related Strain Associated with Peach Yellows in Pennsylvania. Plant Disease, https://doi.org/10.1094/PDIS-06-25-1240-PDN 

Dahan, J., Orellana, G.E., Reyes-Proano, E., Lee, J., and Karasev, A.V. 2025. A novel cogu-like virus identified in wine grapes. Viruses 17 (9): 1175 (https://doi.org/10.3390/v17091175). 

de Souza JO, Klaassen V, Stevens K, Erickson TM, Heinitz C, Al Rwahnih M. 2024. Characterization of Genetic Diversity in the Capsid Protein Gene of Grapevine Fleck Virus and Development of a New Real-Time RT-PCR Assay. Viruses, 16(9). 

Druciarek T., Tzanetakis I.E. 2025. Invisible vectors, visible impact: The role of eriophyoid mites in emaravirus disease dynamics. Virology 606:110478. https://doi.org/10.1016/j.virol.2025.110478 

Fuchs, M., Rwahnih, M. A., Blouin, A. G., Burger, J., Chooi, K.M., Constable, F., Ertunc, F., Fiore, N., Habili, N., Hily, J.-M., Katis, N., Lemaire, O., Maliogka, V. I., Maree, H. J., Minafra, A., Naidu, R.A, Pietersen, G., Saldarelli, P., Schmidt, A.-M., Music. M. Š., Várallyay, É. 2025. A list of eclectic viruses, virus-like diseases and viroids of grapevines that should not be considered for regulatory oversight: a global plea from virologists. Journal of Plant Pathology, 107(2): 847-858. 

Fust C, Lameront P, Shabanian M, Song Y, Abou Kubaa R, Bester R, Maree HJ, Al Rwahnih M, Meng B. 2025. Grapevine leafroll-associated virus 3: a global threat to grapevine and wine industries but a gold mine for scientific discovery. Journal of experimental botany, 76(11): 2985-3000. 

Haegeman A, Foucart Y, De Jonghe K, Goedefroit T, Al Rwahnih M, Boonham N, Candresse T, Gaafar YZA, Hurtado-Gonzales OP, Kogej Zwitter Z, Kutnjak D, Lamovšek J, Lefebvre M, Malapi M, Mavri# Pleško I, Önder S, Reynard JS, Salavert Pamblanco F, Schumpp O, Stevens K, Pal C, Tamisier L, Uluba# Serçe Ç, van Duivenbode I, Waite DW, Hu X, Ziebell H, Massart S. 2024. Correction: Haegeman et al. Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests. Plants 2023, 12, 2139. Plants (Basel, Switzerland), 13(5). 

Hajizadeh M., Ghaderi Zandan N., Koloniuk I., Sierra-Mejia A., Tzanetakis I.E. 2025. Characterization, detection, and prevalence of a novel strawberry crinivirus. Plant Dis. 109:988–991. 

Hardigan, M. A., Finn, C. E., Jones, P. A., Strik, B. C., Peterson, M. E., Bassil, N. V., King, R. M., Wiegand, Z. J., Olaya, C., Martin, R. R., Lee, J., & Lukas, S. B. (2025). ‘Thunderhead’ Erect Primocane Fruiting Blackberry. HortScience, 60(8), 1366–1371. https://doi.org/10.21273/HORTSCI18617-25 

Harper, S, Molnar, C, Nikolaeva, E, Jones, T, and Peter, K. 2024. Draft Genome Sequence of North American Grapevine Yellows phytoplasma strain PDA15. Microbiology. https://mra.msubmit.net/cgi-bin/main.plex?el=A6Nv7CVdN1A3GPKl2F2A9ftdN1dYz2Xow9S7CzQtabJGgZ 

Larrea-Sarmiento AE, Galanti R, Olmedo-Velarde A, Wang X, Al Rwahnih M, Borth W, Lutgen H, Fitch MM, Sugano J, Sewake K, Suzuki J, Wall MM, Melzer M, Hu J. 2024. Characterization of Two Novel Viruses Within a Complex Virome from Flowering Ginger in Hawaii. Plant disease, 108(10): 3001-3009. 

Lavagi-Craddock, I., El-Kereamy, A., Hajeri, S., Lovatt, C. and Vidalakis, G. 2025. Dwarfing of commercial citrus varieties using TsnRNAs: Evaluation of yield, size and tree care from the 1990s commercial and university field trials. Citrograph. Vol. 16:4, Fall 2025 p.32–36. https://citrusresearch.org/citrograph/archive 

Liu, C.-W., Kalish, B., Bodaghi, S., Vidalakis, G., & Tsutsui, H. 2025. A 3D-printed handheld device for quick citrus tissue lysis and nucleic-acid extraction. Advances in Sample Preparation. https://doi.org/10.1016/j.sampre.2025.100199 

Manoharan, B., Qi, S.-S., Vidalakis, G., El-Kereamy, A., Satheesh, V., Elango, D., Dhandapani, V., Dai, Z.-C., & Du, D.-L. (2025). Roles of hormone signaling on defense responses of invasive Sphagneticola trilobata to pathogen and insect herbivore. Physiological and Molecular Plant Pathology. (2025): 102722. https://doi.org/10.1016/j.pmpp.2025.102722 

Mitra, A., Jarugula, S. and Naidu, R.A. 2025. Development of a minireplicon for Grapevine leafroll-associated virus 1 and genetic analyses of sequences in the 5ʹ non-translated region required for replication. Phytopathology 115: 1065-1075. 

Mohammed, Mohammed S., Lahuf, Adnan Abdaljeleel, Jeddoa, Zuhair M., de Souza, Juliana Osse, Al-Rwahnih, Maher. 2025. Survey and high throughput sequencing revealed mixed infections of cucurbit-infecting viruses in zucchini fields in Iraq. Tropical Plant Pathology, 50(1): 67. 

Molnar C., M.K. Shires, A.A. Wright, M.C. Hoskins, S.J. Cowell, E.V. Nikolaeva, R. Knier, M.T. Nouri, B. Black, S.J. Harper. 2024. Putting ‘X’ into context: the diversity of ‘Candidatus Phytoplasma pruni’ strains associated with the induction of X-disease. Plant Dis. 108 (9). 

Nascimento, D. M., Bodaghi, S., Wang, H., Ribeiro-Junior, M. R., Campos, R., Dang, T., Osman, F., Habiger, J., Espindola, A. S., Vidalakis, G., & Cardwell, K. F. 2025. Development and validation of a suite of e-probes for Electronic Diagnostic Nucleic Acid Analysis (EDNA) for 20 graft-transmissible pathogens of citrus using MiFi and blind ring testing among novice users. PhytoFrontiers, 5, 243–253. https://doi.org/10.1094/PHYTOFR-12-24-0140-FI  

Neugebauer, Kerri A., Gillett, Jerri M., Klaassen, Vicki, Miles, Laura A., Rwahnih, Maher Al, Miles, Timothy D. 2025. Occurrence of Grapevine Viruses in Different Cultivars and Regions Within Michigan. Plant Health Progress, 26(2): 155-160. 

Olaya, C., Ohkura, M., Lake A, and Mahaffee, W. 2025. Oregon Clean Plant Center: supporting the berries industry in partnership with the OSU Plant Clinic. Annual Meeting of the American Phytopathological Society, Honolulu, Hawaii. August 2-5, 2025. 

Olaya, C., Reinhold, L., Platt, McK., Peetz, A., Donahue, K., Zasada, I. 2024. Assessment of the distribution of Xiphinema spp and associated nepoviruses in the Pacific Northwest small fruits. https://doi.org/10.1094/PHP-04-24-0034-RS 

Ouro-Djobo A, Obasa K, Oladokun JO, Sétamou M, Al Rwahnih M, Alabi OJ. 2025. Relative occurrence and seasonal variations of wheat-infecting viruses in Texas. Plant disease. 

Osse de Souza J, Erickson TM, Stoddard CS, Almeyda CV, Al Rwahnih M. 2025. Evidence of Rapid Infection of Four Sweetpotato Potyviruses in a Commercial Field in California. Plant disease, 109(2): 308-312. 

Reyes-Proano, E., Knerr, J., Karasev, A.V. 2024. Characterization of birch toti-like virus infecting ornamental European birch. (Abstr.) Phytopathology 114: S1.82. https://doi.org/10.1094/PHYTO-114-11-S1.1. 

Phillips, J., Bodaghi, S., Vidalakis, G., & Blaha, G. 2025. Optimizing qPCR detection of ‘Candidatus Liberibacter asiaticus’: Introducing a new type of internal standard. Plant Disease. https://doi.org/10.1094/PDIS-12-24-2714-RE  

Shires M. K., Molnar, C. Cowell, S. J. Bishop, G. Voelker, J. Thompson, A. A. Nikolaeva, E. Copp, C. Flandermeyer, L. Melton, T. Northfield, T. D. Marshall, A. T. Cooper W. R., Harper S. J. 2025. Alternative Hosts of ‘Candidatus Phytoplasma pruni’ Identified Through Surveys and Vector Gut Content Analysis. Plant Health Progress, 26 (201). 

Sierra-Mejia A., Hajizadeh M., Atanda H.Y., Tzanetakis I.E. 2025. Overcoming the woody barrier: Dodder enables efficient transfer of infectious clones to woody plants. J. Virol. Methods 334:115114. https://doi.org/10.1016/j.jviromet.2025.115114 

Sierra-Mejia A., Villamor D.V.V., Rocha A., Wintermantel W.M., Tzanetakis I.E. 2024. Engineering a robust infectious clone and gene silencing vector from blackberry yellow vein associated virus. Virus Res. 350:199488. https://doi.org/10.1016/j.virusres.2024.199488 

Sierra-Mejia A., Villamor D.V.V., Tzanetakis I.E. 2024. Development and application of an infectious clone and gene silencing vector derived from blackberry chlorotic ringspot virus. Virus Res. 350:199460. https://doi.org/10.1016/j.virusres.2024.199460 

Singh S., Villamor D.V.V., Sharma Poudyal D., Sierra-Mejia A., Tzanetakis I.E. 2025. A systems-based approach to ensure berry crops health status: from the breeder to the field. Eur. J. Plant Pathol. 172:55–73. https://doi.org/10.1007/s10658-024-02985-z 

Stevens KA, Al Rwahnih M. 2024. High-Throughput Sequencing for the Detection of Viruses in Grapevine: Performance Analysis and Best Practices. Viruses, 16(12). 

Tzanetakis I.E., Aknadibossian V., Špak J., Constable F., Harper S.J., Hammond J., Candresse T., Folimonova S.Y., Freitas-Astúa J., Fuchs M., Jelkmann W., Maliogka V.I., Marais A., Martin R.R., Mollov D., Vidalakis G. and another 170 authors. 2025. Streamlining global germplasm exchange: Integrating scientific rigor and common sense to exclude phantom agents from regulation. Plant Dis. 109:736–755. https://doi.org/10.1094/PDIS-04-24-0745-FE 

Vidalakis, G. 2025. The Citrus Clonal Protection Program: Protecting California’s citriculture. Citrograph. Vol. 16:2, Spring 2025 p.50–54. https://citrusresearch.org/citrograph/archive 

Whitfield, A.E., Karasev, A.V., Alabi, O.J., Batuman, O., Cieniewicz, E.J., Fall, M.L., Jacobson, A.L., Pelz-Stelinski, K., Qiu, W., Rayapati, N.A., Reitz, S.R., Turpen, T.H. 2025. Advancing Vineyard Health: Insights and Innovations for Combating Grapevine Red Blotch and Leafroll Diseases. Consensus Study Report, 269 pp. National Academies of Sciences, Engineering, and Medicine. Washington, DC: The National Academies Press. https://doi.org/10.17226/27472.