Brief Summary of Minutes of Annual Meeting

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).

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. 

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. https://doi.org/10.1094/pdis-09-23-1963-re

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). https://doi.org/10.5070/c410161176

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). https://doi.org/10.5070/c411263792

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. https://citrusresearch.org/citrograph/archive

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. https://doi.org/10.1007/s13313-023-00903-1

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 (https://doi.org/10.3390/v15061347).

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 (https://doi.org/10.1094/PDIS-01-24-0034-PDN).  

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. https://iocv.ucr.edu/sites/default/files/2024-01/iocv_news_letter_202307.pdf

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   https://doi.org/10.1016/j.meegid.2023.105538

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. https://doi.org/10.1094/PDIS-06-23-1072-SR

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 https://doi.org/10.1016/j.virol.2024.109991

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. https://doi.org/10.3390/su16146070

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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. https://doi.org/10.3390/chemosensors12060105

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 DOI10.17660/ActaHortic.2024.1388.54

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. https://citrusresearch.org/citrograph/archive

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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 (https://doi.org/10.1007/s00705-024-06067-7).

Singh, S., Stainton, D. and Tzanetakis, I.E. 2024. Development of rapid and affordable virus-mimicking artificial positive controls. Plant Disease 108: 30-34 https://doi.org/10.1094/PDIS-06-23-1072-SR

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  DOI 10.17660/ActaHortic.2024.1388.32

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). https://doi.org/10.5070/c411262591

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. https://citrusresearch.org/citrograph/archive.

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.