Brief Summary of Minutes of Annual Meeting

For the meeting agenda and a photo of the participants on the Zoom call, see the attachment at: https://www.nimss.org/projects/attachment/18819

The multi-state WDC_WERA_TEMP_20 project “Management of Diseases Caused by Systemic Pathogens in Temperate and Sub-Tropical Fruit Crops and Woody Ornamentals” organized virtual annual meeting during April 8^th and 12^th, 2022. The meeting was hosted by Dr. Ioannis Tzanetakis, Professor of Plant Virology and Director, Arkansas Clean Plant Center at the Department of Entomology & Plant Pathology, University of Arkansas, Fayetteville, AR 72701. Dr. Jean-Francois Meullenet, University of Arkansas System Associate VP of the Division of Agriculture, welcomed the attendees followed by a brief overview of the Arkansas Clean Plant Center and the role of research and extension and partnerships between institutions in agricultural sustainability. 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. Dr. Naidu Rayapati, Administrative Advisor from Washington State University, provided a brief account of the WDC_WERA_TEMP_20 project that needs to be revised based on the comments received from the Multistate Research Committee (MRC) of the National Information Management and Support System (NIMSS). Subsequently, there was a Q&A session of the status of the new five-year proposal WERA_TEMP_20 “Management of Diseases Caused by Systemic Pathogens in Fruit Crops and Woody Ornamentals” submitted to Western Association of Agricultural Experiment Station Directors in January 2022. Subsequently, the Western Region’s MRC provided feedback on the proposal and, in view of the importance of the project, recommended for resubmission with appropriate revisions suggested by the MRC and peer-reviewers. Consequently, the proposal is designated as “Western Development Committee” to reflect that the proposal is “Under Review” until MRC approves the project. Based on MRC’s comments, the group discussed critical elements needed for revising the proposal. The writing committee of the project will address the MRC’s comments and concerns and submit a revised version for consideration by the MRC. The project committee has until September 30 to receive final approval of the project. The future venue for the annual meeting in 2023 will be Beltsville, MD, subject to final approval of the project by Western Association of Agricultural Experiment Station Directors. As a special invitee, Dr.  Bret Hess, Executive Director, Western Association of Agricultural Experiment Station Directors, explained the process for becoming an official participant of the multi-state project using the NIMSS system (http://www.nimss.org) and encouraged participants to contact him to assist throughout the process. In addition to research and extension faculty from Land-grant universities, scientists from federal programs (USDA-ARS, USDA-APHIS, etc.), personnel from State Departments of Agriculture, private sector and industry stakeholders can also become official participants of the multi-state projects through NIMSS system by submitting the APPENDIX E through proper channels into the NIMSS system.

An outline of topics presented by participants:

1. Reports:

Oscar Hurtado-Gonzales, USDA APHIS PPQ, Field Ops, Plant Germplasm Quarantine Center

• PGQP status report for fruit trees: new protocols agreed amongst all interested parties to reliably test fruit tree pathogen yet minimizing the time between receiving and releasing material
• Virus diagnostics, new approaches have been implemented using Hi-Plex amplification; allowing for the simultaneous detection of tens of targeted pathogens in a single reaction

Christie Almeyda, North Carolina State University

• Several berry and grape viruses were detected in North Carolina including blueberry scorch and strawberry latent ringspot
• Review of testing capabilities for berries and new virus findings
• Results of field surveys and testing results

Shulu Zhang, Agdia Inc.

• Updates on AmplifyRP and the detection of caulimoviruses in dahlias

Jennifer Nicholson, NCPN coordinator

• National Clean Plant Network funding update and future opportunities
• In depth analyses of the funding for each crop and the special initiatives including economics and education and outreach

Cindy Cooper WSDA

• An update from the National Certification Standards Working Group that included updates on the certification efforts on berries, fruit trees, grapevine and hops from several states across the nation

 Yilmaz Balci and Sage Thompson, USDA APHIS PPQ

• Diagnostic standards for Germplasm under Controlled Import Permit
• Overview and updates to import requirements of fruit trees
• Review of how plants enter the United States
• General discussions on how to mainstream the CIP application process

  Adriana Larrea-Sarmiento, University of Hawaii at Manoa 

• Viral genetic diversity on Ananas germplasm
• Studies on endemic and exotic viruses affecting pineapple
• The pineapple virome is expanding rapidly with viruses found in mixed infections

Alexander Karasev, University of Idaho

• New recombinants of potato virus Y found in tamarillo (Solanum betaceum)
• Data on the virome of an important crop in South America
• PVY recombinants are present in the crop making tamarillo a potential diversity hotspot for the virus

Segun Akinbade, Washington State Department of Agriculture

• Introduction of a new WERA-20 member
• Potential threat of nepoviruses to fruit tree crops in Washington State; epidemiological data on remerging viruses affecting those crops

Irene Lavagi-Craddock, Fatima Osman, University of California 

• Update on NCPN QC initiative. The framework has been approved and a final document will be provided to the stakeholders in May 

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

• Inter-laboratory validation of virus detection on fruit trees using high-throughput sequencing
• Ring tests provided valuable data that can be used to expedite the release time of propagation material

Tobiasz Druciarek, University of Arkansas

• Information on Rose rosette disease with information on additional vectors and possible biocontrol agents; entomopathogenic fungi and predatory mites

Vicki Klaassen, FPS

• Update on the prevalence of grapevine red blotch virus at Russell Ranch Vineyard. The virus is spreading rapidly in Russell Ranch Vineyard whereas movement in the old foundation is relatively slow.

Ioannis Tzanetakis, University of Arkansas

• An update on the phantom agents initiative. Scientists from all over the world are compiling a list of all phantom agents in berries, citrus, fruit trees, grapevine and rose. The first draft will be circulated to the broader community in Fall 2022.

Libby Cieniewicz, Clemson University  

• Research focusing on the ecology of prunus necrotic ringspot virus in southeastern U.S. peach production
• Findings were quite unexpected in some foundation material as infection rate reached 97% in one case   

Scott Harper, Washington State University   

• X-Disease epidemiology: genotypes, vectors and alternate hosts 
• The disease has become the No 1 problem for the cherry industry. The group is working on understanding the phytoplasma dynamics, its vectors and alternative hosts among the weeds found in commercial fields                                                                                            

Alejandro Olmedo-Velarde, University of Hawaii at Manoa

• A reverse genetics system of HGSV2 has been successfully developed and used in experiments involving experimental and natural hosts

Georgios Vidalakis and Stacey Comstock, University of California Riverside

• An update on the CCCP activities
• Preliminary results on citrus indicator reactions to different pathogens using different light intensities and wavelengths

Ekaterina Nikolaeva (Pennsylvania Department of Agriculture)

In 2021, PDA in cooperation with PSU conducted surveys for exotic diseases in orchards and small fruits. Orchard survey targets included Potyvirus Plum pox virus, Asian Pear Blight (Erwinia pyrifoliae), Apple Proliferation (Candidatus Phytoplasma mali), European stone fruit yellow (Ca. Phytoplasma prunorum), Little cherry disease (Little cherry virus 1 and 2), and Almond witches’ broom (Ca. Phytoplasma phoenicium). Small fruit survey targeted Asian pear blight (Erwinia pyrifolia), Nepovirus Tomato black ring virus, Australian Grapevine Yellows (Ca. Phytoplasma australiense), Flavescence Doreé Phytoplasma (Ca. Phytoplasma vitis), and Bois noir Phytoplasma (Ca. Phytoplasma solani). No exotic targets were detected.

PDA continues to operate the Fruit Tree Improvement Program (FTIP), specialized inspection and virus testing program for participating Pennsylvania fruit tree nurseries. All stone fruit nursery material this year was tested for Prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), tomato ringspot virus (ToRSV), and plum pox virus (PPV). A total of 2,297 Prunus samples were processed through the PDA laboratory in 2021, which included samples from registered and common budwood production blocks, registered seed blocks, and certified nursery stock blocks. No PPV was detected in rootstock blocks or in registered source blocks. All blocks met virus-testing requirements for FTIP certification. Very low levels of PDV were detected in nursery stock and common budwood production blocks. ToRSV was only detected in broadleaf weeds found in nursery stock blocks or in common budwood production blocks. PNRSV remains the most commonly found viruses in Prunus in Pennsylvania, although finds in registered source blocks, common budwood production blocks, and nursery production blocks are rare. All virus-positive trees in registered source blocks have been removed.

In 2021, PDA and PSU in cooperation with team continued work on evaluation of known pome virus and viroid distribution on apple trees affected by Rapid Apple Decline (RAD). Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Tobacco Ring Spot Virus (TRSV), Tomato ringspot virus (ToRSV), Apple mosaic virus (ApMV), Apple Luteovirus 1 (ALV1), Citrus Concave Gum-associated Virus (CCGaV), Apple Hammerhead Viroid (AHVd), and Apple scar skin viroid (ASSVd) were included in the investigation. A total of 1,228 samples were collected from nine orchards located in three PA counties. In result, the most common viruses detected on RAD affected trees were ALV-1, ASGV, CCGaV, ASPV, ACLSV, and ToRSV.

Maher Al Rwahnih (University of California, Davis)

 At Foundation Plant Services (FPS), 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. In 2021, the use of HTS played a key role in the US Department of Agriculture-Animal and Plant Health Inspection Service, Plant Protection and Quarantine (USDA-APHIS-PPQ) and California Department of Food and Agriculture (CDFA) approval of FPS’ revised diagnostic testing protocol that replaces biological indexing with a combination of HTS and PCR testing for release of plant material. These approvals are based on years of side-by-side studies comparing HTS analysis to biological indexing which revealed that the performance of the biological indicators was inferior redundant of PCR and HTS testing. FPS scientists have conducted side-by-side studies comparing the efficacy of woody and herbaceous indexing to PCR and HTS testing in Vitis and Prunus diagnostics. The results of these studies indicate that biological indicators give false negative results a significant percentage of the time and do not provide sufficient sensitivity in detecting target viruses or unknown viral pathogens. Similar results were obtained in studies conducted by other collaborators on different crops. Although there are slight variations between the Vitis and Prunus protocols per the horticultural needs of each crop, the length of time from introduction to release of material using the revised protocol is approximately 18-24 months.

FPS has also 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 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. It remains to be determined whether these GLRaV-3/GVA co-infected but asymptomatic vines will develop SVC in the future, suggesting that the missing factor for SVC symptom development is time from infection. We do know that in at least two blocks, GLRaV-3/GVA co-infections are spreading to newly planted certified material, indicating that new infections are occurring. This may also explain the high GLRaV-3/GVA infection rates outside SVC clusters. We will be monitoring these outside vines to determine if they develop SVC symptoms over the next two years. In addition to completing the field survey, we were able to propagate enough Pinot gris vines on nine different root stocks to graft-inoculate these vines this spring and begin monitoring SVC symptom development.

In addition, we are continuing our research on the epidemiology of grapevine red blotch virus (GRBV) which is currently not well understood. In particular, the identity of a primary vector and its role in spread of the virus under field conditions remain 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 this 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. Our current work also indicates that some unknown fraction of early infections are not detected by testing due to uneven GRBV distribution within-vines. If a vector is present and able to transmit GRBV from these vines, secondary spread will occur with the rate determined by factors that are not yet well characterized for GRBV. Our work provide insight on several additional factors. First, new GRBV infections can be detected within a year, as indicated by the first-year testing results for the sentinel vines. Sentinel vine testing also confirmed that GRBV distribution within these vines is uneven, making detection of early infections difficult. Second, analysis of the spatial distribution of GRBV positive vines indicates that the majority of GRBV infections are due to secondary spread within RRV, not as new introductions from the outside. In addition, spatial analysis suggests that spread is occurring via an insect that is more mobile than mealybugs. This could include all the current putative GRBV vectors. While we had hoped to gain insights on a possible vector from the yellow sticky cards, insect counts indicated that all the current suspected vector insects were present at RRV. It is interesting that Spissistilus festinus, the only confirmed GRBV insect vector peaked in September-November 2020, instead of mid-summer, which was the peak time reported in another CA vineyard. We will continue to monitor insects at RRV to determine if this later peak for S. festinus was specific to 2020 or if represents a consistent yearly pattern which could have important implications for the timing of controls measures and sampling for testing. Finally, there were higher Melanoliarus sp. numbers at RRV, but the significance is not clear. In other surveys of CA vineyards, Melanoliarus sp. has been present in very low numbers and only a low percentage have tested positive for GRBV. However, we are not discounting the possibility that this could be an important vector at RRV.

Robert Krueger, National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA

In collaboration with colleagues, we developed an improved reference gene for the sensitive detection of Candidatus Liberibacter asiaticus from plants in diagnostic duplex qPCR and analytical digital droplet PCR (ddPCR) assays. The mitochondrial cytochrome oxidase gene (COX), widely used as a reference, is not ideal because its high copy number can inhibit amplification of small quantities of target genes. The variable copy numbers of COX gene suggested the need for a non-variable, nuclear, low copy, universal reference gene for analysis of HLB hosts. The single-copy nuclear gene, malate dehydrogenase (MDH), developed here as a reference gene, is amenable to data normalization, suitable for duplex qPCR and ddPCR assays. The sequence of MDH fragment selected is conserved in most HLB hosts in the taxonomic group Aurantioideae.

Also, in collaboration with colleagues, we had previously developed a method for long-term preservation of clean stock citrus apical meristems under cryogenic conditions. During the timeframe in question, an additional 40 clean accessions were successfully cryopreserved. Forty-three accessions (over 100 plants) were regenerated to demonstrate trueness to type. In collaboration with a WERA-20 colleague, we will also demonstrate that the phytosanitary status of the cryopreserved meristems is not compromised.

As part of our citrus introduction and sanitation program, 17 accessions were released from State and Federal quarantine and are now available to the user public. 147 clean source accessions were distributed as budwood (1515 budsticks) and 144 accessions were distributed as seed (23,795 seeds) during this time period. These numbers are smaller than normal due to constraints imposed by the pandemic.

Also in collaboration with colleagues, we have implemented assays for phytoplasma and viroid pathogens of date palms.

Alexander Karasev (University of Idaho)

Tamarillo, or tree tomato (Solanum betaceum), is a perennial small tree or shrub species cultivated in subtropical areas for fresh fruit and juice production. In Ecuador, tamarillo orchards are affected by several viruses, with one previously identified as potato virus Y (PVY), however the specific strain composition of PVY in tamarillo was not determined. In 2015 and 2016, eight tamarillo plants exhibiting symptoms of leaf drop, mosaic, and mottled fruit were sampled near Tumbaco and Quito, Ecuador. These “tamarillo” PVY isolates were able to systemically infect tobacco, Nicotiana benthamiana, naranjilla, and tamarillo. Seven of the eight PVY isolates from tamarillo exhibited N-serotype, while one of the PVY isolates studied, Tam15, had no identifiable serotype. One isolate, Tam17, had N-serotype but produced asymptomatic systemic infection in tobacco. In tamarillo, four tamarillo isolates induced mosaic and slight growth retardation and were unable to systemically infect pepper or potato. Tamarillo, on the other hand, was unable to support systemic infection of PVY isolates belonging to the PVY^O and PVY^Eu-N strains. The whole genomes of eight PVY isolates were sequenced from a series of overlapping RT-PCR fragments. Phylogenetically, “tamarillo” PVY isolates were found to belong to the large PVY^N lineage, in a new, ‘Tamarillo’ clade. Recombination analysis revealed that these tamarillo PVY isolates represent at least three novel recombinant types not reported before. The combination of the biological and molecular properties found in these eight PVY isolates suggested the existence of a new, “tamarillo” strain of PVY which may have co-evolved with S. betaceum.

Shulu Zhang (Agdia Inc.)

Viruses and viroids can cause destructive diseases and great losses in economically important crops such as potatoes, grapevines, and cannabis. Agdia Inc., a leading plant diagnostics company, has been utilizing advanced diagnostic technologies to help crop growers and researchers to effectively detect the presence of plant pathogens in their crops and prevent potential crop losses from infection by pathogens. One such technology is recombinase polymerase amplification, a leading isothermal amplification technology, based on which Agdia Inc. has developed its own isothermal amplification platform – AmplifyRP^®. In recent several years, Agdia has developed and commercialized 31 AmplifyRP^® kits. Among them, 29 kits are capable of specifically detecting 28 different pathogens infecting a wide range of crops and 2 Discovery kits can be used for any pathogen. Over the past year, 3 AmplifyRP^® kits were commercialized for Beet curly top virus (BCTV), Fusarium oxysporum (Fo), Ralstonia solanacearum Race 3, Biovar 2 (RsR3B2). These three kits specifically detect BCTV, RsR2B2 or Fo and produce real-time, quantitative results. These test kits are deployable both in laboratories and in the field. They are simple to use, and no thermal cycler and DNA/RNA purification are needed as all reactions works well with plant crude extracts at a constant temperature 39-42°C. The whole assay from sample to result can be completed within 30 minutes and yet it is as sensitive as qPCR or PCR. This isothermal amplification technology AmplifyRP^® has provided a versatile detection tool for rapid detection of many important plant pathogens and helped growers to effectively manage crops and prevent significant economic losses from damages by pathogens. Agdia has also been providing diagnostic products and services to the WERA20 group and collaborating with many members of the group to develop diagnostic kits for numerous important crops such as cherries, grapevines, citrus, or peaches and effectively control/prevent diseases.   

Christie Almeyda (North Carolina State University)

The Micropropagation and Repository Unit (MPRU) at North Carolina State University (NC State) is currently one of the National Clean Plant Network (NCPN) centers that produces, maintains and distributes pathogen-tested G1 material of berry crops (strawberry, blackberry, raspberry and blueberry) and muscadine grapes to industry and researchers in the U.S. The MPRU presently conducts testing for targeted pathogens and therapy for pathogen elimination (heat treatment and meristem-tip culture) and maintains Fragaria, Rubus and Vaccinium G1 (foundation) blocks in vitro, in the greenhouse and the screenhouse. The same methods are applied for muscadine grapes.

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, one company; cleaning imported material, 3 companies). 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, Korean, Japan and Mexico.

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, GLRaV7), Grapevine red blotch virus (GRBV), Grapevine rupestris stem pitting associated virus (GRSPaV), vitiviruses (GVA, GVB), Tobacco Ring Spot Virus (TRSV), and Xyllela fastidiosa. Eighty samples were tested from 8 vineyards in NC (7 Vitis vinifera vineyards and 1 muscadine vineyard) in order to know the incidence of viral pathogens in this area during 2018, 2019 and 2020. GLRaV-3, GRBV and Xyllela fastidiosa were detected during the second year of this survey. GLRaV-3 and GRBV were the most predominant (20/80 each). Only 6 samples were positive for Xyllela fastidiosa. GLRaV-2, GLRaV-3 and GRBV were found in the third year. Grapevine red blotch virus, the causal agent of GRBD, was found in all sample areas and in 34.64% of vines. Grapevine leafroll-associated virus was found in only 12.5% of all samples, indicating that GRBV is more widely distributed in North Carolina than GLRaV. As we continue to collaborate with Dr. Hoffman in this survey, we are also working into cleaning and virus testing the material we currently have at the MPRU (10 NC muscadine cultivars) and new material (5 genotypes) we obtained from the AR breeding program in 2019. 

Scott Harper (Washington State University)

Over the past year our lab has been focused on understanding epidemiology of the causal agents of two very similar diseases affecting cherry and stone fruit production in the Pacific Northwest, Little cherry disease and X-disease. Using sequence, we developed a rapid high-resolution melt screening program to genotype the X-disease phytoplasma, which has enabled us to identify and track the movement of the pathogen in the Pacific Northwest over space and time. We have also identified an expanded alternate host range and vector species for the phytoplasma, which has allowed us to build a model of potential pathogen acquisition and transmission times, and which plant species are involved in the process across the growing season. We are also examining the effect of different cultural controls such as groundcover and orchard floor plant composition and have found that suppression or absence of key biennial weed species reduces overall leafhopper numbers and X-disease phytoplasma positive weeds and trees. We are translating this model into recommendations for growers to manage this disease more effectively and economically. Benefits of this approach include targeted and timed insecticide and herbicide applications to suppress key insect and plant species rather than heavy, broad-scale applications. This will reduce pesticide use and provide better disease control, with reduced impacts on non-target and beneficial species.

One area that I want to emphasize is my work as the lead pathologist of the joint WSU-OSU-USDA Little Cherry Disease task force. This group was established in 2019 in response to my initial research on this disease, and its purpose is to conduct research and extension to understand and develop methods to manage LCD and disseminate results and recommendations to the stakeholder base for implementation in the Pacific Northwest. The research that has and is being conducted by this group is a collaborative effort to understand and describe a pathosystem that has not been researched in detail since the Californian epidemic of the 1980s. I am a PI or co-PI on 12 past, current, or pending grants with other WSU, OSU, and USDA researchers studying topics from disease expression to host range, vector species and vector interaction, transmission and reinfection, and pathogen diversity. Our early collaborative efforts (and the seriousness of the disease) resulted in little cherry being established as a USDA priority area by Congress in 2021.  This led to a $2M in appropriation funds in 2022 for collaborative research between USDA-ARS and WSU on this disease.

We have also developed, validated and published several assays for detection of harmful pathogens in perennial crops, as well as two papers on Little cherry and X-disease phytoplasma biology and tropism, which will aid in optimizing detection and monitoring programs.

Adriana Larrea-Sarmiento, Alejandro Olmedo-Velarde, Michael Melzer, and John Hu (University of Hawaii)

Mealybug wilt of pineapple (MWP) is the most important viral disease affecting pineapple production worldwide. The disease has been associated with infection by members within the genus Ampelovirus, family Closteroviridae, known as pineapple mealybug wilt associated virus (PMWaV) complex. Four species PMWaV-1, PMWaV-2, PMWaV-3, and PMWaV-6 are now species of the PMWaV complex. The identification and molecular characterization of Pineapple secovirus A (PSV-A) [1] and Pineapple secovirus b (PSV-B) [2], both members within the subgenera cholivirus, genus Sadwavirus, were reported in 2020 and 2022, respectively. Analysis of high throughput sequencing (HTS) data from 24 germplasm accessions from the U.S. National Clonal Germplasm Repository (NCGR) in Hilo, Hawaii, and a public domain transcriptome shotgun assembly (TSA) identified two novel sadwaviruses, putatively named pineapple secovirus C (PSV-C) and pineapple secovirus D (PSV-D). These two putative viruses shared amino sequence identities below 80% of the Pro-Pol region compared with their homologs. Furthermore, we are reporting for the first time the complete genome pineapple bacilliform ER virus (PBERV) with 7,485 bp in length. Overall, we discovered a total of 69 viral sequences representing ten 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 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. Given the high occurrence of recombination events, diversity, and discovery of viruses found in Ananas germplasm, the reported and validated RT-PCR assays represent an importance advance for surveillance of viral infections of pineapple.

A reverse genetics system of HGSV2 infects experimental and natural hosts

Hibiscus green spot virus 2 is classified within the Kitaviridae family and genus Higrevirus.  In Hawaii, Hibiscus green spot virus 2 (HGSV2) infection causes leprosis-like symptoms in citrus, and green spot symptoms in Hibiscus spp., including H. arnottianus, a species native to Hawaii. Our group recently demonstrated the transmission of HGSV2 by Brevipalpus mites (Acari: Tenuipalpidae). A reverse genetic system for kitavirids has not been developed yet. This system would help us better understand the basic biology mechanisms of virus-host and virus-mite interactions. Therefore, a robust reverse genetic system of HGSV2 for Agrobacterium-mediated delivery into several natural and experimental hosts was developed. Experimental hosts included Phaseolus vulgaris (common bean), Nicotiana benthamiana, and N. tabacum while natural hosts included Citrus reticulata and H. arnottianus. The HGSV2 reverse genetics system successfully established infection in all the experimental and natural hosts tested with varying efficiency. The robustness and infectivity of the HGSV2 reverse genetics system were corroborated by virus purification and observation of recombinant bacilliform virions under a transmission electron microscope.

Robert Martin and Dimitre Mollov (USDA-ARS)

The program in Corvallis works closely with the University of Arkansas on characterizing novel viruses of berry crops and developing diagnostics.  The programs are working together on virus surveys, characterizing new viruses, and working with State Departments of Agriculture to incorporate new virus information into their certification programs.  Additionally, these two programs are working closely on evaluating High Throughput Sequencing as a suitable replacement for biological indexing, ELISA and PCR testing.  This is a multi-year project so that Vaccinium, Fragaria and Rubus plants with a range of viruses can be tested at multiple time points over two years to evaluate the reliability of HTS in different seasons.  This work is being done in a manner similar to HTS evaluations for Citrus, Grapes, Hops and Tree Fruits.  The other crops are being done by members of WERA-20 and the information on methodology, reliability is compared at the WERA-20 meetings and with APHIS.  The goal is to develop a standard set of SOPs for HTS that would be useful for these crops. Additionally, these two programs work together with the Berry Program at North Carolina State University and APHIS to coordinate the importation of new berry cultivars from overseas.

Stakeholder-oriented Virus database for NCPN crops.

The Corvallis program has taken the lead on developing a virus database for the NCPN crops.  The content development for the database is being done primarily by members of the WERA-20 group, based on the individual’s expertise: Berries (Martin USDA-ARS) and Tzanetakis (University of Arkansas), Citrus (Vidalakis – UC Riverside), Grapes and Roses (Al Rwahnih – UC Davis), Sweetpotato (Clark – Louisiana State University), Tree Fruit and Hops (Harper – Washington State University. The funding for the project is from NCPN.  The beta version of the database is at:  http://virusdb-dev2.westus2.cloudapp.azure.com/

The final version will have updated logos and will be linked to other sites when it is completed in summer of 2022. This will be an ongoing project supported by members of WERA-20, with updates to information in the database reviewed and updated as part of WERA-20 meetings going forward.

Akinbade S.A., Guerra L., Koundal V. and Cooper C. (Washington State Department of Agriculture)

Nepoviruses are one of the most difficult to control or eliminate pathogens once the virus(es) and the vector (nematode) become established in an area. Nepoviruses such as cherry rasp leaf virus (CRLV), tobacco ringspot virus (TRSV) and tomato ringspot virus (ToRSV) are known to occur in Washington State. These viruses if present in any of the nurseries within the WA Fruit Trees Certification Program, will be of great economic concern. Presently, WA Fruit Trees Certification Program have 1,250 linear miles of stoolbeds and about 90,000 registered mother tree which consist of Malus, Pyrus and Prunus species. Soils samples from nurseries where these trees are located have been tested in previous studies and the locations of nematodes capable of transmitting nepoviruses were mapped out. Xiphinema rivesi were identified in some of the nurseries. This study aims to investigate the presence of nematode transmitted viruses in sites where X. rivesi are known to occur in registered blocks of WA Fruit Trees Certification Program and to also assay for the presence of other potential virus-vectoring Xiphinema nematodes.

Over the spring and summer of 2020, soil samples were collected from areas of registered blocks where X. rivesi was earlier identified. Twenty soil cores at 20 feet apart in a row of 400 feet were collected per sample. In places where the rows were longer than 400 feet, the samples were divided into two or three. A total of 179 soil samples were collected from stoolbeds and registered mother blocks. The soil samples were transported in a cooler to the laboratory where they were analyzed for the presence of nematodes. X. rivesi were detected in 112 samples (63%). Total nucleic acids were isolated from 1-5 mostly adult female X. rivesi using RNeasy Plant Mini Kit (Qiagen). Real-time RT-PCR oligos and probe sequences for CRLV, TRSV and ToRSV were obtained from the Clean Plant Center Northwest (CPC-NW) and synthesized by Integrated DNA Technologies, Inc. (IDT). The qRT-PCR assays were performed using the standard protocol provided by CPC-NW and SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen) was used to prepare the reaction. Reaction was performed in Bio-Rad CFX96 Real-Time PCR Thermal Cycler and results were analyzed using CFX Maestro Software. Analysis of the results revealed that none of the X. rivesi tested positive to CLRV, TRSV and ToRSV. Although none of the viruses of concern were present in all the nematodes tested, there is still danger of introduction of any of these viruses into the area where X. rivesi have been detected. Hence, there is need for continuous monitoring of these nurseries for nepoviruses. Also, nematode management strategies have been provided for the participating nurseries to reduce the risk of introduction of nepoviruses.

Randi Jiménez (California Department of Food and Agriculture), Cindy Cooper (Washington State Department of Agriculture), Elizabeth Savory (Oregon Department of Agriculture) , Rachel Knier (Pennsylvania Department of Agriculture), Elizabeth Dorman (Michigan Department of Agriculture & Rural Development), Krishna Puri (Missouri Department of Agriculture)

 We primarily participate with the WERA-20 group as part of a working group of state nursery certification regulators (some of whom have cosigned this letter) organized through the National Clean Plant Network. As state regulators, our primary diseases of concern in nursery crops are caused by viruses and virus-like agents. Furthermore, for several of the states, berries, fruit and nut trees, and grapevines account for the majority of crops within our certification programs.

Over the past year, the working group has been working on harmonization amongst the various state programs and how the different states can update current certification programs based on new knowledge and integration of new technology. A major focus has been how will different technologies be implemented at NCPN Clean Plant Centers and the importance of high-throughput sequencing (HTS). Being able to interact with the virologists in this group makes our jobs within certification easier in that we can communicate directly and get detailed, nuanced information on the diseases we are trying to test for and regulate. It is incredibly important for state regulators to stay up to date on virus diagnostic testing and new viral agents that have recently been identified, and the WERA-20 group is a great resource for the state regulators that need to stay current on these crops. Furthermore, it helps us be aware of emerging pathogens and technologies in our whole region and not just what is going on in our own state.

The NCPN state regulator working group has also devised a national certification standards model. This model will be helpful for USDA and future exports, but it is also important for the state regulators to disseminate this model broadly as well as identify new state participants that will benefit from implementing this model. Participating in WERA-20 is helpful for this kind of outreach. As a WERA-20 participant, I have attended that past two annual meetings. The meetings have given the state regulators a forum to present on how several different states run their certification programs, the national certification standards model as well as a broad view on how virus diagnostic testing helps the states promote and increase the use of clean plants.

Overall, my participation in WERA-20 has been helpful in several different ways in accomplishing goals important for state nursery certification programs. I would like to support the group’s continuation and plan to continue participating in the group at the present time.

Allison Gratz – CFIA Sidney Laboratory – Centre for Plant Health – Tree Fruit Diagnostic program

Tree Fruit update – From 2021-22, 30 new accessions were accepted for testing from non-approved foreign sources and domestic breeding programs, and 32 accessions completed testing.  During the same period, approx. 35 samples from Canadian approved foreign certification programs in USA, Germany, the Netherlands and Belgium were submitted by CFIA inspection staff and tested.  Pathogen detection was accomplished using ELISA, RT-PCR, PCR, and herbaceous and woody bioassays.  Virus elimination efficiencies were seen when plants were a) strategically subjected to different lengths of time at 37C dependent on the virus(es) targeted for elimination and b) strategically subjected to either in vitro or in vivo thermotherapy.  In vivo thermotherapy followed by shoot tip grafting produced plants faster, but some pathogens were only reliably eliminated using in vitro thermotherapy followed by meristem culture. 

Canadian Fruit Tree Export Program (CFTEP) – The Generation 1 (G1) repository at the CPH holds approx. 500 Malus, Pyrus, Prunus, and Cydonia spp. accessions. From 2021/22, propagative material from approx. 100 accessions were distributed within Canada, and to the USA, Australia, New Zealand and Chile.  Currently, generation level 1, 2 & 3 Prunus material is tested for PDV, PNRSV, CLRV (cherries) or PDV, PNRSV, PPV (non-cherries) every two or three years.  Composited samples of G2 & G3 material are collected under supervision of CFIA inspection staff and tested at the CPH using ELISA.

Ioannis Tzanetakis (University of Arkansas)

In collaboration with colleagues from Oregon and California, all of which participate in WERA-20 we have made progress on the characterization and population structure of several berry viruses, including two in strawberry, two in blueberry and three in blackberry. This information has been used in the development of CRISPR diagnostics that can be 10,000 more sensitive than (RT)-PCR.

Rose rosette has been a focus on the Arkansas program for over a decade and important progress has been made in virus epidemiology and biocontrol.  A new vector has been identified and experiments have verified that a single individual is able to transmit the virus. A fungus belonging to the genus Meira has been identified and used to control the virus vector with promising results.

Work on the presence of predatory mites present in roses was completed with identification of several species that can predate on eriophyid mites, vectors of the rosette virus.  Three hundred twenty-one (321) predatory mite individuals have been recovered from collected samples. DNA has been isolated from all predatory mites using a non-destructive method. Mite-specific primers amplifying the ITS1 region of the ribosomal DNA have been developed and used for barcoding with twenty-one (21) species of predatory mites identified. Among these the most commonly encountered were Typhlodromalus peregrinus and Amblyseius andersoni, both belonging to the family Phytoseiidae.

1. peregrinus has been reported in 18 countries including the US. In the past it was generally found on citrus, blackberry and solanaceous plants and associated with many insect and mite species by different researchers. Aleyrodidae, Coccidae and Tetranychidae were evaluated as optimal prey for this species, however more recent studies suggest that T. peregrinus is also an effective predator of thrips and eriophyoid mites. It can also develop on various kind of pollen, making it a suitable candidate for further tests in Phyllocoptes control. A. andersoni is also a generalist that can feed and reproduce on many species of mites and small insects, pollen, honeydew, plant juices and fungal spores.
2. andersoni is one of the most commonly used species in augmentative biological control. In Europe, A. andersoni was found as the most frequent species in commercial nursery rose production systems, which suggests that this species thrives well on roses. Studies on roses in Europe showed that A. andersoni is a good candidate for preventing spider mite outbreaks, as it easily survives without spider mites. As both predatory mite species can survive on other sources of food, including thrips, pollen, and fungal spores they can survive longer on plants, thus preventing possible outbreaks of the vectoring mites. Interestingly, both species were most prevalent on rose rosette symptomatic plants as well on plants inhabited by the vectoring mite. Changes induced by the virus and presence of the vector seems to create conditions suitable for both predatory mites.

1. Alabi, O.J., Diaz-Lara, A., Erickson, T.M. and Al Rwahnih, M., 2021. Olea europaea geminivirus is present in a germplasm repository and in California and Texas olive (Olea europaea L.) groves. Archives of virology, 166(12), pp.3399-3404.
2. Beaver-Kanuya E, Szostek SA, Harper SJ. (2022) Detection of Malus and Pyrus-infecting viroids by real-time RT-PCR. Journal of Virological Methods, 300: 114395
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7. Davis TJ, Gomez MI, Harper SJ, Twomey M (2021) The economic impact of Hop stunt viroid and certified clean planting materials. Hortscience, 56(5): 1471-1475
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29. Wilson, H., Hogg, B.N., Blaisdell, G.K., Andersen, J.C., Yazdani, A.S., Billings, A.C., Ooi, K.L.M., Soltani, N., Almeida, R.P., Cooper, M.L. and Al Rwahnih, M., 2022. Survey of Vineyard Insects and Plants to Identify Potential Insect Vectors and Noncrop Reservoirs of Grapevine Red Blotch Virus. PhytoFrontiers™, 2(1), pp.66-73.
30. Wright AA, Shires M, Beaver C, Bishop GM, DuPont ST, Naranjo R, Harper SJ (2021) The effect of Candidatus Phytoplasma pruni infection on sweet cherry fruit. Phytopathology, In Press. https://doi.org/10.1094/PHYTO-03-21-0106-R
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