Brief Summary of Minutes of Annual Meeting

• Naidu Rayapati, Washington State University,

Dan Edward Veloso Villamor/Yannis Tzanetakis (University of Arkansas)

     In collaboration with colleagues, several of which participate in WERA-20, we are working on the characterization and population structure of several viruses in strawberry (rhabdoviruses), blueberry (luteo, carlavirus and vitivirus) and blackberry (allexi-, ifla- and reovirus). This information is used in the development of detection protocols that have the ability to detect the vast majority of isolates that are circulating in small fruit crops in the United States. One of the highlights of the year is the completion of the comparison of conventional methods to high throughput sequencing (HTS), another collaboration between WERA-20 participants. The comparison was done using berry selections with known virus profiles. The results revealed the following trend:

1. Virus detection by HTS and PCR were nearly identical with the exception of some viruses missed by HTS and detected by PCR and vice-versa.
2. Known viruses in some samples were not detected by HTS and PCR in all seasons.
3. HTS detected four novel viruses for which no PCR test is available. The presence of these viruses was validated by overlapping PCR to confirm authenticity. These viruses are the following: (1) a luteovirus in Vaccinium, a nucleorhabdovirus in Fragaria, and a potexvirus and coguvirus-like in Rubus).

Overall, these results suggest the use of HTS for virus detection should be done at least two growing seasons to allow detection of low tittered virus(es). Additionally, the failure of HTS to detect some viruses can be addressed by increasing the amount of sequence reads for each sample. This can be done by either decreasing the amount of multiplex samples per sequencing lane or using a different platform with higher sequencing output (i.e. Novaseq). 

Maher Al Rwahnih (UC-Davis: CA state report)

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 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 recent years, the fruit tree program at FPS has experienced significant growth in response to industry demand. In 2016, FPS acquired a Controlled Import Permit (P588) to facilitate the introduction, quarantine, and release of imported Prunus for the fruit tree industry. In 2020, we were successful in obtaining USDA-APHIS approval to revise the permit. In the past, regulations have required the grafting of candidate selections to four Prunus biological indicators (P. persica ‘GF 305’, P. avium ‘Canindex’, P. avium ‘Bing’, and P. serrulata ‘Kwanzan’) that were observed for symptom development in a greenhouse for at least one full growing season. If no disease symptoms were observed, the selection might be eligible for full quarantine release. This process frequently needed to be repeated if the imported selection underwent virus therapy, sometimes adding years to the quarantine process. We conducted side-by-side studies comparing HTS analysis to biological indexing which revealed that the performance of the biological indicators was inferior. Furthermore, since reliable laboratory tests were available to test for known pathogens detected by Canindex and Kwanzan, the removal of these two indicators that were redundant to the PCR and HTS tests was proposed. A new protocol for fruit tree introduction and testing was developed in coordination with Dr. Scott Harper at the Clean Plant Center Northwest (CPCNW, Prosser, Washington) to adopt the new testing approach and harmonize the standard operating procedures of the two Prunus quarantine programs. USDA APHIS PPQ and the CDFA approved the new protocol with the removal of Canindex and Kwanzan as biological indicators. Under the new protocol, plants are subjected to greenhouse indexing and molecular testing at two different time points by PCR and HTS. Plants may be released if all test results demonstrate plants are free of viruses or virus like agents. The culmination of this work and collaboration is the release of 20 selections, with an anticipated release of 11 additional selections in late summer 2020. The full release of new plant material under the new protocol will only take eight months to one year if it enters the program free of target viruses. Fruit tree nurseries and growers will greatly benefit from the release of material under this protocol as highly anticipated material be available to nurseries sooner, allowing them to begin their propagation and distribution of quality material to growers.  

The ‘Development and validation of real time quantitative PCR assays for the detection of fruit tree viruses’ study evaluated the broad-range detection capacity of currently available real-time RT-PCR assays for Prunus-infecting viruses and developed new assays when current tests were inadequate or absent. Available assays for 15 different viruses were exhaustively evaluated in silico to determine their capacity to detect virus isolates deposited in GenBank. During this evaluation, several isolates deposited since the assay was designed exhibited nucleotide mismatches in relation to the existing assay’s primer sequences. In cases where updating an existing assay was impractical, we performed a redesign with the dual goals of assay compactness and comprehensive inclusion of genetic diversity. The efficiency of each developed assay was determined by a standard curve. To validate the assay designs, we tested them against a comprehensive set of 87 positive and negative Prunus samples independently analyzed by high throughput sequencing.  As a result, the real-time RT-PCR assays described herein successfully detected the different viruses and their corresponding isolates. To further validate the new and updated assays a Prunus germplasm collection was surveyed. The sensitive and reliable detection methods described here will be used for the large-scale pathogen testing required to maintain the highest quality nursery stock. 

Last year, we reported the discovery of two novel vitiviruses, “grapevine virus L” (GVL) and “grapevine virus M” (GVM). Vitiviruses are ssRNA(+) viruses in the family Betaflexiviridae (subfamily Trivirinae). This year we report the description of a novel virus detected by high-throughput sequencing (HTS) in a sample of grapevine (Vitis vinifera) cv. Kizil Sapak (sample/isolate 127) that originated from Turkmenistan. The complete genome of the virus, tentatively named “grapevine Kizil Sapak virus” (GKSV), is 7,604 nucleotides in length, excluding the poly(A) tail. The genome organization of GKSV, encoded genes, and sequence domains are typical for members of the family Betaflexiviridae, specifically those belonging to the subfamily Trivirinae. Phylogenetic analysis placed GKSV within the subfamily Trivirinae, in the same clade as fig latent virus 1 (FLV-1) but distinct from the clades formed by members of other genera. A comparative analysis of GKSV-127 with the HTS-derived sequences obtained from two additional isolates showed that they are genetic variants of the same virus species. Based on current ICTV species and genus demarcation criteria, and the results of the sequence and phylogenetic analyses, we propose that GKSV and FLV-1 represent a new genus within the subfamily Trivirinae. 

Relatively few negative sense (ns)RNA viruses have been associated with infection in plants, including agricultural crops. However, in the last few years, the use of high throughput sequencing (HTS) has allowed the identification of new nsRNA viruses in plants. For example, the first nsRNA viruses identified and transmitted in grapevine, Grapevine Muscat rose virus (GMRV) and grapevine Garan dmak virus (GGDV), were only recently discovered because of HTS. The genomes of both viruses were comprised of three segments each containing a unique gene: RNA-dependent RNA polymerase (RdRp), nucleocapsid protein (NP) and movement protein (MP). Based on sequence identity and phylogenetic analysis, GMRV and GGDV represent new members of the family Phenuiviridae; most phenuiviruses are linked to diseases in vertebrates and are vectored by arthropods. Similarly, HTS was used to characterize nsRNA viruses in apple and citrus associated with different diseases. The discovery of these two nsRNA viruses in grapevine and the parallel report of additional viruses with this type of genetic material in other perennial crops suggests a more extensive distribution. Consequently, the characterization of nsRNA viruses in plants via HTS will remain an active area of research for some time. 

Little cherry disease (LCD), associated with little cherry virus-1 (LChV-1) or -2 (LChV-2), is a common problem of cherries which occurs worldwide, causes unmarketable fruit and often results in tree or orchard removal. Most of the new cherry rootstocks used in cherry production are interspecific Prunus hybrids which introduces an increased risk of an adverse reaction (hypersensitivity) to some viruses. Hypersensitive reactions exhibit graft union gum exudation, premature abscission, and tree death within one or two growing seasons and have been shown to occur in Prunus when infected with prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV). FPS has been evaluating the effects of LChV-1 and LChV-2 on 15 different popular Prunus rootstocks. Rootstocks were t-bud grafted with a scion variety from the same accession in June 2018.  In 2019, bud take was recorded in the spring and rootstock vegetation above the scion buds was tested by RT-qPCR to confirm successful virus transmission in the fall. Two years of budtake and tree performance observations will be recorded and evaluated. Rootstocks will be then be rated for sensitivity to LChV-1 and LChV-2, and this information will be shared with growers and nurseries to assist in making rootstock selection decisions. 

While HTS remains a powerful new technology with significant benefits, there are technical challenges associated with the technology that warrants the establishment of guidelines for its use in plant certification and quarantine programs. We have begun efforts in a collaborative project with the APHIS Plant Germplasm Quarantine Program (PGQP) and Center for Plant Health Science & Technology (CPHST) in Beltsville Maryland to coordinate the development of minimum basic requirements for the adoption of HTS technologies, including nucleic acid extractions, library preparation, depth of sequencing and bioinformatics, for the detection of viral pathogens. Both labs have optimized several HTS pipelines and data analysis protocols, but an inter-laboratory validation of these protocols is still needed. The goal of this project is to validate an HTS protocol (TruSeq Stranded Total RNA, Ribo-depleted) using the NextSeq Illumina platform via an inter-laboratory comparison among three laboratories (FPS, CPHST, and PGQP). The project will culminate in the preparation of a validation report for each sample panel evaluated. The validation report would be used as a guideline for the preparation of SOPs or another ISO-type document that will include minimum acceptable criteria for quality control (QC). In addition, these results will be used as an example for preparing a workflow (guidelines) for the validation of HTS protocols on other crops. Successful completion of this project will, 1) set up the stage for further HTS protocols verification/validation for other specialty crops; 2) provide a mechanism for the evaluation of validation results for faster HTS protocol acceptance; 3) increase stakeholders confidence in HTS used for regulated pathogen detection in the certification programs by minimizing the risk of false negatives; 4) increase regulators confidence on the reliability and potentials of HTS for detection of regulatory pathogens and ultimately; 5) expedite the release of foreign quarantined and domestic propagative plant material to stakeholders. Clean plant material for clean nursery stock will ultimately facilitate international and domestic movement of nursery material and will safeguard the industry from exotic plant pathogens. 

Allison Gratz (the CFIA Sidney Laboratory, Canada)

Quarantine and Diagnostic Activities - The CPH continues to test non-certified tree fruit and grapevine material (from both non-approved foreign sources routed directly to CPH and domestic programs).  Likewise, the CPH carries out some regulatory testing for virus and virus-like diseases of small fruit (berries). The testing requirements for imports and exports are determined on a case-by-case basis depending on the origin of the material and requirements of the importing country.  The CPH also tests samples taken from grapevine and tree fruit shipments imported from Canadian approved foreign certification programs from various sources, including the United States, France, Germany, and the Netherlands. 

Tree Fruit Program Overview & Update - From 2014 to 2019, the diagnostics program tested 254 new Malus, Pyrus, and Prunus spp. accessions from non-certified sources in Canada (BC, SK, MB, ON, PQ, NS), USA (WA, CA, OH), Czech Republic, France, Germany, Hungary, Italy, Spain, and Switzerland. Of these, 17% were infected with a virus or viroid that required elimination prior to placement in the G1 repository (ACLSV, ASGV, ASPV, CGRMV, CLRV, CNRMV, CVA, LCV-1, PBNSPaV, PDV, PLMVd, PNRSV, unknown (bioassay positive)).  

The average length of time to complete testing of non-certified tree fruit samples submitted since 2008 was 3.8 years (Prunus spp.), 4.3 years (Malus spp.) or 4.7 years (Pyrus spp.), with an extra two years required if virus elimination was needed.  The annual number of samples received during this period varied from 14 to 44 (average of 29) with the number of shipments ranging from 6 to 14 (average of 10).  

The purpose of the CPHs Generation 1 (nuclear level) repository is to supply Canadian industry with virus tested propagative material, and also to support Canadian industry by maintaining selections from domestic breeding programs at a level to meet import regulations of major trading partners.  Currently it houses 484 Malus, Pyrus, Cydonia, and Prunus accessions.  Permission must be obtained from sponsors in order to access the material:  90% are sponsored by Canadian breeding programs (primarily Agriculture and Agri-Food Canada (AAFC), University of Guelph and University of Saskatchewan), or Canadian industry; 1% are sponsored by international companies, and the remainder are publicly available for program use.  

From 2014 to 2020, about 40, 000 buds were distributed from the repository with 63% in volume (50% of orders) distributed to Canadian clients; 25% of buds (34% of orders) to American clients, and the remainder distributed to other international destinations.  

Other accomplishments - The CPH has hosted scientists from various domestic and international facilities. Typically, these visits have been based around collaborative research/diagnostic interests and projects, whereby methods and information has been shared. Some examples include representatives from the Animal and Plant Health Inspection Service of the United States Department of Agriculture, Vineland Research and Innovation Center, PhytoDiagnostics, University of Victoria, Brock University, AAFC, and CFIA scientists as per the lab-exchange initiative. 

Staff at the CPH are involved in various plant-health related groups and committees including:

• North American Plant Protection Organization (NAPPO) working group to review RSPM 35 "Guidelines for the Movement of Stone and Pome Fruit Trees and Grapevines into a NAPPO Member Country";
• NAPPO panel for the development of Next Generation Sequencing standards for plant virus diagnostics;
• Canadian Grapevine Certification Network
• BC Plant Protection Advisory council for grapevines;
• International Council for the Study of Virus and other Graft Transmissible Diseases of Fruit Crops
• International Council for the Study of Viruses and Virus-like Diseases of Grapevine
• Food and Agricultural Organization International Plant Protection Convention Technical Panel on Diagnostic Protocols ;
• Plant Health Quadrilateral (Canada, USA, New Zealand and Australia) Working Groups: “DNA barcoding”; “Diagnostic Collaboration” and “Managing regulatory issues arising from new diagnostic technologies”
• Genomics Research and Development Initiative;
• Collaborative projects as part of the European Phytosanitary Research Coordination Network 

Alejandro Olmedo-Velarde/ Mike Melzer (University of Hawaii)

Flat mites are minute polyphagous arachnids that belong to the Tenuipalpidae family. Brevipalpus and Tenuipalpus are considered the most relevant genera in the family because they contain quarantine pests and the former have members able to transmit plant viruses. Brevipalpus-transmitted viruses (BTVs) cannot infect systemically their hosts and rather are limited to local lesions where their mite vectors fed. BTVs can be further classified to BTV-C and BTV-N according to the where they replicate in the cytoplasm and nucleus, respectively. BTVs infect citrus, coffee, passion fruit, and ornamentals. Both BTV-C and BTV-N contain causal agents of citrus leprosis, a disease of economic importance for the American Citrus production and whose causal agents are quarantine pests for USDA-APHIS. Among the BTV-N causing citrus leprosis, orchid fleck virus (OFV) is a cosmopolitan virus whose orchid and citrus strains have been associated to the disease. In February 2020, citrus trees showing leprosis-like symptoms on leaves and stems were observed in an abandoned orchard on Hawaii Island. RT-PCR assays revealed the presence of a BTV-N using a universal BTV-N primer set and RNA extracted from rough lemon and mandarin trees. Sequencing show the identity of the BTV-N was OFV. The identity of OFV was corroborated by USDA-APHIS-CPHST laboratory and additionally the identity of the OFV was further determined to be the orchid strain 2 of OFV. An ongoing response plan is being implemented to eradicate this outbreak as a collaborative effort among the University of Hawaii, Hawaii Department of Agriculture and USDA-APHIS-PPQ. In 2019, viral-like symptoms resembling by those caused by BTV-C were observed in passion fruit in Honolulu. High throughput sequencing (HTS) on libraries constructed from dsRNA revealed the presence of citrus leprosis virus C2 (CiLV-C2) among other plant viruses found infecting passion fruit. The identity of the Brevipalpus mite transmitting CiLV-C2 was putatively determined to be B. yothersi using bean common as indicator hosts, 28S rDNA barcoding and RT-PCR. In 2018, viral-like symptoms were observed in papaya fruits only from papaya trees on the Hawaii Island. Symptoms differed by those caused by papaya ringspot virus (PRSV) on fruits, and PRSV-like symptoms on leaves were absent. HTS from a dsRNA library revealed the presence of abundant contigs showing low similarity, below 50% protein identity, to Virgaviridae, Kitaviridae, and Negeviruses members. Considering the similarity of contigs to Kitaviridae members, being BTV-C members, and common presence of Brevipalpus mites feeding on papaya fruits in Hawaii, Brevipalpus mites might represent a putative vector.

Adriana Larrea-Sarmiento/John Hu (University of Hawaii)

The complete genomic sequence of a novel member of the family Secoviridae was determined by high-throughput sequencing (HTS) of a pineapple accession obtained from the National Plant Germplasm Repository (NPGR) in Hilo, Hawaii. The predicted genome of the putative virus was composed of two RNA molecules of 6,128 and 4,161 nucleotides in length, excluding the poly-A tails. Each genome segment contained one large open reading frame (ORF). BLASTx analysis of the two contigs showed that RNA1 shared 35% identity with dioscorea mosaic-associated virus of the proposed subgenus “Cholivirus” and strawberry mottle virus of the proposed subgenus “Stramovirus”, and RNA2 shared 26% identity with dioscorea mosaic associated virus and chocolate lily virus A of the proposed subgenus “Cholivirus”. These related viruses are currently classified as Sadwavirus members within the family Secoviridae. These results support the placement of this new virus as a putative member of the genus Sadwavirus, family Secoviridae. The name “pineapple secovirus A” (PSV-A) is proposed for this putative new virus infecting pineapple. Two sets of primers designed based on the HTS-derived sequences were used in tandem to detect the presence of PSVA in pineapple. The presence of this new virus in pineapple has been confirmed by RT-PCR and Sanger sequencing from six samples collected in Oahu-Hawaii. Additional testing carried out in China, Australia and pineapple accession samples from different countries retrieved from the USDA-Agricultural Research Service (USDA-ARS-NPGR) at the Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center (PBARC) in Hilo suggests a cosmopolitan distribution of the new Sadwavirus. Two isometric virions particles (A1-A2) were reported in Australia back to 2002 infecting pineapple which suggests the presence of more sadwavirues infecting pineapple. These results support the placement of PSV-A as a putative member of the genus Sadwavirus, family Secoviridae. Further research is needed to identify the biological vector of PSVA, investigate whether PSVA is related to the two isometric viruses infecting pineapple in Australia, and determine if this putative new pineapple virus is involved in the etiology of mealybug wilt of pineapple (MWP).

Shulu Zhang (Agdia, Inc.)

Early detection and effective control of plant pathogens is very important to prevent their widespread resulting in serious economic losses in crops like cherries, citruses, grapes, hops, peaches, or plums. Agdia utilizes a leading isothermal amplification technology called recombinase polymerase amplification (RPA) and has developed AmplifyRP® tests for rapid and accurate detection of nucleic acids from many plant pathogens. So far, Agdia has commercialized 23 AmplifyRP® kits including 21 AmplifyRP® kits specific to single species/strains of diverse pathogens and 2 AmplifyRP® Discovery kits suitable for any pathogen. Among the pathogen-specific kits, there are 7 kits in AmplifyRP® Acceler8® format, 11 kits in AmplifyRP®  XRT format and 3 kits in AmplifyRP®  XRT+ format. Eight AmplifyRP® XRT kits have been commercialized during the past 12 months and are specific to the following pathogens listed below: 

Grapevine red blotch virus (GRBV) from the genus Grablovirus in the family Geminiviridae is an emerging virus disease of grapevines (Cieniewicz et al. 2020b, Fuchs 2020).  Limited information is available on the spread dynamics of GRBV in vineyards.  We investigated red blotch disease progress in three vineyards with a disparate initial inoculum prevalence (Cieniewicz et al. 2019a).  Secondary spread was documented in two vineyards in California but not in a vineyard in New York.  Increase in annual disease incidence was unrelated to the estimated initial source of inoculum at planting but populations of Spissistilus festinus were absent in the New York vineyard, low in one of the two California vineyards and moderate in the second California vineyard.  These results illustrated a differential disease progress in distinct vineyard ecosystems and suggested that GRBV spread dynamics in vineyards could be related to vector abundance (Cieniewicz et al. 2019a).  Furthermore, middle-row vineyard cover crop samples collected from GRBV-infected California vineyards, particularly legume species which are preferred hosts of S. festinus, tested negative for GRBV, suggesting a minimal role, if any, as inoculum reservoirs for GRBV spread (Cieniewicz et al. 2019a). 

No information is available on the genetic relatedness of S. festinus from vineyards in California and other crops in other regions of the United States.  To studied the diversity of S. festinus populations, we collected specimens from various crops and geographic locations in the United States, and characterized fragments of the mitochondrial cytochrome C oxidase 1 (mt-COI) gene and the nuclear internal transcribed spacer 2 (ITS2) region by polymerase chain reaction and sequencing (Cieniewicz et al. 2020a).  Maximum-likelihood and Bayesian analyses of the mt-COI and ITS2 sequences yielded similar phylogenetic tree topologies, revealing two distinct genetic S. festinus lineages with all of the specimens from California comprising one phylogenetic clade, alongside a single GenBank entry from Arizona, and all of specimens from the Southeastern United States comprising a statistically-supported distinct clade, regardless of host and year of collection.  These results suggest the existence of two genotypes within S. festinus in the United States (Cieniewicz et al. 2020a).  The only distinct morphological trait between the two genotypes was a less elevated pronotum in the representative specimens from California, compared to the representative specimens from the Southeastern United States.  Since this phenotypic feature is inconspicuous, a diagnostic polymerase chain reaction targeting a variable region of the mt-COI fragment was developed to reliably distinguish between the specimens of the two genotypes of S. festinus and to facilitate their specific identification (Cieniewicz et al. 2020a. 

Christie Almeyda (North Carolina State University)

The Micropropagation and Repository Unit (MPRU) at North Carolina State University (NCSU) 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. While cleaning up berry crops, the following viruses were detected on blueberries: Blueberry latent virus (BBLV) and Blueberry red ring spot virus (BRRV). Blackberry yellow vein-associated virus (BYVaV), Blackberry virus E (BlVE) and Citrus concave gum-associated virus (CCGaV-like) were detected on blackberries. Not long ago, 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. 

In partnership with Dr. Hoffmann (NCSU strawberry and grape extension specialist), the MPRU was able to collaborate with its diagnostics capacity for virus surveys on grapes. Various NC grower fields were tested to validate the establishment of molecular testing at the MPRU using protocols previously developed by Foundation Plant Services (FPS), UC-Davis in collaboration with Dr. Maher Al Rwahnih. The MPRU now has the capacity of testing for 10 pathogens affecting grapes using quantitative RT-PCR. Targeted pathogens were selected based on importance and prevalence in the region. The pathogens currently being tested are Grapevine leaf roll viruses (GLRaV-2, GLRaV-3, GLRaV-4, GLRaV-7), Grapevine red blotch virus (GRBV), Grapevine rupestris stem pitting associated virus (GRSPaV), vitiviruses (GVA, GVB), Tobacco Ring Spot Virus (TRSV), and Xyllela fastidiosa. 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. 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. The third and last year of the survey will define if there is a clear pattern of these pathogens occurring in NC. 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 muscadine cultivars) and new material (5 genotypes) we recently obtained from the AR breeding program.

Ruhui Li, USDA-ARS

Blackcuurant revision virus was found in the Ribes germplasm of USDA-ARS. An improved method was developed for the detection for a citrus virus. Three different new viruses were identified from epiphyllum cactus. Collaborative Research projects include the following examples: Joseph Foster (USDA-APHIS-PGQP): Viruses infecting stone fruits and small fruits; Ekaterina Nikolaeva (Pennsylvania Department of Agriculture) and Kari Peter (Pennsylvania State University): Etiology of Rapid Apple Decline; Benjamin Gutierrez (USDA-ARS) and Margarita Bateman (USDA-APHIS): Viruses infecting fruit trees; Mengji Cao (Southwest University of China): Citrus and camellias; Liping Wu (Nachang University of China): Camellias; Luping Zheng (Fujian Agricultural and Forestry University) and Joseph Postman (USDA-ARS): Ribes spp. 

Oscar Hurtado-Gonzales: (USDA-APHIS)

Alexander Karasev (University of Idaho)

Wine grape production in Idaho occurs on approximately 1,300 acres, predominately in Canyon County in the Southwest, and Nez Perce County in the Northwest. In the course of the GLRaV-3 testing of wine grapes in southern Idaho, plants of two grapevine cultivars were found to harbor a novel genetic variant of GLRaV-3, named ID45, which exhibited ≤80% nucleotide sequence identity level to the known GLRaV-3 isolates in its most conserved HSP70h gene. The ID45 variant caused no foliar symptoms in ‘Cabernet Sauvignon’ in the fall, and was demonstrated to have poor reactivity to commercial virus-specific antibodies. The entire 18,478-nt genome sequence of the GLRaV-3-ID45 was determined using a combination of high-throughput and conventional Sanger sequencing, and demonstrated to have typical organization for the genus Ampelovirus (family Closteroviridae), with only 70 to 77% identity level to the GLRaV-3 genomes from other established phylogroups. We concluded that ID45 represented a new phylogenetic group IX of GLRaV-3. Database search using ID45 nucleotide sequence as a query suggested that this novel ID45 variant is present in at least one other grape-growing state in the U.S., in California, and in Brazil. An RT-PCR based test was developed to distinguish ID45 from the predominant, GLRaV-3 phylogroup I found in Idaho in single and mixed infections. In September of 2014-2015, a survey of wine grapes was conducted in Canyon and Nez Perce counties of Idaho for the presence of GRBV. Three grapevines were found positive by PCR producing the DNA fragment of expected size, 720-bp; all three positive samples came from a single vineyard in Canyon county, from the same wine grape cultivar, Syrah. To expand the GRBV survey, 434 random grapevine samples collected in 2009-2011 in 14 vineyards in Canyon, Elmore, Ada, and Nez Perce counties were re-analyzed for the presence of GRBV; six additional GRBV-positive samples coming from two additional vineyards (Canyon county) and two additional grapevine cultivars, Merlot and Petite Sirah, were identified by PCR. The five whole genomes for these GRBV isolates were sequenced in DNA plasmids by Sanger methodology, and subjected to phylogenetic analysis. All whole genomes sequenced were assigned to clade 2 of GRBV, most closely related to a group of GRBV isolates from Washington state. This phylogeny of the Idaho GRBV isolates suggests the introduction of GRBV to Idaho from the same infected source, probably through infected planting material. 

Ekaterina Nikolaeva (Pennsylvania Department of Agriculture)

Pennsylvania Department of Agriculture in cooperation with Penn State University conducted 2019 PPA 7721 funded surveys for exotic diseases in orchards and small fruits. Orchard survey targets included Potyvirus Plum pox virus, Asian Pear Blight (Erwinia pyrifoliae), Asiatic brown rot (Monilia polystroma), Apple brown rot (Monilinia fructigena), Apple Proliferation (Candidatus Phytoplasma mali), European stone fruit yellow (Ca. Phytoplasma prunorums), 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, but we did confirm presence of Ca. Phytoplasma pyri (16SrX, Apple Proliferation group) in plum tree.

Data gathering were continued a Rapid Apple Decline syndrome. In 2019, PDA and PSU team visited 34 apple orchard blocks in 17 PA farms located in Adams, Bedford, Berks, Columbia, Franklin, Luzerne, and Northumberland counties. The orchards were inspected for the symptoms and signs of Rapid Apple Decline (RAD). The main characteristics included presence of dead and declining young dwarf trees, wilting, unseasonal tree discoloration and defoliation, dark brown cankers around graft union, and presence of green suckers. A total of 993 samples was tested in PDA lab for presence of Apple luteovirus 1 (ALV1) via conventional RT PCR. More than 200 samples within the same group were also tested for the presence of other viruses known to infect apple trees, including apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV) apple stem pitting virus (ASPV), tomato ringspot virus (ToRSV), and apple mosaic virus (ApMV). In result, 30% tested trees were found positive for ALV-1, 19.6% for ToRSV, 8.9% for ApMV, 14.1% for ACLSV, 54.3% for ASGV and 25.2% for ASPV. Similar to previous year results, ALV1 was detected on a wide range of apple varieties, including Aztec Fuji, Buckeye Gala, Crimson Crisp, Fuji, Gala, Golden Delicious, Honeycrisp, and Jonagold. Most of the ALV1 positive trees (97%) were grafted on M9 rootstocks while only few positive trees were found on Bud 9 (1.5%), Bud 10 (1.5%) and G11 (1.1%). A total of 57 samples (total RNAs) was subjected to Illuminia RNA sequencing by USDA ARS lab. RNA reads of the samples were de novo assembled and resulted contigs were blasted against three local databases retrieved from NCBI GenBank. Four known apple viruses, including ACLSV, ALV1, ASGV and ASPV were detected in the samples in different combinations. A new virus (Bunyaviridae), Citrus concave gum-associated virus (CCGaV), was also identified in the majority of the samples. The genetic diversities were found very low among ALV1 and CCGaV isolates, while high genetic variations occurred among three latent viruses, especially ASPV. The work on determination of ALV1 pathogenicity was started in August 2018 and continued in 2019. Three hundred M9 rootstocks (not grafted) trees from different suppliers were evaluated for the presence of ALV1. ALV1 positive rootstocks were used for grafting with ALV1 negative budwoods. In July 2019, we revisited grafted trees to evaluate virus transmission to the scion of the grafted trees and possible symptom development. In result, ALV1 was detected in 52.8% scions of grafted trees. That confirmed USDA ARS data that ALV1 can be transmitted from rootstocks to the budwood through graft union.

PDA continues to operate the Fruit Tree Improvement Program (FTIP), a specialized virus-tested fruit tree certification program. Three nurseries have been participating in the FTIP last year. Over 3,500 samples were tested for viruses of concern, including Prunus Necrotic Ringspot Virus (PNRSV), Prune Dwarf Virus (PDV), Tomato Ringspot Virus (ToRSV), and Plum Pox Virus (PPV). No PPV was detected in rootstock blocks or in registered source blocks. PNRSV (2.4%) remains the most commonly found virus in Prunus in PA nurseries. The occurrence of PDV (0.6%) and ToRSV (0.2%) in registered blocks and nursery production blocks remain low. All blocks met virus-testing requirements for FTIP certification.

Scott J. Harper (Washington State University)

The Harper lab is one of the major participants in the Washington & Oregon Little cherry disease task force, providing the pathology component in collaboration with entomology, horticulture and extension researchers to understand the pathology and etiology of this disease. The Clean Plant Center Northwest (CPCNW) has been continuing the previous year’s collaboration with Foundation Plant Services (FPS) at UC Davis, on the identification of pathogens present in diseases of unknown etiology. The CPCNW has been active in protecting US agriculture from harmful pests and diseases, and during the reporting period processed and released a total of 59 pome and stone fruit, 3 grapevine, and 7 hop virus-tested cultivars to industry.

Lauri Guerra (WA Department of Agriculture)

Regular Testing of Registered Prunus trees - Normally we test every 3 years all plants by Elisa for ILAR, CLRV and PPV and on the other 2 years we test half in the lab for Ilar and CLRV by Elisa and the other half in the field by Shirofugen indexing.

Testing scheme for G2 trees when sourcing G3 trees - One round of greenhouse bio-indexing (dormant material) and one round of lab testing (actively growing material), using PCR or qPCR when available  - positives confirmed by sequencing. All testing would be completed before trees are ready to transplant to Registered blocks. If a positive is found, nurserymen can propagate from another source, missing one year only, instead of 3 years, as before.Before we would run twice in the greenhouse, skipping a year. Indicators used for Greenhouse indexing. Malus and Pyrus - Russian, Radiant, Spy 227, Micromalus, Virginia Crab, Nouveau Poiteau, Lord Lambourne (experimenting also Geneva 16 to verify its usefullnes as an indicator), Prunus - Bing, Sam, Kwanzan, Canindex, GF-305, Tilton, Shiro, Tomentosa.

Nepovirus vectors survey - Finished testing all registered blocks for the presence of nepovirus vectors (2293 soil samples – 20 cores per samples, totaling ~45,000 cores). The only vector nematode identified in some blocks was Meloidogyne rivesi. A proposal was presented and approved for Farm Bill funding, to identify on these sites if there is the presence of ToRSV, TRSV and CRLV.

Arunabha Mitra/Naidu A. Rayapati (Washington State University)

Viral diseases are one of the significant concerns to sustainability of the grape and wine industry in Washington State that contributes an estimated $6 billion to the State’s economy. Grapevine leafroll disease (GLD) continues to be the most insidious and widely distributed in Washington vineyards compared to other viral diseases. Vineyard surveys have indicated the occurrence of Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-2, GLRaV-3 and GLRaV-4 in Washington vineyards, with GLRaV-3 being the most predominant and economically important than other GLRaVs. During the past few years, we have been studying the genetic diversity of GLRaVs for a better understanding of their role in GLD epidemiology and to implement robust strategies for management of the disease in vineyards. In previous studies, we have reported the presence of molecularly divergent isolates of GLRaV-1 and GLRaV-2 in vineyards (Phytopathology 100 [2010]: 698-707 and Phytopathology 101 [2011]:1446-1456). In recent years, we have extended these studies to examine the molecular variability of natural populations of GLRaV-3 and GLRaV-4. Total RNA preparations were made from representative samples collected from wine grape cultivars that tested positive for GLRaV-3 and subjected to high-throughput sequencing (HTS) to generate near-complete viral genome sequences. A global phylogenetic analysis of these sequences revealed the presence of distinct variants of GLRaV-3 in Washington vineyards that aligned with six genetic variant groups, designated as I, II, III, V, VI, and IX, previously reported from other grapevine-growing regions. Among them, GLRaV-3 isolates belonging to variant group I were found to be predominant in Washington vineyards. Using a combination of HTS and Sanger sequencing, the complete genome of three strains of GLRaV-4 (strain 4, strain 5 and strain 9) was determined to be 13,824 nucleotides (nt), 13,820 nt and 13,850 nt, respectively. An analysis of their genome sequences in comparison with GLRaV-4 strains (strain 4, strain 5, strain 6, strain 9, strain Pr, strain Car, and strain Ob) reported from different grapevine-growing regions revealed intraspecies recombination among a few strains of the virus. Overall, these results expanded our current understanding of the genetic diversity of GLRaVs and provided a foundation to gain insights into the epidemiology of GLD for implementing sustainable disease management strategies in vineyards

Cindy Cooper (WA Department of Agriculture)

 “Harmonizing State Certification and Quarantine Programs, presented by members of the National Planting Stock Certification Standards Working Group”

The National Planting Stock Certification Standards Working Group was created in 2017, with a grant from APHIS, to create an ongoing opportunity to communicate with USDA on issues effecting state level certification programs and plant movement. The goals of the group are:

1. Communication between state regulators, researchers, and federal counterparts, to improve and promote state level pathogen-tested planting stock certification programs;
2. To work toward understanding and harmonizing program standards between states;
3. Collaborate on rewriting and standardizing the national model certification standards developed for NCPN crops by the tier 2 committees;
4. Mentor emerging state certification programs through sharing combined group knowledge.

We hope to become a permanent committee within the National Plant Board, or another established parent group, and will seek on-going funding to facilitation and face-to-face meetings.

Regulators from Washington, Oregon, California, Michigan, Pennsylvania and New York are currently participating, and we invite regulators from other states to join us.

The group has identified the common elements included in the national model standards for certification, such as definitions of the limited generation scheme, acceptable G1 sources, site selection criteria, isolation distances, frequency and timing of inspections, sampling and testing, and a list of diseases for which planting stock is monitored under certification. We are conducting a gap analysis comparing state program standards to the national model standard and identifying missing or disparate content, with the goal of harmonizing wherever possible.

Each participating state gave an overview of established certification programs they maintain, crop programs that have become inactive, and new crops for which rules are being established. WA, OR, CA, PA and NY have active fruit tree certification programs. NY, WA, OR, CA and MI have active grapevine programs. Several states are developing rules for blueberry and hop certification.  Funding for certification programs and lab testing varies from state to state, with some having established assessments on sales of planting stock to draw from, though most state programs are fee-for-service, paid for by the participating nurseries.  Likewise, lab capacity is varied, with some states utilizing university labs to conduct molecular testing. All six states utilize molecular testing, but none currently have High Throughput Sequencing capacity. Only Washington and California continue to conduct field or greenhouse bio indexing as part of their testing regime. State regulators feel HTS it is an important tool for diagnostics and support its development for use by clean plant centers.

State Departments of Agriculture utilize the clean plant centers as a designated source of G1 material for certification programs.  Clean plant centers also provide testing services and will clean up material that nurseries want to bring into a certification program.

The National Clean Plant Network (NCPN) is a program consisting of plant pathogen ‘clean plant’ services by clean plant centers to 7 crop taxa; including fruit trees (stone and pome fruits), grapes, hops, berries (primarily Fragaria, Rubus, and Vaccinium) citrus, roses, and sweet potato.  The stated purpose of the program indicates that NCPN is “… a ‘Network’ of clean plant centers and allied programs; located at universities, U.S. gov’t agencies, and non-profit entities; with the mission of diagnosing plant pathogens in ‘mother’ (nuclear stock) plants; and in applying therapeutics; to ‘clean’ these plants and maintain them in foundations; in preparation for their acquisition and increase and use by industry; and to engage in program governance and special initiatives.

The program is administratively nested in USDA.  It was established by the Farm Bill of 2008 as an experimental initiative.  Under the Farm Bill of 2014, NCPN was reauthorized, made permanent, given a baseline funding of not less than $5.0 million annually, and given a reaffirmation of the programs purposes.

NCPNs Special Initiatives represent an exciting venue under which program participants engage across tradition specialty crop lines to engage with each other in advancing the Network and their own centers organizationally and administratively.  5 special initiatives were highlighted this year at WERA-20 including:

• Updating an NCPN Plan for 2020-2024.
• The new plans intention is to update, validate, and circumscribe the Network.
• The plan has 3 goals; program operations, governance, and special initiatives.
• Program values are also highlighted, including quality, service, connectivity, empowerment, and sustainability.
• The plan also highlighted significant other special initiatives that could be pursued by NCPN such as international collaboration, succession management, and focus on critical issues.
• The draft is in final form ready for broader review by members.

NCPN Education/Outreach/Communications:

• A team of about 60 persons representing NCPNs first special initiative.
• The team produces NCPN outreach tools such as brochures, websites, newsletters, and various fact sheets and has been vital to advancing the visibility and stature of the program.
• NCPN currently has a series of new Communications Plans developed by its members in a series of meetings in Portland, OR and Sacramento, CA. The plans are being reviewed by NCPN to ascertain how best to implement them.  Core to the plans is a recommendation for NCPN to establish a formal communications Directorate within the program.

NCPN Economics Initiative:

• A newly formed team of about 40 members led for NCPN by Cornell Univ. economists and meeting at that location last year to formally launch the program.
• The team and its associated leadership are assessing existing clean plant program economic studies, analyzing gaps in knowledge, and planning a roadmap forward.
• The NCPN Governing Board has provided the team with funding to support added economic studies as identified by the members.

 NCPN Quality Initiative:

• A newly formed team of about 40 members led for NCPN by the University of Riverside in collaboration with select leaders from the National Plant Diagnostic Network and USDA.
• Meetings to establish program direction were recently held at Riverside, CA with an added training of NCPN technicians in select principles of quality being conducted at the USDA/APHIS/PPQ laboratory in Beltsville, MD.
• The team is looking at quality in all aspects of NCPN, including scientific laboratories, governance, recordkeeping, program foundations, and communications.
• Select progress includes the establishment of an NCPN Quality Steering Committee, a team to initiate a draft of laboratory Quality Standards, proposals for added clean plant center staff training, and NCPN support for Quality Managers at clean plant centers.

 NCPN Scientific Information Sharing and Hight Throughput Sequencing (HTS):

• NCPN continues to support the gathering and work of scientists interested in sharing information about diagnostics and new technologies, such as HTS.
• Of particular interest to NCPN is the establishment of linkages between NCPN clean plant centers and Federal/State regulators gathering to discuss efficiencies that might allow for the smoother and more rapid movement of clean plant material.

The Network also reported-out on critical and emerging issues that it’s facing in FY 2020 including the impacts of COVID-19 on NCPN (such as the need to extend or adjust existing clean plant center agreements for an added year to compensate for activity changes or slowdowns; program exploration into new governance, networking, and organizational paradigms; continue efforts to accurately circumscribe the programmatic boundaries of NCPN; NCPN foundations and how they might change over time under pressure from vectors of pathogens; and new crops / new centers and criteria for their entry into the Network.

Adiputra, J., Jarugula, S. and Naidu, R.A. 2019. Intra-species recombination among strains of the ampelovirus Grapevine leafroll-associated virus 4. Virology Journal 16: 139.

Al Rwahnih, M., Alabi, O.J., Hwang, M.S., Stevens, K. and Golino, D., 2019. Identification and genomic characterization of grapevine Kizil Sapak virus, a novel grapevine-infecting member of the family Betaflexiviridae. Archives of Virology, 164(12), pp.3145-3149.

Alabi, O.J., Gaytan, B.C., Al Rwahnih, M., Villegas, C., 2020. A description of the possible etiology of the cilantro yellow blotch disease. Plant Disease. https://doi.org/10.1094/PDIS-09-19-1958-SC

Appel, D.N., Alabi, O., McBride, S.A., Al Rwahnih, M. and Pontasch, F., 2019. The Incidence of Grapevine Viruses in Four Texas Blanc du Bois Vineyards. Plant Health 2019.

Beaver-Kanuya, E., & Harper, S. J. (2019). Detection and quantification of four viruses in Prunus pollen: Implications for biosecurity. Journal of virological methods, 271, 113673.

Beaver-Kanuya, E., & Harper, S. J. (2020). Development of RT-qPCR assays for the detection of three latent viruses of pome. Journal of Virological Methods, 278, 113836.

Bennypaul, H., I. Abdullahi, M. W. Harding, and C. Neeser (2019). First Detection of Wheat streak mosaic virus in Two Perennial Weed Species, Agropyron cristatumand Hordeum jubatum subsp. intermedium, in Canada. Plant Disease 2019 103:6, 1441-1441

Bennypaul, H., I. Abdullahi, M. W. Harding, and R. Aboukhaddour (2019). First Detection of European Isolates of Wheat streak mosaic virus in Canada. Plant Disease 2019 103:6, 1442-1442

Brewer, E., Cao, M., Gutierrez, B.L., Bateman, M., Li, R. 2020. Discovery and molecular characterization of a novel trichovirus infecting sweet cherry. Virus Genes. https://doi.org/10.1007/s11262-020-01743-7.

Britt, K., Gebben, S., Levy, A., Al Rwahnih, M., Batuman, O., 2020. The Detection and Surveillance of Asian Citrus Psyllid (Diaphorina citri)-Associated Viruses in Florida Citrus Groves. Frontiers in Plant Science, 10: 1687. https://doi.org/10.3389/fpls.2019.01687

Chingandu, N., Jarugula, S., Movva, A. and Naidu, R.A. 2020. “The absence of grapevine red blotch virus in Washington’s certified grapevine nurseries” at the Washington Winegrowers Association Annual Meeting, Convention & Trade Show, March 2-5, 2020, Kennewick, WA.

Cieniewicz, E., Flasco, M., Brunelli, M., Onwumelu A., Wise, A. and Fuchs, M.F. 2019a. Differential spread of grapevine red blotch virus in California and New York vineyards. Phytobiomes Journal, 3:203-211.

Cieniewicz, E., Poplaski, V., Brunelli, M., Dombroswkie, J. and Fuchs, M. 2020a. Two distinct Spissistilus festinus genotypes in the United States revealed by phylogenetic and morphological analyses. Insects, 11:80; DOI:10.3390/INSECTS11020080.

Cieniewicz, E., Wise, A., Smith, R., Cooper, M, Martinson, T. and Fuchs, M. 2019b. Studies on red blotch ecology inform disease management recommendations. Wine Business Monthly, March issue, pp. 92-102.

Cieniewicz, E.J., Qiu, W., Saldarelli, P., Fuchs, M. 2020b. Seeing is believing: Lessons from emerging viruses in grapevine. Journal of Plant Pathology, https://doi.org/10.1007/s42161-019-00484-3.

Davenport B., Groth-Helms D., Li R., Zhang S. (2019): Development of a real-time duplex isothermal assay for the detection of Tobacco rattle virus and an endogenous internal RNA control in ornamental hosts. IX International Symposium on New Ornamental Crops. September 30 - October 3, 2019, Guadalajara, Mexico.

Davenport B., Li R., Zhang S. (2019): Isothermal detection for Dickeya and Clavibacter michigenesis subsp. sepedonicus, two prominent potato tuber pathogens. Australasian Plant Pathological Society Meeting, November 25–28, 2019, Melbourne, Victoria, Australia.

Diaz-Lara, A., Brisbane, R.S., Aram, K., Golino, D. and Al Rwahnih, M., 2019. Detection of new vitiviruses infecting grapevine in California. Archives of virology, 164(10), pp.2573-2580.

Diaz-Lara, A., Golino, D., Preece, J.E. and Al Rwahnih, M., 2020. Development of RT-PCR degenerate primers to overcome the high genetic diversity of grapevine virus T. Journal of Virological Methods, p.113883.

Diaz-Lara, A., Klaassen, V., Rowhani, A., Stevens, K., Hwang, M., Golino, D.A. and Al Rwahnih, M., 2019. Comparison of newly developed ELISA and RT-PCR assays for the detection of all known genetically diverse variants of GLRaV-3. Plant Health 2019.

Diaz-Lara, A., Klaassen, V., Stevens, K., Hwang, M., Golino, D.A. and Al Rwahnih, M., 2019. Improved detection of fruit tree viruses and viroids by real-time quantitative PCR. Plant Health 2019.

Diaz-Lara, A., Martin, R.R., Al Rwahnih, M., Vargas, O.L. and Rebollar-Alviter, Á., 2020. First evidence of viruses infecting berries in Mexico. Journal of Plant Pathology, 102(1), pp.183-189.

Diaz-Lara, A., Navarro, B., Di Serio, F., Stevens, K., Hwang, M.S., Kohl, J., Vu, S.T., Falk, B.W., Golino, D. and Al Rwahnih, M., 2019. Two Novel Negative-Sense RNA Viruses Infecting Grapevine Are Members of a Newly Proposed Genus within the Family Phenuiviridae. Viruses, 11(8), p.685.

Diaz-Lara, A., Stevens, K., Klaassen, V., Golino, D. and Al Rwahnih, M., 2020. Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp. Plants, 9(2), p.273.

Druciarek, T., Lewandowski, M. and Tzanetakis I.E. 2019. A new, sensitive and efficient method for taxonomic placement in the Eriophyoidea and virus detection in individual eriophyoids. Experimental and Applied Acarology 78: 247-261.

Fuchs, M. 2020. Grapevine red blotch virus. In: Invasive Species Compendium and Crop Protection Compendium, CABI International, Wallingford, Oxfordshire, United Kingdom, in press.

Fuchs, M. 2020. Grapevine viruses: A multitude of diverse species with simple but poorly adopted management solutions in the vineyard. Journal of Plant Pathology, in press.

Green, J.C., Rwahnih, M.A., Olmedo-Velarde, A., Melzer, M.J., Hamim, I., Borth, W.B., Brower, T.M., Wall, M. and Hu, J.S., 2020. Further genomic characterization of pineapple mealybug wilt-associated viruses using high-throughput sequencing. Tropical Plant Pathology 45:64-72.

Hadaway, K., Kogan, C., Jarugula, S. and Naidu, R.A. 2019. “Elucidating differences in red leaf symptoms produced by biotic and abiotic stresses in grapevines” at the IEEE Women in Engineering Leadership Summit, July 30, 2019, Richland, WA (Received best poster award)

Hamim, I. Wayne B. Borth · Michael J. Melzer · Jon Y. Suzuki · Marisa M. Wall,· John S. Hu 2019. Occurrence of tomato leaf curl Bangladesh virus and associated subviral DNA molecules in papaya in Bangladesh: molecular detection and characterization. Archives of Virology 164:1661-1665

Hamim, I., Al Rwahnih, M., Borth, W.B., Suzuki, J.Y., Melzer, M.J., Wall, M.M., Green, J.C., Hu, J.S., 2019. Papaya Ringspot Virus Isolates from Papaya in Bangladesh: Detection, Characterization, and Distribution. Plant Disease, 103(11): 2920-2924.

Hamim, I., Maher Al Rwahnih, Wayne B. Borth, Jon Y. Suzuki, Michael J. Melzer, Marisa M. Wall, James C. Green, and John S. Hu 2019 Papaya ringspot virus isolates from papaya in Bangladesh: detection, characterization and distribution. Plant Disease 103:2920-2924.

Hoffmann M, Talton W, Nita M, Jones T, Al Rwahnih M, Sudarshana MR, and Almeyda C. 2020. First Report of Grapevine red blotch virus, the Causal Agent of Grapevine Red Blotch Disease, in Vitis vinifera in North Carolina. PDIS-07-19-1539-PDN.

Hoffmann, M., Talton, W., Nita, M., Jones, T.J., Al Rwahnih, M., Sudarshana, M.R. and Almeyda, C.V., 2019. First Report of Grapevine red blotch virus, the causal agent of Grapevine Red Blotch Disease in Vitis vinifera in North Carolina. Plant Disease, (ja).

James D., Phelan, J., Sanderson, D. (2019). Detection by high throughput sequencing and molecular characterization of complexes of fabviruses infecting Staccato® sweet cherry (Prunus aviam) in Canada. Canadian Journal of Plant Pathology.

Jarugula, S., Adegbola, R., Mitra, A., Chingandu, N., Sekhar, T., Swamy, P., Bagewadi, B. and Naidu, R.A. 2020. “Rogueing symptomatic vines for controlling viral diseases in vineyards” at the Washington Winegrowers Association Annual Meeting, Convention & Trade Show, March 2-5, 2020, Kennewick, WA. (Poster presentation received First place under the People Choice and 3rd place under Professional Category).

Katsiani, A., Stainton, D., Lamour, K. and Tzanetakis, I.E. 2020. The population structure of Rose rosette virus in the United States. Journal of General Virology 101, in press

Lan, P., Tian, T., Pu, L., Rao, W., Li, F., Li, R. 2019. Characterization and detection of a new badnavirus infecting Epiphyllum spp. Archives of Virology. https://doi.org/10.1007/s00705-019-04237-6.

Larrea-Sarmiento, A., Alejandro Olmedo-Velarde, James C. Green, Maher Al Rwahnih, Xupeng Wang, Yun‑He Li, Weihuai Wu, Jingxin Zhang, Tracie Matsumoto Brower, Marisa Wall and John S. Hu 2020, Identification and complete genomic sequence of a novel sadwavirus discovered in pineapple (Ananas comosus) Archives of Virology https://doi.org/10.1007/s00705-020-04592-9

Larrea-Sarmiento, A., X. Wang, W. B. Borth, R. P. Barone, A. Olmedo-Velarde, M. J. Melzer, J. S. K. Sugano, R. Galanti, J. Y. Suzuki, M. M. Wall, and J. S. Hu 2019 First report of bean common mosaic virus infecting flowering ginger (Alpinia purpurata) in Hawaiʻi. Plant Disease https://doi.org/10.1094/PDIS-06-19-1264-PDN

Li R., Davenport B., Zhang S., Schuetz K., Bai T.T., Fu A.G., Zheng S.J. (2020): Development of a simple, rapid, and sensitive AmplifyRP isothermal assay for detection of Fusarium oxysporum f. sp. cubense Tropical Race 4. Plant Health 2020, Denver, Colorado, USA.

Li R., Davenport B., Zhang S., Schuetz K., Ling K.S. (2020): Development of a triplex AmplifyRP molecular assay for a reliable detection of Tomato brown rugose fruit virus. Plant Health 2020, Denver, Colorado, USA.

Liu, H., Wu, L., Zheng, L., Cao, M., Li, R. 2019. Characterization of three new viruses of the family Betaflexiviridae associated with camellia ringspot disease. Virus Research. https://doi.org/10.1016/j.virusres.2019.197668.

Liu, Q., Xuan, Z., Wu, Y., Li, M., Zhang, S., Wu, D., Li, R., Cao, M. 2019. Loquat is a new natural host of apple stem grooving virus and apple chlorotic leaf spot virus. Plant Disease. https://doi.org/10.1094/PDIS-04-19-0721-PDN.

Maree, H.J., Blouin, A.G., Diaz-Lara, A., Mostert, I., Al Rwahnih, M. and Candresse, T., 2020. Status of the current vitivirus taxonomy. Archives of Virology, 165(2), pp.451-458.

Martínez-Lüscher, J., Plank, C.M., Brillante, L., Cooper, M.L., Smith, R.J., Al-Rwahnih, M., Yu, R., Oberholster, A., Girardello, R. and Kurtural, S.K., 2019. Grapevine red blotch virus may reduce carbon translocation leading to impaired grape berry ripening. Journal of agricultural and food chemistry, 67(9), pp.2437-2448.

Mitra, A., Jarugula, S., Donda, B., Jordan, E. and Naidu, R.A. 2019. “Elucidating the genetic diversity of Grapevine leafroll-associated virus 3 for managing grapevine leafroll disease in vineyards” at the Northwest Center for Small Fruits Research 2019. December 2-4, 2019, Ferndale, WA.

Mitra, A., Jarugula, S., Donda, B., Jordan, E. and Naidu, R.A. 2019. “Genetic diversity of Grapevine leafroll-associated virus 3 in Washington State vineyards” at the 2019 American Phytopathological Society Annual Meeting, August 3-7, 2019, Cleveland, OH. (Received “Phytobiomes Student Poster Award” from the scientific journal ‘Phytobiomes’ at the American Phytopathological Society).

Naidu, R.A. “Status of Grape Viruses in Washington: Update on Research Progress” and “Virus Testing: How to sample, benefits of testing, interpreting results, economics of testing” at the Washington Advancements in Viticulture and Enology annual research seminar. February 19, 2020, Prosser, WA.

Nikolaeva E.V., Knier R., Molnar C., Peter K., Jones T., and Costanzo S. 2019. First Report of Strawberry (Fragaria × ananassa) as a Host of a ‘Candidatus Phytoplasma americanum’-Related Strain in the United States. Plant Disease V. 104, N 2. P.560.

Olmedo-Velarde Alejandro, Adam C. Park, Jari Sugano, Janice Y. Uchida, Michael Kawate, Wayne B. Borth, John S. Hu, and Michael J. Melzer 2019. Characterization of Ti ringspot-associated virus, a novel emaravirus associated with an emerging ringspot disease of Cordyline fruticosa (L.) Plant Disease https://doi.org/10.1094/PDIS-09-18-1513-RE

Olmedo-Velarde, A., Roy, A., Belanger, C.A., Watanabe, S., Hamasaki, R.T., Mavrodieva, V.A., Nakhla, M.K., Melzer, M.J. (2019). First Report of Tomato Chlorotic Dwarf Viroid Infecting Greenhouse Tomato in Hawaii. Plant Disease 103(5): 1049.

Pechinger, K., Chooi, K. M., MacDiarmid, R. M., Harper, S. J., & Ziebell, H. (2019). A new era for mild strain cross-protection. Viruses, 11(7), 670.

Peng, L., Wu, L., Grinstead, S.C., Kinard, G.R., Li, R. 2019. Molecular characterization and detection of two novel carlaviruses infecting cactus. Archives of Virology. https://doi.org/10.1007/s00705-019-04279-w.

Sanderson, D., & James, D. (2019). Analysis of the genetic diversity of genome sequences of variants of apple hammerhead viroid. Canadian Journal of Plant Pathology.

Thekke-Veetil, T., Ho, T., Postman, J. D., and Tzanetakis, I. E. 2020. Comparative analysis of a new blackcurrant waikavirus with other members of the genus. Eur. J. Plant Pathol., in press

Thompson, B.D., Dahan, J., Lee, J., Martin, R.R., and Karasev, A.V. 2019. A novel genetic variant of Grapevine leafroll-associated virus-3 (GLRaV-3) from Idaho grapevines. Plant Disease 103: 509-518 (http://dx.doi.org/10.1094/PDIS-08-18-1303-RE).

Thompson, B.D., Eid, S., **Vander Pol, D., Lee, J., and Karasev, A.V. 2019. First report of grapevine red blotch virus in Idaho grapevines. Plant Disease 103: 2704 (http://dx.doi.org/10.1094/PDIS-04-19-0780-PDN).

Wang, Y., Wang, Q., Yang, Z., Li, R., Liu, Y., Li, J., Li, Z., Zhou, Y. 2020. Development of a sensitive and reliable reverse transcription-droplet digital polymerase chain reaction (RT-ddPCR) assay for the detection of Citrus tristeza virus. European Journal of Plant Pathology. https://doi.org/10.1007/s10658-019-01920-x.

Wright, A. A., Cross, A. R., & Harper, S. J. (2020). A bushel of viruses: Identification of seventeen novel putative viruses by RNA-seq in six apple trees. Plos One, 15(1), e0227669.

Wright, A. A., Cross, A. R., & Harper, S. J. (2020). A bushel of viruses: Identification of seventeen novel putative viruses by RNA-seq in six apple trees. Plos One, 15(1), e0227669.

Wu, L., Du, T., Liu, H., Peng, L., Li, R. 2019. Complete genomic sequence of tea-oil camellia associated deltapartitivirus, a novel virus from Camellia oleifera. Archives of Virology. https://doi.org/10.1007/s00705-019-04429-0.

Wu, L., Liu, H., Bateman, M., Komorowaka, B., Li, R. 2019. First identification and molecular characterization of apricot symptomless virus. Archives of Virology. https://doi.org/10.1007/s00705-019-04401-y.

Zhang S., Davenport B., Li R., Matousek J., Groth-Helms D., Schuetz K. (2020): Development of an isothermal AmplifyRP XRT assay for rapid real-time detection of Hop stunt viroid through recombinase polymerase amplification. Plant Health 2020, Denver, Colorado, USA.

Zhang, S., Yang, L., Ma, L., Tian, X., Li, R., Zhou, C., Cao, M. 2020. Virome of Camellia japonica: discovery and molecular characterization of new viruses of different taxa in camellias. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2020.00945.

Zheng, L., Chen, M., Li, R. 2020. Camellia ringspot associated virus 4, a proposed new foveavirus from Camellia japonica. Archives of Virology. https://doi.org/10.1007/s00705-020-04655-x.

Zurn, J.D., Ho, T., Li, R., Bassil, N.V., Tzanetakis, I., Martin, R.R., Postman, J.D. 2019. First report of Blackcurrant reversion virus in Ribes nigrum germplasm in the United States. Plant Disease. 103:1051. https://doi.org/10.1094/PDIS-03-18-0526-PDN.