SAES-422 Multistate Research Activity Accomplishments Report

Sections

Basic Information
Additional Info

Status: Approved

Basic Information

Project No. and Title: WERA20 : Virus and Virus-Like Diseases of Berries, Fruit and Nut Trees, and Grapevines.
Period Covered: 10/01/2017 to 09/30/2018
Date of Report: 08/30/2018
Annual Meeting Dates: 06/27/2018 to 06/29/2018

Al Rwahnih, Maher (malrwahnih@ucdavis.edu) University of California-Davis; Almeyda, Christie (cvalmeyd@ncsu.edu) North Carolina State; Balci, Yilmaz (Yilmaz.Balci@aphis.usda.gov) USDA-APHIS; Cooper, Cindy (ccooper@agr.wa.gov) Washington State Department of Agriculture; Di Bello, Patrick (patrick.dibello@oregonstate.edu) Oregon State University; Gratz, Allison (allison.gratz@inspection.gc.ca) Canadian Food Inpsection Agency – Sidney; Guerra, Lauri (lguerra@agr.wa.gov) Washington State Department of Agriculture; Guzman, Melinda (guzmanme@oregonstate.edu) Oregon State University; Harper, Scott (scott.harper@wsu.edu) Washington State University-Prosser; Hu, John (johnhu@hawaii.edu) University of Hawaii; Hurtado-Gonzales, Oscar (Oscar.Hurtado-Gonzales@aphis.usda.gov) USDA-APHIS; Jones, Robert P (Robert.P.Jones@aphis.usda.gov) USDA-APHIS; Keller, Karen (karen.keller@ars.usda.gov) USDA-ARS; Kinard, Gary (Gary.Kinard@ARS.USDA.GOV) USDA-ARS; Lake, Amanda (amanda.lake@ars.usda.gov) USDA-ARS; Lavagi, Irene (irenela@ucr.edu) University of California-Riverside; Li, Ruhui (Ruhui.Li@ARS.USDA.GOV) USDA-ARS; Lichens Park, Ann (apark@nifa.usda.gov) USDA-NIFA; Lutes, Lauri Ann (Lauri.Lutes@oregonstate.edu) Oregon State University; Martin, Bob (Bob.Martin@ars.usda.gov) USDA-ARS; Mavrodieva, Vessela (Vessela.A.Mavrodieva@aphis.usda.gov) USDA-APHIS; Nakhla, Mark (Mark.K.Nakhla@aphis.usda.gov) USDA-APHIS; Olaya, Cristian, Washington State University; Postman, Joseph (Joseph.Postman@ARS.USDA.GOV) USDA-ARS; Poudyal, Dipak (dpoudyal@oda.state.or.us) Oregon Department of Agriculture; Puckett, Josh (jmpuckett@ucdavis.edu) University of California-Davis; Rabindran, Shailaja (Shailaja.Rabindran@aphis.usda.gov) USDA-APHIS; Rayapati, Naidu (naidu.rayapati@wsu.edu) Washington State University; Rosenbaum, Robin (rosenbaumr@michigan.gov) Michigan Department of Agriculture; Roy, Avijit (avijit.roy@aphis.usda.gov) USDA-APHIS; Rudyj, Erich (Erich.S.Rudyj@aphis.usda.gov) USDA-APHIS; Salati, Raquel (raquelsalati@eurofinsUS.com) Eurofins BioDiagnostics; Shiel, Pat (patrick.j.shiel@aphis.usda.gov) USDA-APHIS-PPQ; Sudarshana, Mysore (Sudi) (mrsudarshana@ucdavis.edu) USDA-ARS; Talton, Win (lwtalton@ncsu.edu) North Carolina State University; Trujillo, Sarah (sarah.g.trujillo@aphis.usda.gov) USDA-APHIS; Villamor, Dan Edward (dvvillam@uark.edu) University of Arkansas; Wei, Alan (apwei@agri-analysis.com), Agri-Analysis; Welliver, Ruth (rwelliver@pa.gov) Pennsylvania Department of Agriculture; Zhang Shulu (shulu@agdia.com) AGDIA

Brief Summary of Minutes of Annual Meeting

Meetings were held at the Courtyard by Marriott in Corvallis, Oregon from June 27-29. Thirty-three people attended the field trip on June 27. We first visited TRECO Oregon Rootstock & Tree Co. near Woodburn, OR which produces about 8 million apple rootstock trees per year. The showed the trimming, sorting, packing and cold storage facilities and then we visited stool beds. The stool beds ranged from newly planted to 40 year old beds. We then visited North American Plants Co. near McMinnville, OR, which is a large tissue culture facility that sells about 16 million finished plants per year, mostly in 2inch pots. The participants enjoyed these tours of large scale agriculture, where they may get 2-5 plants from a clean plant center and bulk them up for commercial sales. We then had lunch in McMinnville followed by a tour of the Ribes collection at the National Clonal Germplasm Repository where we were able to see Ribes with multiple virus infections, many of which were discovered by members in attendance. From there we did a tour of the NCGR facilities, and finished with a tour of the blueberry germplasm collection.

The meeting sessions began with a welcome by Dr. Naidu Rayapati from Washington State University on behalf of Dr. Scott Hulbert and Dr. Hanu Pappu, Administrative Advisors for the WERA20 group. Dr. Ann Lichens-Park, NIFA representative for the WERA20 group, gave an overview of funding opportunities available at NIFA related to the work being done by this group on the second day of the meetings.

There was an overview of the National Certification Harmonization efforts presented by Dr. Erich Rudyj from APHIS with updates on Nursery Pilot projects being overseen by the Oregon and Washington State Departments of Agriculture. This was followed by research and state reports from: North Carolina, Canadian Food Inspection Station, Arkansas, California, Hawaii, Washington and Oregon and research reports from Oregon, Washington, and ARS, updates on new tests available from Agdia and Agri-Analysis. There were also several discussion sessions. There was a session on Quality Control with talks by APHIS on how to prepare reference material for testing labs and ARS on how to identify a positive in qPCR, and on a ring test being done across multiple laboratories for blueberry, caneberry and strawberry viruses. There was then a discussion on High Throughput Sequencing as a tool for diagnosis and how to standardize the technology as a tool for quarantine and certification and how to get the funding to validate HTS as an acceptable test method. Finally there was a discussion on developing a database for the viruses of the crops covered by WERA-20 and NCPN where we had invited the developers to address questions or concerns on which information would be public and what would be limited.

At the business meeting we decided the next meeting would be held in Geneva, New York and organized by Marc Fuchs.

Meeting Agenda:

Agenda for WERA-20 Meetings June 27-29, 2018

Location: Courtyard by Marriott (400 SW 1st St.)

June 27, Field Trip

Pick-up at hotels at 8:00 AM, depart at 8:10 AM for TRECO Oregon Rootstock & Tree Co.

Arrive at TRECO at 9:20

TRECO Facility and Field tour 9:20-10:30

Depart TRECO at 10:30

Travel to North American Plant

Arrive North American Plant 11:30

Tour North American Plant 11:30 to 12:30 (Large commercial tissue culture company)

12:30 Depart for lunch in McMinnville Oregon

Lunch 12:45 to 2:00

Depart for Corvallis, OR at 2:00

Arrive Ribes Collection 3:00, see at Black currant reversion and Tomato ringspot virus

Arrive at National Clonal Germplasm Repository 3:45

Tour NCGR

5:00 Return to hotel

Dinner on your own

June 28 – Meetings at the Courtyard by Marriott in the Cascade/Willamette rooms

8:00 – 8:30 Registration

8:30 – 9:00 Welcome and Introductions

9:00 Nursery Certification – Overview of Certification Harmonization Efforts at the National Level - Erich Rudyj and Cindy Cooper

10:00 Break

10:20 Updates on Certification Programs in Oregon, Dipak Poudyal

10:40 NCPN-Berries: North Carolina Update, Christie Almeyda

11:00 Update CFIA-Sidney, B.C., Laboratory for Plant Health, Allison Gratz

11:20 Arkansas Report, Dan Villamor

11:40 California State Report, Maher Al Rwahnih

12:00 – 1:00 Lunch

1:00 Nanobody and its use against grapevine viruses, Alan Wei

1:20 AmplifyRP®: a versatile diagnostic tool for rapid detection of plant pathogens, Shulu Zhang

1:40 What’s a positive in qPCR? Mysore Sudarshana

2:00 Comparability of diagnostic testing between labs through use of reference materials and standard test conditions” and/or “Quality Management concepts in laboratory settings, Pat Shiel

2:20 Further characterization of PMWaVs in Hawaii, John Hu

2:40 Sweet cherry virus survey in Oregon, Host-specificity and diversity of ToRSV on tree and small fruits proposal, Lauri Ann Lutes

3:00 Break

3:30 Ring test of viruses in berry crops, Bob Martin

3:50 Grapevine viruses in Washington State, Naidu Rayapati

4:10 Disease trends in fruit trees in Washington State, Scott Harper

4:30 Business meeting

5:00 Finish for the day

5:45 Travel to Lumos Winery for Dinner

Return to hotels about 9:00 PM

June 29 – Meetings at the Courtyard by Marriott in the Cascade/Willamette rooms

8:00 Implementing Next Generation Sequencing Technologies in PGQP-Field Operations, Oscar Hurtado-Gonzales

8:20 Molecular characterization and detection of two viruses infecting blackcurrant, Ruhui Li

8:40 NCPN-Berries Update – Corvallis

9:00 Virus database for NCPN crops. Bob Martin, Carrie Hertel, Mark Clemens

10:00 Break

10:30 Validation of High Throughput Sequencing for Plant Release from Quarantine– How do we position ourselves to be successful in the SCRI. Maher Al Rwahnih, Mark Nahkla

11:30 Other topics for discussion?

12:00 Close Meeting.

Accomplishments

Plant Health Program, Oregon Department of Agriculture administrates pathogen-tested certification programs for clonally propagated planting materials such as fruit trees and grapevines, performs statewide survey of state and federal level quarantine or regulated pathogens, and provides diagnostic service to nurseries and growers. In 2017-2018, following major activities related to certification programs were completed.

Major accomplishment:

Oregon Department of Agriculture Report

Twenty-eight nurseries participated in Oregon's virus ornamental and fruit tree certification program in 2017. These nurseries grow registered plants (either G2 or G3 or both) of Malus (apples and crabapples), Prunus (fruiting and ornamental cherries, fruiting and ornamental plums, peaches, apricots, etc.), Pyrus (domestic pears, Asian pears, and flowering pears), and Cydonia (quince). Registered plants are inspected and tested regularly for viruses of economic importance.

Malus, Pyrus, and Cydonia scions and rootstocks are tested for three latent viruses, Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV). In fall of 2017, a total of 4685 samples from 25 nurseries were tested for these viruses. All the samples tested negative to ACLSV and ASGV but eight samples were found ASPV positive. Delimitation surveys were conducted in these positive sites and infected materials were eradicated.

Oregon has been a key stakeholder to implement a multiyear project to create a regional grapevine virus certification program by harmonizing quarantines and programs for grapevine nursery stock certification with neighboring states (Idaho and Washington) in the Pacific Northwest. Grapevine quarantine pests were compared and a common pest list was developed. The Idaho State Department of Agriculture (ISDA), Oregon Department of Agriculture (ODA), Washington State Department of Agriculture (WSDA), and Washington Wine Industry Foundation (WWIF) worked closely with other stakeholders in the grapevine and wine industries to develop harmonized rules for registration and certification of grapevine stock program for the Pacific Northwest.

A pilot study was performed based on these registration and certification standards to determine whether Oregon grapevine nurseries currently meet the standards developed for the PNW or not. Three grapevine nurseries participated in the pilot study in the fall of 2017. A detailed questionnaire inquiring about the source of mother plants (G2 and G3) and describing the requirements for registered blocks in fields, greenhouses/screenhouses, or containers, certified nursery stocks (G4), and best management practices was used. In general, the three nurseries met most of the requirements as outlined in the draft standards. Nurseries were also inspected for diseases and samples were collected for testing for indicator pathogens. A total of 59 leaf samples were collected from each nursery to test for Grapevine red blotch virus, Grapevine leaf roll-associated virus 3, and Xylella fastidiosa. All these samples tested negative to these pathogens.

Three caneberry nurseries participated in the pilot study to assess similarities and differences between their current practices and the standards based on the State Level Model Regulatory Standard (SLMRS) proposed by the National Clean Plant Network (NCPN). The three nurseries have different nursery stock production systems – Nursery 1 grows stock as plantlets, Nursery 2 grows stock in containers, and Nursery 3 has field-grown stock. Nurseries were assessed for source of mother stocks (G2 or G3), nursery locations for growing mother (G2 or G3) and nursery stocks (G4), and adherence to best management practices.

All three nurseries meet the SMLRS standards with a few exceptions. The three nurseries source their mother stocks (G2 or G3) from a Clean Plant Center to varying degrees. Nursery 1 obtains G1 materials from Clean Plant Center. Nursery 2 uses G2 materials and certified materials from other states as mother stocks. Nursery 3 uses plantlets obtained from G2 sources and field grown canes of unknown G level as mother stocks. All the nurseries satisfied requirements for location or sites for producing G2, G3, or G4 materials. Nurseries varied in their adherence to best management practices described by the SMLRS. Nursery 2 and 3 did not meet the requirement for isolation distance from uncertified Rubus spp., and primocane and flowering cultivars. Nursery 1 and 2 grew nursery stock in soil-less media. Nursery 3 produces field-grown stock; nematode testing was not performed prior to planting as required by the standards.

A total of 59 samples were collected and tested for five economically important canary viruses, Black raspberry necrosis virus (BRNV), Raspberry bushy dwarf virus (RBDV), Raspberry leaf mottle virus (RLMV), Strawberry necrotic shock virus (SNSV), and Tomato ringspot virus (ToRSV). RBDV, SNSV and ToRSV were tested by ELISA and BRNV and RLMV were tested using virus-specific reverse transcriptase PCR. All of the nursery samples tested negative for BRNV, SNSV, and ToRSV. Samples from Nursery 1 and 2 tested negative for RLMV and RBDV. Of the samples obtained from Nursery 3, 15% and 42% were positive for RLMV and RBDV, respectively. In addition, soil samples were collected from Nurseries 2 and 3 to test for the presence of nematodes. ToRSV, is vectored by Xiphenema americanum. X. americanum nor any other plant parasitic nematodes was found in samples from Nursery 2. In Nursery 3, Xiphinema spp. were not observed but some plant parasitic nematodes were found in low densities.

Arkansas report

In collaboration with colleagues, several of which participate in WERA-20 we are working on the characterization and population structure of several viruses in blueberry, currant and blackberry. This information is used in the development of detection protocols that have the ability to detect the vast majority of isolates that circulate in the United States

We have open channels of communication with both industry and regulators to optimize the guidelines so as to be implemented in the near future. We are holding meetings with state regulators to harmonize certification guidelines among states and commodities.

Validation of High Throughput Sequencing as a method for quarantine or certification testing – in collaboration with USDA-ARS Corvallis, OR. We have compared HTS as a detection method with a combination of bioassays, ELISA and PCR for 10 samples each of Strawberry, Blueberry and Rubus. In all cases with one exception, the HTS detected all viruses detected by conventional means. With HTS we also identified several new viruses in each of the crops. Two of the viruses in blueberry have been validated by PCR testing. This project will continue next year.

Cornell Report

Grapevine red blotch virus. An economic study estimated the cost of GRBV to range from $2,210 to $68,550/ha when initial infected rates and quality penalties for suboptimal fruit composition are high. Based on these estimates, roguing symptomatic vines and replanting with clean vines derived from virus-tested stocks minimize losses if virus incidence is low to moderate (below 30%), while a full vineyard replacement should be pursued if disease incidence is higher, generally above 30%.

Surveys of GRBV and wild Vitis virus 1 (WVV1) in free-living Vitis species in northern California and New York from 2013 to 2017 showed the presence of these two grabloviruses in 28% (57 of 203) of samples from California but in none (0 of 163) from New York. The incidence of GRBV was significantly higher in free-living vines from California counties with high compared to low grape production, and in samples near (< 5km) to compared to far (> 5km) from vineyards. These results suggested a directional spread of GRBV inoculum predominantly from vineyards to free-living Vitis species. WVV1 incidence was also significantly higher in areas with higher grape production acreage. However, in contrast to GRBV, no differential distribution of WVV1 incidence was observed with regard to distance from vineyards. Two distinct phylogenetic clades were identified for both GRBV and WVV1 isolates from free-living Vitis species, although the nucleotide sequence variability of the genomic diversity fragment was higher for WWV1 than GRBV. Additionally, evidence for intra-specific recombination events was found in WVV1 isolates and confirmed in GRBV isolates. The prevalence of grabloviruses in California free-living vines highlights the need for vigilance regarding potential virus inoculum sources in order to protect new vineyard plantings and foundation stock vineyards in California.

For GFLV, a system of binary plasmids based on the two genomic RNAs of GFLV strains F13 and GHu was designed and optimized parameters to maximize systemic infection frequency in Nicotiana benthamiana via agroinoculation were investigated. The genomic make-up of the inoculum, the identity of the co-infiltrated silencing suppressor, and temperature at which plants were maintained significantly increased systemic infection.

Using a reverse genetics approach, the RNA2-encoded protein 2A^HP of GFLV strain F13, particularly to its 50 C-terminal amino acids, was shown to carry the determinant of necrotic lesions on inoculated leaves of Nicotiana occidentalis (Martin et al., 2018). The necrotic response showed hallmark characteristics of a genuine hypersensitive reaction, such as the accumulation of phytoalexins, reactive oxygen species, pathogenesis-related protein 1c and hypersensitivity-related (hsr) 203J transcripts. Transient expression of the GFLV-F13 protein 2A^HP fused to an enhanced green fluorescent protein (EGFP) tag in N. occidentalis by agroinfiltration was sufficient to elicit a hypersensitive reaction. In addition, the GFLV-F13 avirulence factor, when introduced in GFLV-GHu, which causes a compatible reaction on N. occidentalis, elicited necrosis and partially restricted the virus.

Collaborative research

Research at Cornell was collaborative during this reporting period with efforts between (i) Cornell and Washington State University, and extension educators in New York and California, and (ii) Cornell and The Ohio State University, (iii) Cornell and UC-Davis, (iv) Cornell and industry researchers, and (v) Cornell and scientists in France. Research was also interdisciplinary with the involvement of virologists, entomologists, geneticists, viticulturists, quantitative epidemiologists, biologists and agriculture economist.

Other accomplishments

Efforts to reinstate a grape certification program in New York are under way. It is anticipated that the first NY certified material will be available in 2019 or 2020.

APHIS-PPQ Report

For us in the regulatory realm, we rely on high quality research to inform our decisions and serve our stakeholders. My contributions to the effort are in alignment of research efforts with regulatory programs to identify, characterize, and mitigate threats to U.S. agriculture and natural resources. Last year, we helped organize and deliver a workshop at the national American Phytopathological Society meeting on assay method validation and use. We have written a review paper submitted for publication in Plant Health Progress.

We feel that this paper will contribute to general understanding of method validation by the research community and will inform the current and future development of methods, especially High Throughput Sequencing (HTS). In addition to this, PPQ is also heavily involved in working with the international plant testing community to learn and harmonize assays for smooth and transparent international trade. Over the last year, we have invited researchers like Mahar Al Rwahnih and Mike Rott to update us on the latest technologies. We of course maintain contact with other researchers like you to help provide subject matter expertise on specific pathogens or situations.

Impact: The impact of PPQ on WERA-20 is significant. Many of the new organisms characterized by the WERA-20 researchers are of US regulatory concern. We appreciate the openness and cooperation in working with us to balance the needs of furthering the scientific knowledge of these organisms with our obligations to protect US agriculture and natural resources from the threats of outbreak and possible damage of these organisms
Collaboration: PPQ is also heavily involved in working with the international plant testing community to learn and harmonize assays for smooth and transparent international trade. We have invited researchers like Mahar Al Rwahnih and Mike Rott to update us on the latest technologies. We also fund WERA-20 projects that are related to PPQ’s mission and collaborate with researchers on projects that impact our mission.
Other accomplishments: We seek to further our interactions with WERA-20 research community in the areas of assay harmonization and inter-laboratory coordination of testing to demonstrate direct comparability and trend tracking of results for overall program improvement. We have developed tools for plant diagnosticians to further these efforts and we look forward to working with the WERA-20 to showcase these tools and explore their applicability to diagnostic testing done by the WERA-20.

Washington State University Report

Virus diseases affecting fruit quality and vine health are one the highest research priorities for the grape and wine industry in Washington State. In response, the grape virology program (http://wine.wsu.edu/virology/ ) is conducting fundamental and applied research in a holistic manner to generate science-based knowledge for mitigating negative impacts of virus diseases in vineyards. Vineyard surveys have consistently shown that Grapevine leafroll-associated virus 3 (GLRaV-3) is more common across the state vineyards than Grapevine red blotch virus (GRBV). Thus, GLRaV-3 is considered as a significant constraint for advancing vineyard health and productivity. Due to similar symptoms produced by leafroll and red blotch diseases in red-fruited wine grape cultivars, molecular diagnostic assays are essential for reliable identification of GLRaV-3 and GRBV in vineyards. Studies in commercial vineyards have shown that both GLRaV-3 and GRBV can cause significant impacts on fruit yield and grape quality in several red-fruited wine grape cultivars. Of the two nepoviruses (Tobacco ring spot virus [TRSV] and Grapevine fanleaf virus [GFLV]) causing fanleaf degeneration and decline symptoms, TRSV was found in one commercial vineyard, whereas GFLV was detected in two commercial vineyards located in distinct geographic locations. Studies have shown that TRSV can be spread by the dagger nematode, Xiphinema rivesi. Conversely, the dagger nematode species currently present in Washington soils (except X. index, the known vector of GFLV) are unlikely to spread GFLV. Thus, a combination of planting virus-tested cuttings and implementing nematode control are necessary for the management of TRSV. In contrast, roguing infected vines and replanting with virus-tested cuttings would help preventing the spread of GFLV in vineyards. Virus indexing of grapevines in Certified Mother blocks in registered nurseries revealed the absence of GRBV and very low incidence of GLRaV-3. Thus, registered nurseries in Washington State appears to be free from red blotch virus. The research-based knowledge was shared with grape and wine industry stakeholders and regulatory agencies via presentations at grower meetings and workshops for broader dissemination of research outcomes.

Foundation Plant Services (UC-Davis) 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, 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.

Progress has also been made in improving the efficiency of diagnostic laboratory assays for Grapevine leafroll-associated virus 3 (GLRaV-3). GLRaV-3 has an exceptional number of highly diverse variants. Recent studies based on genome-wide phylogenetic analysis demonstrated that GLRaV-3 variants can be divided into eight distinct groups, six of which have been identified in California. This level of genetic diversity makes it almost impossible to identify a conserved region common to all isolates for design of a single qPCR-based assay. Up to now, FPS has dealt with this problem by designing variant-specific assays, six to date. However, it isn’t feasible to test large numbers of vines using six different assays. FPS embarked on a study to survey and analyze GLRaV-3 genetic variants and work towards improving the RT-qPCR assay design. FPS reconstructed the near complete genomes of 34 GLRaV-3 isolates, including three new divergent variants using HTS and incorporated new genetic data into a more complete characterization of genetic variation across GLRaV-3 variants. A small but highly conserved region was identified that was then used to construct a single new RT-qPCR assay, referred to as FPST, for detecting all GLRaV-3 variants characterized to date. When compared with previous GLRaV-3 assays, FPST was the one RT-qPCR assay that detected ALL variants obtained to date. The assay was further validated with 2452 samples obtained from USDA National Clonal Germplasm Repository in Winters, CA, the Davis Virus Collection at UC-Davis, the FPS pipeline of foreign and domestic introductions, selected vineyards in the main grape-growing areas of California, and samples provided by international collaborators. Further verification of the FPST assay was obtained by testing the above population with a new GLRaV-3 Enzyme-linked immunosorbent assay (ELISA) kit. This new ELISA kit has detected all known GLRaV-3 variants characterized to date and the side-by-side comparison indicated that all samples testing positive by the FPST RT-qPCR assay also tested positive by the new ELISA kit. Finally, the new assay is being shared with stakeholders, thus benefiting growers, researchers, and diagnostic labs involved in the grapevine industry in the US and around the world.

New viral discoveries were made in pistachio trees. Pistachio trees from the National Clonal Germplasm Repository (NCGR) and selected orchards in California were surveyed for viruses and virus-like agents by HTS. Analyses of 60 trees, Pistacia vera and clonal UCB-1 hybrid rootstock (P. atlantica × P. integerrima), identified a novel virus found in the NCGR that showed low amino acid sequence homology (~42%) to members of genus Ampelovirus, family Closteroviridae and was provisionally named “Pistachio Ampelovirus A” (PAVA). A putative viroid, provisionally named “Citrus bark cracking viroid-pistachio” (CBCVd-pis), was also found in the NCGR and showed approximately 87% similarity to Citrus bark cracking viroid (CBCVd, genus Cocadviroid, family Pospiviroidae). Both pathogens were graft transmissible to healthy UCB-1 plants. A field survey of 123 trees from commercial orchards found no incidence of PAVA, but five (4%) samples were infected with CBCVd-pis. Of 675 NCGR trees, 16 (2.3%) were positive for PAVA and 172 (25.4%) were positive for CBCVd-pis by reverse-transcription polymerase chain reaction.

A novel RNA virus was detected in grapevine cultivar ‘Kizil Sapak’ by HTS and tentatively named “grapevine virus J” (GVJ). The full genome of GVJ is 7,390 nucleotides in length, which comprises five open reading frames (ORFs), including a 20K ORF (ORF 2) between the replicase (ORF 1) and the movement protein (ORF 3) genes. According to the level of sequence homology and phylogenetics, GVJ is proposed as a new member of the genus Vitivirus (subfamily Trivirinae; family Betaflexiviridae), with the closest characterized virus being grapevine virus D (GVD). Further work is needed to identify the mechanisms by which GVJ is transmitted. Field surveys and biological studies are underway to determine the prevalence of GVJ in commercial vineyards and to assess its effect on vine performances. In addition, the incidence of GVJ in other selections introduced to FPS and the USDA National Clonal Germplasm Repository in Winters, CA, will be determined using the newly developed RT-PCR assay.

A geminivirus was identified for the first time in Prunus spp. This novel virus, provisionally named Prunus geminivirus A (PrGVA), was identified by Illumina sequencing from an asymptomatic plum tree. PrGVA was subsequently confirmed by rolling cycle amplification, cloning, and Sanger sequencing of its complete genome (3,174 to 3,176 nucleotides) from an additional 18 (9 apricot and 9 plum) field isolates. Apart from the nonanucleotide motif TAATATT↓AC present in its virion strand origin of replication, other conserved motifs of PrGVA support its geminiviral origin. PrGVA shared highest complete genome (73 to 74%), coat protein amino acid (83 to 85%) and rep-associated amino acid (74%) identities with Grapevine red blotch virus (GRBV). PrGVA was graft but not mechanically transmissible. Quantitative polymerase chain reaction screening of Prunus spp. in the National Clonal Germplasm Repository collection using newly designed primers and probes revealed 69.4% (apricot), 55.8% (plum), and 8.3% (cherry) incidences of PrGVA. PrGVA is proposed as a novel member of the genus Grablovirus based on its close genome and phylogenetic relationship with GRBV.

Rose rosette disease (RRD) of roses is widely distributed in the eastern U.S. RRD is caused by Rose rosette virus (RRV), which is vectored by the eriophyid mite (Phyllocoptes fructiphilus Keifer). California is one of the main producers of roses in the U.S. with a wholesale value of US$ 17.6 million in 2016. In August 2017, symptoms suggestive of RRD were observed on two roses (cvs. Veterans’ Honor and Brilliant Pink Iceberg) in a commercial nursery in Wasco, CA. A total of 415 samples, representing many rose cultivars were collected from the nursery and tested for RRV by RT-qPCR assay. Only the two symptomatic plants were positive for RRV. Plants were also microscopically examined and the P. fructiphilus mite was observed and recovered from several plants throughout the nursery. In June 2018, a separate incident occurred where three garden roses (unknown cultivars) planted in two neighboring homes in Bakersfield, CA, about 50 km from the 2017 finds, were identified with RRD symptoms. Leaves from all three plants tested positive for RRV by RT-qPCR and were also infested with P. fructiphilus mites. To confirm these results HTS was performed on all five symptomatic plants and analysis revealed that the RRV genomes from the two nursery plants sampled in 2017 were 100% identical to one another. The RRV genomes from the three homeowner plants sampled in 2018 were 100% identical to each other, but not to the nursery samples. These results suggest that the samples from these two locations are likely two separate introductions of RRV. To our knowledge, this is the first detection of RRV causing RRD in CA.

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 begun evaluating the effects of LChV-1 and LChV-2 on 16 different popular Prunus rootstocks. All rootstocks will be grafted with a scion variety from the same accession. Observations of budtake and tree performance will be recorded and evaluated for two years. Rootstocks will 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.

An interactive relationship between vitiviruses and grapevine leafroll viruses was characterized in grapevine. Grapevine viruses A and B (GVA and GVB) were found more frequently in the presence of co-infecting Grapevine leafroll associated viruses (GLRaV-1, −2 or −3) than in their absence. The titers of the vitiviruses in co-infection with leafroll viruses were found to be higher than were their titers in the absence of leafroll virus infection. The occurrence of vitivirus-associated stem-pitting symptoms was correlated with leafroll virus co-infection. Specific pairing associations on the species level were found between different viti- and leafroll virus species: GVB was associated preferentially with GLRaV-2; GVA was associated preferentially with GLRaV-1 and GLRaV-3. In contrast to the increase in vitivirus titer seen with leafroll virus co-infection, the incidence and titer of grapevine leafroll virus appeared to be unaltered by vitivirus co-infection.

FPS has also worked collaboratively with James Schoelz at the University of Missouri to conduct a state-wide survey of about 400 grapevines samples for common grapevine viruses. Several viruses were detected, including Grapevine red blotch associated virus. The results of this study will help with certification and to establish a clean plant program.

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 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.

USDA-ARS Corvallis Report

Developing reliable testing virus protocols is critical for certification, quarantine and studies on virus epidemiology. We have coordinated a ring test among 6-9 laboratories (depending on crop) for blueberries, strawberries and caneberries. We collected 10-12 samples from each crop, most with known virus infections and several healthy plants for each crop. The tissues were powdered in liquid nitrogen, aliquoted and shipped on dry ice to the various laboratories. Each lab was sent a list of primer pairs to use for the RT-PCR testing along with parameters for running the PCRs, annealing temps, number of cycles, amplicon size. This was completely blind, some of the primers pairs should have been negative in all tests, while others should have given positive results in multiple samples. Some samples had mixed infections, some single infections. Each lab was asked to run the tests, and send back the result. The testing was consistent across most laboratories, but there did appear to be some contamination issues. We are repeating these tests again this year, and will use screw on vials for the samples rather than Eppendorf tubes with snap lids since opening these could cause a release of some of the fine powdered tissue. We also did bioassays on the samples in our lab.

We are continuing our work on Raspberry leaf curl disease and now have five viruses from native Rubus species in the northeastern U.S. and eastern Canada where the disease was common in the first half of the 20^th century. We are propagating a cultivar of red and black raspberry and a blackberry that were widely grown in that region at the time the disease was prevalent. We obtained from the National Clonal Germplasm Repository and in the process of inoculating these plants with various combinations of the viruses detected in native and commercial raspberries in the northeast.

We are also working with the harmonization of certification standards for the three major berry crops and grapevines. In addition, we are taking the lead on developing a virus database for the viruses of crops in the National Clean Plant Network. This project is in cooperation with WERA-20 members that are directors of NCPN centers for the various crops (Berries, Citrus, Grapevines, Hops, Roses, Sweetpotatoes and Tree Fruit). The structure of the database is being developed in collaboration with the Center for Applied Software and Systems at Oregon State University.

AGDIA Report

Early detection and effective control of plant pathogens is very important to prevent their wide spread resulting in serious economic losses in crops like cherries, citruses, grapes, hops, peaches or plums. Agdia's AmplifyRP^® utilizes a leading isothermal amplification technology called recombinase polymerase amplification (RPA) and has recently become a versatile diagnostic tool for rapid and accurate detection of nucleic acids in all types of plant pathogens. So far, Agdia has commercialized 13 pathogen-specific AmplifyRP^® kits in one of the three formats (Acceler8^®, XRT or XRT⁺) currently available. Among them, 5 AmplifyRP^® kits including AmplifyRP^® XRT⁺ for Xylella fastidiosa (Xf) are newly released since 2017 WERA-20 meeting. AmplifyRP^® XRT⁺ for Hop stunt viroid (HSVd) has also been developed. In addition to the 13 AmplifyRP^® kits, Agdia offers two AmplifyRP^® Discovery kits that can be used for any pathogens. Agdia has recently acquired the intellectual properties right for AmpliFire and started to offer this portable, battery-operable fluorescence reader. Overall, AmplifyRP^® is simple, as sensitive as PCR or qPCR, and field-deployable. No thermal cycler and DNA/RNA purification are needed as all reactions works well with plant crude extracts at an isothermal temperature 39°C.

Impacts

At FPS, we now have in-house virus testing employing the latest HTS technology using a verified, established protocol. Our work emphasizes the importance of establishing biological significance of novel viruses discovered by HTS. This has become the more demanding work for viruses discovered by HTS. APHIS has approved provisional release of material based on HTS, which can greatly shorten the time for getting a new cultivar to a nursery.
Side-by-side biological indexing and lab indexing with HTS is being done for Grapevines, Berries and Tree Fruit crops and will continue until we have enough corroborative evidence to support the findings of HTS. At that point we will prepare a white paper proposing the adoption of HTS as an acceptable means of virus testing for quarantine and certification. This will be presented to APHIS and from there to trading partners and Plant Protection Organizations.
Harmonization of certification programs across states for the Berries, Grapes, Tree fruit and nut crops have progressed this past year. These are joint efforts by State Depts. of Agriculture, the nursery industry and researchers. Draft model standards have been developed and piloted for several crops (blueberry, tree fruit, and caneberry). There is also a project to standardize grape certification in the Pacific Northwest (OR, WA, ID) and this standard is moving forward towards regulation in the three states.
The directors of the NCPN centers are all involved with WERA-20 and make use of information presented at WERA-20 to improve their testing programs. These directors are also involved in developing a virus database for the WERA-20 crops with a focus on information for regulators, nurseries and growers. Updating the information in this database is expected to become a dedicated activity of the WERA-20 group at their annual meetings.
The impact of PPQ on WERA-20 is significant. Many of the new organisms characterized by the WERA-20 researchers are of US regulatory concern. We appreciate the openness and cooperation in working with us to balance the needs of furthering the scientific knowledge of these organisms with our obligations to protect US agriculture and natural resources from the threats of outbreak and possible damage of these organisms.
PPQ is also heavily involved in working with the international plant testing community to learn and harmonize assays for smooth and transparent international trade. We have invited researchers like Mahar Al Rwahnih and Mike Rott to update us on the latest technologies. We also fund WERA-20 projects that are related to PPQ’s mission and collaborate with researchers on projects that impact our mission.
PPQ seeks to further our interactions with WERA-20 research community in the areas of assay harmonization and inter-laboratory coordination of testing to demonstrate direct comparability and trend tracking of results for overall program improvement. We have developed tools for plant diagnosticians to further these efforts and we look forward to working with the WERA-20 to showcase these tools and explore their applicability to diagnostic testing done by the WERA-20.

Publications

WERA-20 Publications 2017/2018

Adiputra, J., Kesoju, S.R., Naidu, R.A. 2018. The relative occurrence of Grapevine leafroll-associated virus 3 and Grapevine red blotch virus in Washington State vineyards. Plant Disease (in press).

Al Rwahnih, M., Alabi, O. J., Westrick, N. M., and Golino, D. 2018. Prunus geminivirus A: A Novel Grablovirus Infecting Prunus spp. Plant Disease. 102:1246–1253.

Al Rwahnih, M., Rowhani, A., Westrick, N., Stevens, K., Diaz-Lara, A., Trouillas, F. P., et al. 2018. Discovery of Viruses and Virus-Like Pathogens in Pistachio using High-Throughput Sequencing. Plant Disease. 102:1419–1425.

Cieniewicz, E., Pethybridge S., Gorny, A., Madden, L., Perry, K.L., McLane, H. and Fuchs, M. 2017a. Spatiotemporal spread of grapevine red blotch-associated virus in a California vineyard. Virus Research 230:59-62.

Cieniewicz, E., Pethybridge S.J., Loeb, G.M., Perry, K.L. and Fuchs, M. 2018b. Insights into the ecology of grapevine red blotch virus in a diseased vineyard. Phytopathology 108:94-102.

Cieniewicz, E., Thompson, J.R., McLane, H., Perry, K.L., Dangl, G.S., Corbett, Q., Martinson, T., Wise, A., Wallis, A., O’Connell, J., Dunst, R., Cox, K. and Fuchs, M. 2018a. Prevalence and diversity of grabloviruses in free-living Vitis spp. Plant Disease, https://apsjournals.apsnet.org/doi/pdf/10.1094/pdis-03-18-0496-re.

Coletta-Filho, H.D., Francisco, C.S., Lopes, J.R.S., Muller, C. and Almeida, R.P.P. 2017. Homologous recombination and Xylella fastidiosa host-pathogen associations in South America. Phytopathology 107: 305-312.

Cornara, D., Cavalieri, V., Dongiovanni, C., Altamura, G., Palmisano, F., Bosco, D., Porcelli, F., Almeida, R.P.P. and Saponari, M. 2017. Transmission of Xylella fastidiosa by naturally infected Philaenus spumarius (Hemiptera, Aphrophoridae) to different host plants. Journal of Applied Entomology 141: 80-87.

Cornara, D., Saponari, M., Zeilinger, A.R., de Stradis, A., Boscia, D., Loconsole, G., Bosco, D., Martelli, G.P., Almeida, R.P.P. and Porcelli, F. 2017. Spittlebugs as vectors of Xylella fastidiosa in olive orchards in Italy. Journal of Pest Science 90: 521-530.

Daugherty, M.P., Zeilinger, A.R. and Almeida, R.P.P. 2017. Conflicting effects of climate and vector behavior on the spread of a plant pathogen. Phytobiomes 1: 46-53.

Di Bello, P.L., Laney, A.G., Druciarek, T., Ho, T., Gergerich, R.C., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. 2016. A novel Emaravirus is associated with redbud yellow ringspot disease. Virus Res. 222:41-47.

Diaz-Lara, A., Golino, D., and Al Rwahnih, M. 2018. Genomic characterization of grapevine virus J, a novel virus identified in grapevine. Archives of Virology. 163:1965–1967.

Finn, C.E., Strik, B.C., Yorgey, B.M., Peterson, M.E., Jones, P.A., Lee, J. and Martin, R.R. 2018. ‘Columbia Giant’ thornless trailing blackberry. HortScience 53:251-255.

Finn, C.E., Strik, B.C., Yorgey, B.M., Peterson, M.E., Jones, P.A., Lee, J. and Martin, R.R. 2018. ‘Columbia Sunrise’ thornless trailing blackberry. HortScience 53:256-260.

Francisco, C.S., Ceresini, P.C., Almeida, R.P.P. and Coletta-Filho, H.D. 2017. Spatial genetic structure of coffee-associated Xylella fastidiosa populations indicates that cross-infection does not occur with sympatric citrus orchards. Phytopathology 107: 395-402.

Giampetruzzi, A., Saponari, M., Almeida, R.P.P., Essakhi, S., Boscia, D., Loconsole, G. and Saldarelli, P. 2017. Complete genome sequence of the olive-infecting strain Xylella fastidiosa subsp. pauca De Donno. Genome Announcements 5: e00569-17.

Giampetruzzi, A., Saponari, M., Loconsole, G., Boscia, D., Savino, V.N., Almeida, R.P.P., Zicca, S., Landa, B.B., Chacón-Diaz, C. and Saldarelli, P. 2017. Genome-wide analysis provides evidence on the genetic relatedness of the emergent Xylella fastidiosa genotype in Italy to isolates from Central America. Phytopathology 107: 816-827.

Hassan, M., Di Bello, P.L., Keller, K.E., Martin, R.R., Sabanadzovic, S. and Tzanetakis, I.E. 2017. A new, widespread emaravirus discovered in blackberry. Virus Research 235:1-5.

Herrbach, E., Alliaume, A., Prator, C.A., Daane, K.M., Cooper, M.L. and Almeida, R.P.P. 2017. Vector transmission of grapevine-leafroll associated viruses. In: B. Meng et al. (eds.), Grapevine Viruses: Molecular Biology, Diagnostics and Management. DOI 10.1007/978-3-319-57706-7_24.

Ho, T., Harris, A., Katsiani, A., Khadgi, A., Schilder, A. and Tzanetakis, I.E. 2018. Genome sequence and detection of Peach rosette mosaic virus. Journal of Virological Methods 254: 8-12.

Jimenez, J., Webster, C.G., Moreno, A., Almeida, R.P.P., Blanc, S., Fereres, A. and Uzest, M. 2017. Fasting alters aphid probing behaviour but does not universally increase the transmission rate of non-circulative viruses. Journal of General Virology 98: 3111-3121.

Kandel, P.P., Almeida, R.P.P., Cobine, P.A. and De La Fuente, L. 2017. Natural competence rates are variable among Xylella fastidiosa strains and homologous recombination occurs in vitro between subspecies fastidiosa and multiplex. Molecular Plant-Microbe Interactions 30: 589-600.

Koloniuk, I., Thekke-Veetil, T., Reynard, J.S., Mavrič, I.P., Přibylová, J., Brodard, J., Kellenberger, I., Sarkisova, T., Špak, J., Lamovšek, J. and Massart, S., 2018. Molecular Characterization of Divergent Closterovirus Isolates Infecting Ribes Species. Viruses 10: 369

Labroussaa, F., Ionescu, M., Zeilinger, A.R., Lindow, S.E. and Almeida, R.P.P. 2017. A chitinase is required for Xylella fastidiosa colonization of its insect and plant hosts. Microbiology 163: 502-509.

Li, R., Fuchs, M., Perry, K.L., Mekuria, T. and Zhang, S. 2017. Development of a fast AmplifyRP Acceler8 diagnostic assay for grapevine red blotch-associated virus. Journal of Plant Pathology 99:657-662.

Lutes, L. A., and Pscheidt, J. W. 2018. First Report of Cherry leaf roll virus on Sweet Cherry in Oregon. Plant Disease. 102:691–691.

Maree, H. J., Fox, A., Al Rwahnih, M., Boonham, N., and Candresse, T. 2018. Application of HTS for routine plant virus diagnostics: State of the art and challenges. Front. Plant Sci. 9 Available at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01082/full [Accessed July 12, 2018].

Martin, I., Vigne, E., Berthold, F., Komar, V., Lemaire, O., Fuchs, M. and Schmitt-Keichinger, C. 2018. The fifty distal amino acids of the 2A^HP homing protein of grapevine fanleaf virus elicit a hypersensitive reaction on Nicotiana occidentalis. Molecular Plant Pathology 19:731-743.

Martin, R.R. and Tzanetakis, I.E. 2018. High risk blueberry viruses by region in North America; Implications for certification, nurseries, and fruit production. Viruses 10:342 ; doi:10.3390/v10070342

Osterbaan, L., Schmitt-Keichinger, C. Vigne, E. and Fuchs, M. 2018. Optimal systemic grapevine fanleaf virus infection in Nicotiana benthamiana following agroinoculation. Journal of Virological Methods 257:16-21.

Pandey, B., Naidu, R.A. and Grove, G.G. 2018. Detection and analysis of mycovirus‑related RNA viruses from grape powdery mildew fungus Erysiphe necator. Archives of Virology 163: 1019-1030.

Perry, K.L., McLane, H., Thompson, J.R. and Fuchs, M. 2018. A novel grablovirus from non-cultivated grapevine (Vitis sp.) in North America. Archives of Virology 163:259-262.

Prator, C.A., Kashiwagi, C.M., Voncina, D. and Almeida, R.P.P. 2017. Infection and colonization of Nicotiana benthamiana by Grapevine leafroll-associated virus 3. Virology 510: 60-66.

Ricketts, K.D., Gómez, M.I., Fuchs, M.F., Martinson, T.E., Smith, R.J., Cooper, M.L., Moyer, M. and Wise A. 2017. Mitigating the economic impact of grapevine red blotch: Optimizing disease management strategies in U.S. vineyards. American Journal of Enology and Viticulture 68:127-135.

Rowhani, A., Daubert, S., Arnold, K., Al Rwahnih, M., Klaassen, V., Golino, D. and Uyemoto, J.K., 2018. Synergy between grapevine vitiviruses and grapevine leafroll viruses. European Journal of Plant Pathology, pp.1-7.

Setiono, F.J., Chatterjee, D., Fuchs, M., Perry, K.L. and Thompson, J.R. 2018. The distribution and ability to detect grapevine red blotch virus in its host depends on time of sampling and tissue type. Plant Disease, https://apsjournals.apsnet.org/doi/pdf/10.1094/pdis-03-18-0450-re.

Thekke-Veetil, T. and Tzanetakis, I.E. 2017. Development of reliable detection assays for blueberry mosaic- and blackberry vein banding- associated viruses based on their population structures. Journal of Virological Methods 248: 191-194.

Thekke-Veetil, T., Ho, T., Postman, J.D., and Tzanetakis, I.E. 2017. Characterization and detection of a novel idaeovirus infecting black currant. European Journal of Plant Pathology 149: 751-757.

Tzanetakis, I.E. and Martin, R.R. 2017. A systems-based approach to manage strawberry virus diseases. Canadian Journal Plant Pathology 39:5-10.

Vargas-Ascencio, J. Perry, K.L., Wise, A. and Fuchs, M. 2017. Detection of Australian grapevine viroid in Vitis vinifera in New York. Plant Disease 101:848.

Voncina, D., Rwahnih, M.A., Rowhani, A., Gouran, M. and Almeida, R.P.P. 2017. Viral diversity in autochthonous Croatian grapevine cultivars. Plant Disease 101: 1230-1235.

Weiland, J.E., Benedict, C., Zasada, I.A., Scagel, C.R., Beck, B.R., Davis, A., Graham, K., Peetz, A., Martin, R.R., Dung, J.K.S., Gaige, A.R. and Thiessen, L. 201X. Late summer disease symptoms in western Washington red raspberry fields associated with co-occurrence of Phytophthora rubi, Verticillium dahliae, and Pratylenchus penetrans, but not Raspberry bushy dwarf virus. Plant Disease 102:938-947.

Wistrom, C.M., Blaisdell, G.K., Wunderlich, L.R., Botton, M., Almeida, R.P.P. and Daane, K.M. 2017. No evidence of transmission of grapevine leafroll-associated viruses by phylloxera (Daktulosphaira vitifoliae). European Journal of Plant Pathology, 147: 937-941.

Yepes, L.M. Cieniewicz, E., Krenz, B., McLane, H., Thompson, J.R., Perry, K.L. and Fuchs, M. 2018. Causative role of grapevine red blotch virus in red blotch disease. Phytopathology 108:902-909.

Zeilinger, A.R., Rapacciuolo, G., Turek, D., Oboyski, P.T., Almeida, R.P.P. and Roderick, G.K. 2017. Museum specimen data reveal emergence of a plant disease may be linked to increases in vector population. Ecological Applications 27: 1827-1837.

Zongoma, A.M., Dangora, D.B., Al Rwahnih, M., Bako, S.P., Alegbejo, M.D. and Alabi, O.J., 2018. First Report of Grapevine yellow speckle viroid 1, Grapevine yellow speckle viroid 2, and Hop stunt viroid Infecting Grapevines (Vitis spp.) in Nigeria. Plant Disease, 102(1), pp.259-259.

Zongoma, A.M., Dangora, D.B., Al Rwahnih, M., Bako, S.P., Alegbejo, M.D. and Alabi, O.J., 2018. First Report of Grapevine leafroll-associated virus 1 Infecting Grapevines (Vitis spp.) in Nigeria. Plant Disease, 102(1), pp.258-258.

Book Chapters:

Burger, J., Maree, H.J., Gouveia, P., and Naidu, R.A. 2017. Grapevine leafroll-associated virus 3. In: Meng B., Martelli G., Golino, D., Fuchs M (eds.), Grapevine Viruses: Molecular Biology, Diagnostics and Management. Springer, Cham, Switzerland, pages 167-195. (Book Chapter)

Cieniewicz, E.J., Perry, K.L. and Fuchs, M. 2017b. Grapevine red blotch virus: Molecular biology of the virus and management of the disease. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng, B., Martelli, G.P., Golino, D.A. and Fuchs, M.F. (eds). Springer Verlag, pp. 303-314.

Fuchs, M. and Lemaire, O. 2017. Novel approaches for virus disease management. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng, B., Martelli, G.P., Golino, D.A. and Fuchs, M.F (eds). Springer Verlag, pp. 599-621.

Fuchs, M. Schmitt-Keichinger, C. and Sanfaçon, H. 2017. A renaissance in nepovirus research provides new insights into their molecular interface with hosts and vectors. In: Advances in Virus Research, M. Kielian, K. Maramorosch, T.C Mettenleiter and M.J Roosinck (eds.), pp. 61-105.

Golino, D., Fuchs, M., Al Rwanih, M., Farrar, K., and Martelli, G.P. 2017b. Regulatory aspects of grape virology: Certification, quarantine and harmonization. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng, B., Martelli, G.P., Golino, D.A. and Fuchs, M.F. (eds). Springer Verlag, pp. 581-598.

Golino, D., Fuchs, M., Sim, S., Farrar, K. and Martelli, G. 2017a. Improvement of grapevine planting stock through sanitary selection and pathogen elimination. In: grapevine viruses: molecular biology, diagnostics and management. Meng, B., Martelli, G.P., Golino, D.A. and Fuchs, M.F. (eds). Springer Verlag, pp. 561-579.

Naidu, R.A. 2017. Grapevine leafroll-associated virus 1. In: Meng B., Martelli G., Golino, D., Fuchs M (eds.), Grapevine Viruses: Molecular Biology, Diagnostics and Management. Springer, Cham, Switzerland, pages 127-139. (Book chapter)

Rowhani, A., Daubert, S.D., Uyemoto, J.K., Al Rawhnih, M. and Fuchs, M. 2017. American nepoviruses. In: Grapevine Viruses: Molecular Biology, Diagnostics and Management. Meng, B., Martelli, G.P., Golino, D.A. and Fuchs, M.F (eds). Springer Verlag, pp. 109-126.

Schilder, A., Hall, H.K., Tzanetakis, I.E. and Funt, R.C. 2017. Diseases, viruses, insects, and weeds of blackberries and their hybrids. Pp.202-244. In: Funt, R.C. and Hall H.K. (Ed) Blackberries and their hybrids. Wallingford, UK: CAB International

Tzanetakis, I.E. and Martin R.R. 2017. Production of high health plants for nuclear stock. Pp. 93-100. In: Funt, R.C. and Hall H.K. (Ed) Blackberries and their hybrids. Wallingford, UK: CAB International

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