Source: UNIVERSITY OF ARKANSAS submitted to NRP
PLANT VIRUS EPIDEMIOLOGY AND CONTROL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1002361
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Mar 7, 2014
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703
Performing Department
Plant Pathology
Non Technical Summary
Viral diseases account for significant losses every year; estimated in the billions of dollars. Control of emerging and re-emerging diseases is a challenging undertaking as knowledge on the virus epidemiology is limited or non-existent. The situation becomes more complicated because significant number of the diseases that have emerged in the last 15-20 years are caused by virus complexes rather than individual viruses. The examples of strawberry decline, blackberry yellow vein and raspberry crumbly fruit and decline come to mind, where all three diseases are caused by an array of viruses, several of which were unknown few years ago and have cost growers directly and consumers indirectly over 100 million dollars in the past decade.The number of new viruses in economic crops will likely increase as agriculture moves to new, uncharted, production areas. This practice and the predicted climate change towards warmer and drier growing conditions, circumstances that favor vector movement and survival, elevate the possibility for the emergence of new diseases. In addition, the global movement of propagation material leads to the dispersal of exotic viruses or novel strains of endemic viruses to new areas, altering the agricultural and natural ecosystems with unpredictable consequences. The serious consequences to growers, consumers and the environment associated with the free movement of planting material can be ameliorated or even eliminated with the development of new technologies that would allow for the rapid, sensitive and most importantly, universal detection of all viruses, known and new, infecting the propagation material.This project will tackle emerging disease problems in Arkansas, US and global agriculture by studying virus epidemiology and gene function using laboratory and field-based approaches. It will develop protocols based on next generation sequencing that will allow the assessment of multiple plants simultaneously and detect all viruses, making the interception of exotic viruses possible and providing the nursery industry with a powerful tool in the quest for virus-free propagation material. As a result, growers will have access to better quality, high yielding material able to produce sustainably for longer periods of time and the consumer will enjoy better quality product at better prices as the supply chain will not be threatened by diseases as those aforementioned.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121129110170%
2121820110110%
2122199110120%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1129 - Berries and cane fruits, general/other; 2199 - Ornamentals and turf, general/other; 1820 - Soybean;

Field Of Science
1101 - Virology;
Goals / Objectives
Goals:Minimize disease losses in field and specialty crops.Objectives:1a. Study virus epidemiology and develop disease control strategies1c. Study gene function in eriophyid mite-transmitted virusesDevelop next generation methodologies to mainstream detection in clonally-propagated crops.Objectives:2a. Develop protocols for mainstreaming sample processing2b. Develop interactive, on-line tools for analysis to allow for data manipulation without the need for a highly specialized bioinformatician.
Project Methods
Study virus epidemiology and develop control strategies for virus diseasesThe laboratory has successfully used double-stranded RNA, total nucleic acids and small interfering RNAs as templates for discovery of viruses or assessment of virus population structures. The dsRNA, total nucleic acid and siRNA extraction protocols have been described in detail in previous papers.In more detail, dsRNA or total nucleic acids are reverse transcribed and subjected to degenerate oligonucleotide-primed PCR as described before. For siRNA, adapters are ligated using RNA ligase followed by RT-PCR using primers corresponding to the adapter sequences. Amplicons are processed using the 454 (dsRNA and total nucleic acids) or Illumina (siRNAs) platforms. The primer sequences are trimmed off and bioinformatics analysis follows. Sequences are subjected to BLASTn to determine whether samples are infected with known viruses and eliminate any host material. The remaining sequences are assembled into contigs using Velvetor for small data sets, CAP3 and analyzed using BLASTx. The BLASTx analysis yields virus protein orthologs. Sequences with inconclusive classification are screened using ORF and those containing large open reading frames are compared against the Conserved Domain Database (CDD) to determine whether the putative proteins have structural similarities to virus proteins. The putative virus datasets are used to develop primers and confirm infection.For known and new viruses alike, a reliable detection test is a prerequisite for any epidemiological study. Unlike cellular organisms, viruses tend to have population structures, formed by multiple isolates and strains. Studying the population structure of a virus provides an insight into its diversity, and this knowledge can be used to develop tests able to detect all available virus isolates. Detection techniques could be immunological (ELISA or immunoblots) with antibodies developed against the most representative isolate in the population or molecular (PCR and its variations) based on conserved genomic areas. With a reliable detection tool the prevalence of the virus will be reliable, minimizing the possibility of false negatives or positives. If the virus is prevalent and hypothesized to be the causal agent or associated with a disease then work on putative vectors will follow. Based on sequence analysis we can hypothesize the identity of a virus vector (e.g. a subgroup 1 crinivirus is expected to be transmitted by Trialeurodes spp.) and initiate work on transmission for different types of vectors as previously described. If the 'best guess' is not a vector then other putative vectors will be assessed based on their presence in the field. If the causal agent of the disease is a single virus then pathogenicity and fulfillment of Koch's postulates can be completed using the methodologies described for Soybean vein necrosis virus and Rose rosette virus. In the case of virus complexes such as BYVD, a vector may act as a bottleneck and only allow a single species to be transmitted. After obtaining pure culture of each virus it is possible to reconstruct the disease and determine all the components needed for symptom development.Field monitoring to determine when viruses are being vectored during the season is critical and essential in the implementation of a control strategy. This will be done using sentinel plants and sticky cards in successive steps. Trap plants will be placed in fields where disease is present and left for 7, 15, or 30 days depending on the mode of transmission of the virus (non-persistence, semi-persistent, persistent) and then replaced with a subsequent set of plants throughout the growing season. Plants will be tested for all the viruses involved in disease and reconstruct the timeline of infection. Measures can then be implemented to minimize disease incidence. For example, if the virus is vectored by aphids in a non-persistent manner and transmission occurs early in the season then agricultural oils can be used to reduce transmission, whereas chemical sprays may have resulted in higher disease incidence. In the case of persistent transmission targeted chemical treatments at the time of the vector movement in the field may give excellent results as they can eliminate the vectors before they are able to transmit the virus. If the vector is moving slowly in the field such as soilborne vectors targeted treatment at the center of dispersal may lead to elimination of the virus in the field. In the case of aerial vectors, control may be possible by treating a specific area of the field or areas adjacent to a field (ex. boarder rows) that will eliminate the vector.Another component of virus epidemiology are the alternative hosts that function as reservoirs and may play a major role in pathogen dispersal. After the identification of alternative hosts and the vectors they harbor it may be that targeted sprays on the reservoir rather than the crop plant can reduce the incidence of disease. Targeted pesticide use can minimize spread of the virus, the incidence of the disease, cost to the grower and environmental impact since limited sprays are needed to control disease.Study gene function in emerging viruses transmitted by eriophyid mitesGiven that some mite-transmitted viruses replicate in their mite vectors, it is important to study the role of virus proteins during cell entry and develop strategies to block the process without altering the vector physiology.Gene function studies of negative-sense RNA viruses such as emaraviruses will be done by agroinfiltration and gene expression in-planta. In the case of positive-strand RNA viruses we will develop infectious clones and manipulate gene expression as previously described.Develop next generation methodologies to mainstream detection in clonally-propagated crops.The proposed project aims to provide a sensitive, reliable and cost-effective alternative to the industry standards using next generation sequencing. The protocols presented in Objective 1 can also be applied here. In order to make the methodology more cost effective at the reverse transcription (454) or ligation step (Illumina) each sample can be barcoded using a signature oligo, unique for each sample.This objective aims to develop an automated bioinformatics pipeline with an internet interface able to mine data of all known viruses but also determine the presence of novel viruses. In more detail: Users will log onto a website and submit their data. The program will trim adapters, eliminate host material, construct contigs and run the remaining sequences through BLAST and CDD. Results will be returned to the submitter in the form of an email and data presented as individual files for each step of the process.

Progress 03/07/14 to 09/30/18

Outputs
Target Audience:Producers, state and federal regulators, industry (nurseries- propagators), master gardeners,researchers working on berries; soybean, ornamentals and mycoviruses Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two PhD studentsand oneMS student as well asand three PhD scientists haveworked on the projects during the reporting period. How have the results been disseminated to communities of interest?National and regional scientific meetings National and regionalstakeholer (producer)meetings On-line reporting (ncpnberries.org) Conference proceedings Industry publications Peer-reviewed publications Book chapters What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Soybean: There are significant discoveries associated with soybean vein necrosis virus during the past year: - Kudzu is an asymptomatic, systemic host for the virus that may act as a green-bridge for summer infections - The western flower thrips, the most important vector for orthotospoviruses, is not a vector of the virus - The virus is not seed-transmissible in Arkansas breeding accessions - Accessions with dense pubescence provides tolerance to the soybean thrips - Transmission can be lowered to ~ 30% of the control using peptides that bind to thrips cell receptors - The virus, confined to thrips feeding areas in soybean, becomes systemic once plants are co-infected with bean pod mottle virus, possibly the most important soybean virus Blueberry: Developed a national map of the prevalence of all the major viruses affecting the crop Discovered two new viruses, one infecting a breeding accession about to be released as a named cultivar. The interception of the latter has saved the entity over one million dollars by not propagating/recalling infected plants Currants: Extended research portfolio to include currants. Have discovered two new viruses that are widespread across Europe, having implications on the movement of plants across continents Diagnostics: Improved diagnostics for major quarantine viruses allowing for the easier movement of plants across state and country lines

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 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
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Di Bello, P.L., Thekke-Veetil, T., Druciarek, T. and Tzanetakis, I.E. 2018. Transmission attributes and resistance to rose rosette virus. Plant Pathology 67: 499-504.
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: 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.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Shan, H., Pasin, F., Tzanetakis, I.E., Simón-Mateo, C., García, J.A. and Rodamilans, B. 2018. Truncation of a P1 leader proteinase facilitates potyvirus replication in a non-permissive host. Molecular Plant Pathology 19: 1504-1510.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: 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
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhou, J. Aboughanem-Sabanadzovic, N., Sabanadzovic, S. and Tzanetakis, I.E. 2018. First report of soybean vein necrosis virus infecting kudzu (Pueraria montana) in the United States of America. Plant Disease 102: 1674.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Koloniuk, I., Thekke-Veetil, T., Reynard, J.S., Mavri?, I.P., P?ibylová, J., Brodard, J., Kellenberger, I., Sarkisova, T., Špak, J., Lamovšek, J., Massart, S., and Tzanetakis I.E. 2018. Molecular Characterization of Divergent Closterovirus Isolates Infecting Ribes Species. Viruses 10: 369
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Maes, P., ⿦⿦Tzanetakis, I.E.,⿦.and Kuhn, J.H. 2018. Taxonomy of the family Arenaviridae and the order Bunyavirales. Archives of Virology 163: 2295⿿2310
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Thekke-Veetil, T., Ho, T., Postman, J.D., Martin, R.R. and Tzanetakis, I.E. 2018. A Virus in American Blackcurrant (Ribes americanum) with Distinct Genome Features Reshapes Classification in the Tymovirales. Viruses, 10: 406.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Producers, state and federal regulators, industry (nurseries- propagators), mastergardeners,researchers working on berries, soybean and ornamentals and mycoviruses Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two PhD and a Masters student as well as three postdoctoral associates have worked on the projects during the reporting period. How have the results been disseminated to communities of interest?National and regional scientific meetings International, national and regionalstakeholer (producer)meetings On-line reporting (ncpnberries.org) Conference proceedings Industry publications Peer-reviewed publications Book chapters What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We are working on expanding Virfind, a virus detection and discovery pipeline so as to detect all pathogens of a particular crop is a single assay. The program is expanding in eriophyid mite research, focusing of virus transmission attributes. This is not only because of the work on Rose rosette virus (RRV), but also because of the identification of a widespread, eriophyid-transmitted virus infecting blackberry. The virus, Blackberry leaf mottle associated virus (BLMaV), is a new member of the genus Emaravirus and a close relative to RRV. Unfortunately, BLMaV is widespread and only second in prevalence to Blackberry yellow vein associated virus. In Arkansas, BLMaV is present in more than 50% of the samples tested and we hypothesize that is the missing link in the onset of blackberry yellow vein disease. We are continuing the work on RRV; finalizing the population structure of the virus based on the complete genome sequences of over 100 isolates collected across the United States. This information will enable us to identify 'hot spots' in the genome and tie them to the different symptomology observed in some cultivars. He have developed a detection protocol able to detect all circulating isolates and we are finalizing a protocol what allows for the simultaneous detection of RRV and its mite vector in a single assay. We are tackling Lemoine's disease of peony, a disorder described more than 100 years ago. We have identified Cycas necrotic stunt virus, a virus never reported in the Western hemisphere in 90% of the more than 200 accessions tested and we are in the process of fulfilling Koch's postulates, work that will ameliorate a major constrain for Arkansas growers when it comes to movement of propagation material across state lines.

Publications

  • Type: Book Chapters Status: Published Year Published: 2017 Citation: 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
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: 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
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Martin R.R. and Tzanetakis, I.E. 2017. Introduction to diseases caused by viruses and virus-like agents. Pp. 70-71. In: Martin, R.R., Ellis, M.A., Williamson, B. and Williams, R.N. (Ed) Compendium of Raspberry and Blackberry Diseases and Insects 2nd Edition. APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Tzanetakis, I.E., Susaimuthu, J., Sabanadzovic S., and Martin R.R. 2017. Blackberry Yellow Vein Disease Complex (BYVD). Pp. 71-75. In: Martin, R.R., Ellis, M.A., Williamson, B. and Williams, R.N. (Ed) Compendium of Raspberry and Blackberry Diseases and Insects 2nd Edition. APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: MacFarlane, S.A., Tzanetakis, I.E., Halgren, A.B. and Martin, R.R. 2017. Raspberry mosaic disease complex. Pp. 75-78. In: Martin, R.R., Ellis, M.A., Williamson, B. and Williams, R.N. (Ed.) Compendium of Raspberry and Blackberry Diseases and Insects 2nd Edition. APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Tzanetakis, I.E. 2017. Blackberry virus F. Pp. 80. In: Martin, R.R., Ellis, M.A., Williamson, B. and Williams, R.N. (Ed.) Compendium of Raspberry and Blackberry Diseases and Insects 2nd Edition. APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Quito-Avila, D.F., Tzanetakis, I.E. and Martin, R.R. 2017. Raspberry latent virus. Pp. 84-85. In: Martin, R.R., Ellis, M.A., Williamson, B. and Williams, R.N. (Ed) Compendium of Raspberry and Blackberry Diseases and Insects 2nd Edition. APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Tzanetakis, I.E. and Martin, R.R. 2017. Strawberry necrotic shock virus. Pp. 90-91. In: Martin, R.R., Ellis, M.A., Williamson, B. and Williams, R.N. (Ed) Compendium of Raspberry and Blackberry Diseases and Insects 2nd Edition. APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Martin, R.R. and Tzanetakis, I.E. 2017. Other viruses and virus-like agents. In: Pp. 91-93. In: Martin, R.R., Ellis, M.A., Williamson, B. and Williams, R.N. (Ed) Compendium of Raspberry and Blackberry Diseases and Insects 2nd Edition. APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Garc�a, M. L., Dal B�, E., da Gra�a, J. V., Gago-Zachert, S., Hammond, J., Moreno, P., Natsuaki, T., Pall�s, V., Navarro, J.A., Reyes, C.A. and Luna, G.R., Sasaya, T., Tzanetakis, I.E., Vaira, A.M., Verbeek, M. and ICTV Report Consortium. 2017. Ophioviridae in ICTV 10th Report. https://talk.ictvonline.org/ictv-reports/ictv_online_report/negative-sense-rna-viruses/w/ophioviridae
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Garc�a, M. L., Dal B�, E., da Gra�a, J. V., Gago-Zachert, S., Hammond, J., Moreno, P., Natsuaki, T., Pall�s, V., Navarro, J.A., Reyes, C.A. and Luna, G.R., Sasaya, T., Tzanetakis, I.E., Vaira, A.M., Verbeek, M. and ICTV Report Consortium. 2017. ICTV Virus Taxonomy Profiles: Ophioviridae. Journal of General Virology 98:1161-1162.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Thompson, J.R., Dasgupta, I., Fuchs, M., Iwanami, T., Karasev, A.V., Petrzik, K., Sanfa�on, H., Tzanetakis, I. E., van der Vlugt, R., Wetzel, T., Yoshikawa, N., and ICTV Report Consortium. 2017. Secoviridae in: ICTV 10th Report. https://talk.ictvonline.org/ictv-reports/ictv_online_report/positive-sense-rna-viruses/picornavirales/w/secoviridae
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 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.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 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.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Thompson, J.R., Dasgupta, I., Fuchs, M., Iwanami, T., Karasev, A.V., Petrzik, K., Sanfa�on, H., Tzanetakis, I. E., van der Vlugt, R., Wetzel, T., Yoshikawa, N., and ICTV Report Consortium. 2017. ICTV Virus Taxonomy Profiles: Secoviridae. Journal of General Virology 98: 529-531.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: 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.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Gress, J., Smith, S. and Tzanetakis I.E. 2017. First report of citrus leaf blotch virus in peony in the USA. Plant Disease 101: 637
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Tzanetakis, I.E. and Martin, R.R. 2017. A systems-based approach to counter strawberry virus diseases. Canadian Journal of Plant Pathology 39: 5-10.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Thekke-Veetil, T., Khadgi, A., Johnson, D.T., Burrack, H., Sabanadzovic, S. and Tzanetakis, I.E. 2017. First report of raspberry leaf mottle virus in blackberry in the United States. Plant Disease 101: 265
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Shahid, M.S., Aboughanem-Sabanadzovic, N., Sabanadzovic, S. and Tzanetakis, I.E. 2017. Genomic characterization and population structure of a badnavirus infecting blackberry. Plant Disease 101: 110-115
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Tzanetakis, I.E., Gergerich, R.C. and Martin, R.R. 2017. National Blueberry Certification Scheme in the United States. Acta Horticulturae 1180: 221-224
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 1. Thekke-Veetil, T., Ho T., Postman, J.D. and Tzanetakis, I.E. 2017. Discovery of a novel plant virus with unique genome features from black currant. International Council for the Study of Virus and other Graft Transmissible Diseases of Fruit Crops (ICVF) proceedings pp. 22
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Thekke-Veetil, T., Ho T., Polashock, J., and Tzanetakis, I.E. 2017. Identification of a new vitivirus from blueberry. International Council for the Study of Virus and other Graft Transmissible Diseases of Fruit Crops pp. 106
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: Stetina. T.J., Rothrock C.S., Spurlock, T.N. and Tzanetakis I.E. 2017. Transmission of mycoviruses of R. solani and effects on fungal growth. APS annual meeting abstract
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: Thekke-Veetil, T., Ho T., Postman, J.D. and Tzanetakis, I.E. 2017. Molecular characterization of a new member of the genus Waikavirus. APS annual meeting abstract
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2017 Citation: 3. Katsiani, A.T., Lamour, K., and Tzanetakis, I.E. 2017. Unveiling the population structure of rose rosette virus. APS annual meeting abstract


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Producers, state and federal regulators, industry (nurseries- propagators), mastergardeners,researchers working on berries; soybean and ornamentals Changes/Problems:None at this point What opportunities for training and professional development has the project provided?Two PhD students and four postdoctoral associates have worked on the projects during the reporting period. Two postoctoral associates have moved to tenure/non tenure track positions in academia. How have the results been disseminated to communities of interest?National and regional scientific meetings National and regionalstakeholer (producer)meetings On-line reporting (ncpnberries.org) Conference proceedings industry publications Peer-reviewed publications Book chapters What do you plan to do during the next reporting period to accomplish the goals?Continue the work already in progress

Impacts
What was accomplished under these goals? The blackberry virome has expanded to include a new member of the Caulimiviridae that can incorporate in the host genome. This has major implications on the production of clean plants for propagation as once incorporated there are no available technologies to eliminate it from the germplasm. For this reason material may need to be eliminated from breeding and nursery production pipelines. Rosette is the most important disease of rose in North America. We concluded the studies on transmission attributes and identified a genotype that is resistant to the virus. This knowledge will be used in integrated approaches for mite control whereas the resistance genotype may be the basis for control strategies either as material planted in areas with high disease incidence or used in breeding programs to incorporate resistance to new cultivars We have evidence of allopatric speciation of emaraviruses, an emerging plant virus group. This work has been of utmost importance not only because of the implication in virus evolution but most importantly because there is proof that the mite vectors can transmit in species unrelated to the original host of the virus. We have met with state and federal regulators and completed the pilot studies for the national certification program for blueberry. Three states have successfully complied with the guidelines and are moving forward with formulating language to implement the new rules we developed. In the bioinformatics arena, Virfind, the bioinformatics pipeline for virus detection and discovery is used globally (more than 250 users and 2500 runs) by academics, industry, regulators and federal labs.The plant virology laboratory at the University of Arkansasalso provides support to industry, academia and federal laboratories when it comes to bioinformatics analyses.

Publications

  • Type: Book Chapters Status: Published Year Published: 2016 Citation: 1. Tzanetakis, I.E. and Martin, R.R. 2016. Blueberry latent spherical virus. Pp. 60, In: Polashock, J.J., Caruso, F.L., Averill, A.L. and Schilder A.C. (Ed) Compendium of Blueberry, Cranberry and Lingonberry Diseases and Pests 2nd Edition, APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: 2. Tzanetakis, I.E. and Martin, R.R. 2016. Blueberry latent virus. Pp. 60, In: Polashock, J.J., Caruso, F.L., Averill, A.L. and Schilder A.C. (Ed) Compendium of Blueberry, Cranberry and Lingonberry Diseases and Pests 2nd Edition, APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: 3. Tzanetakis, I.E. and Martin, R.R. 2016. Blueberry virus A. Pp. 60-61, In: Polashock, J.J., Caruso, F.L., Averill, A.L. and Schilder A.C. (Ed) Compendium of Blueberry, Cranberry and Lingonberry Diseases and Pests 2nd Edition, APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: 4. Martin, R.R. and Tzanetakis, I.E. 2016. Blueberry mosaic virus. Pp. 64, In: Polashock, J.J., Caruso, F.L., Averill, A.L. and Schilder A.C. (Ed) Compendium of Blueberry, Cranberry and Lingonberry Diseases and Pests 2nd Edition, APS Press, St. Paul, MN.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: 5. Tzanetakis, I.E. and Martin, R.R. 2016. Blueberry Certification. Pp. 154-156, In: Polashock, J.J., Caruso, F.L., Averill, A.L. and Schilder A.C. (Ed) Compendium of Blueberry, Cranberry and Lingonberry Diseases and Pests 2nd Edition, APS Press, St. Paul, MN.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Martin, R.R., Constable F. and Tzanetakis, I.E. 2016. Quarantine regulations and the impact of modern detection methods. Annual Review of Phytopathology 54: 189205
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: 6. Thekke-Veetil, T., Ho, T., Moyer, C., Whitaker V.M. and Tzanetakis, I.E. 2016. Detection of Strawberry necrotic shock virus using conventional and TaqMan� quantitative RT-PCR. Journal of Virological Methods 235:176-181.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: 7. 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 Research 222:41-47.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Thekke-Veetil, T. and Tzanetakis, I.E. 2016. First report of strawberry polerovirus-1 in strawberry in the United Stated. Plant Disease 100: 867
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Ho, T., Quito-Avila, D., Keller, K.E., Postman, J.D., Martin, R.R. and Tzanetakis, I.E. 2016. Evidence of sympatric speciation of elderberry carlaviruses. Virus Research 215: 72-75
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: 10. Aboughanem-Sabanadzovic, N., Tzanetakis, I.E., Lawrence, A., Stephenson, R.C. and Sabanadzovic, S. 2016. A novel ilarvirus is the causal agent of ligustrum associated with privet necrotic ringspot disease in the southern United States. Phytopathology 106: 87-93
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: " Di Bello, P.L., Ho, T. and Tzanetakis, I.E. 2015. The evolution of emaraviruses is becoming more complex: seven segments identified in the causal agent of Rose rosette disease. Virus Research 210: 241-244.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Gergerich, R.C., Tzanetakis, I.E. and Martin, R.R. 2016. Towards a national certification scheme for Rubus in the United States. Acta Horticulturae 1133: 483-486
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shahid, M.S., Tzanetakis, I.E. Aboughanem-Sabanadzovic, N. and Sabanadzovic, S. 2016. An integrating badnavirus infects blackberry. Acta Horticulturae 1133: 507-510.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: 5. Thekke-Veetil, T., Polashock, J., Plesko, I.M., Keller, K.E., Martin, R.R., Ho, T. and Tzanetakis, I.E. 2016. Blueberry mosaic associated virus - a putative new member of the Ophioviridae. Acta Horticulturae 1117: 103-110.
  • Type: Other Status: Published Year Published: 2016 Citation: Tzanetakis I.E. Clean plants, new detection technologies and National Certification standards. Small Fruit News, Vol. 16 (1) January 2016.
  • Type: Other Status: Published Year Published: 2016 Citation: 2. Di Bello, P.L., Laney, A.G., Druciarek, T., Ho, T., Gergerich, R.C., Karen E. Keller, Robert R. Martin and Tzanetakis I.E. 2016. Redbud yellow ringspot-associated Emaravirus. International Committee for the Taxonomy of Viruses proposal


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Stekeholders including producers and nurserymen (berries, ornamentals and soybean), state and federal regulators (berries and ornamentals, scientists (berries, ornamentals and soybean)and the general public (ornamentals) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Four graduate students (two PhD and two MS) and fivePhD scientists working full or part time are part of this project. There have also been several undergraduates that are participating in research associated with this project andone of them has expressed their interest in continuing for a graduate degree. Graduate students and postdocs have presented their work in national and regional meetings. Two postdoctoral associated have secured sought-after positions in academia (tenure track faculty) and industry. How have the results been disseminated to communities of interest?The results of this research has been presented to the stakeholders as peer-reviewed publications, articles in the popular press and presentation in scientific and grower meetings. The stakeholders are eager to learn and our group provides them with high quality knowledge that they use to improve their livelihoods and make better decisions, in the farm or their local nursery. What do you plan to do during the next reporting period to accomplish the goals?We will continue using the same mode of action as there are no major pitfalls identified and research is moving at an accelerated pace.

Impacts
What was accomplished under these goals? A study on the blackberry virosome at a field and regional level (Southeastern United States) was completed in 2015. We have a better understanding of virus and vector movement in the regional level, whereas in the field level we were able to pinpoint the time of infection for several of the most common blackberry viruses. During this study we identified five new viruses and observed their movement in the field between seasons. We have evidence of sympatric speciation of five carlaviruses, members of one of the most important plant virus genera, in elderberry. This work has been of utmost importance not only because of the implication in virus evolution but most importantly because there is proof that the newly emerged viruses can infect and cause disease in field settings in members of the genus Rubus. Work on Rhizoctonia solani mycoviruses has led to the discovery at least 20 new virus species. An important and exciting discovery is the presence of Rhizoctonia solani RNA virus 1 (RsRV1) in an anastomosis group (AG) 4 isolate. This virus has previously only been found in a chinese AGI-1A isolate associated with slow growth and reduced fungal pathogenicity. This discovery provides evidence that R. solani mycoviruses can infect different anastomosis groups elevating their potential as biocontrol agents in field settings. In the bioinformatics arena, Virfind, the bioinformatics pipeline for virus detection and discovery is used globally by academics, industry, regulators and federal labs. Virfind is one of the three pipelines used in the proficiency testing part of the European Union Cost Action FA 1407 'Application of next generation sequencing for the study and diagnosis of plant viral diseases in agriculture'. The plant virology laboratory also provides support to industry, academia and federal laboratories when it comes to bioinformatics analyses. The draft guidelines for all berry certification programs have been completed and I will move forward with their implementation through my appointment as interim Director of the NCPN Education and Outreach group. This project has saved growers millions of dollars either directly by providing better managing strategies for disease control or indirectly by providing high qulity berry and rose propagation material which is performing better and is productive for a longer period of time in the field. The potential of mycovirus as biocontrol agents of a significant plant pathogen as Rhizoctonia only enchances the goal of this project.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Ho, T. and Tzanetakis, I.E. 2014. Developing a virus detection and discovery pipeline using next generation sequencing. Virology 471-473:54-60
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Martin, R.R. and Tzanetakis I.E. 2015. Control of virus diseases of berry crops. Advances in Virus Research 91:271-309.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Gergerich, R.C., Welliver, R., Gettys, S., Osterbauer, N.K., Kamenidou, S., Martin, R.R., Golino, D., Eastwell, K., Fuchs, M., Vidalakis, G. and Tzanetakis, I.E. 2015. Safeguarding fruit crops in the age of agricultural globalization. Plant Disease 99: 176-187.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Thekke-Veetil, T., Polashock, J.J., Marn, M.V., Plesko, I.M., Schilder, A.C., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. 2015. Population structure of blueberry mosaic associated virus: Evidence of reassortment in geographically distinct isolates. Virus Research 201: 79-84.
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: MacFarlane, S., McGavin W. and Tzanetakis I.E. 2015. Virus-testing by PCR and RT-PCR amplification in berry fruit. Methods in Molecular Biology - Plant Pathology 1302: 227-248
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Ho, T., Martin. R.R. and Tzanetakis, I.E. 2015. Next Generation Sequencing of Elite Berry Germplasm and Data Analysis Using a Bioinformatics Pipeline for Virus Detection and Discovery. Methods in Molecular Biology - Plant Pathology 1302: 301-313
  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Zhou, J., Domier, L. Sabanadzovic, S. and Tzanetakis I.E. 2015. Soybean vein necrosis virus. Pp. 124-125, In: Hartman, G.L., Rupe, J.C., Sikora E.J., Domier L.L.,Davis J.A. and Steffey, K.L. (Ed) Compendium of Soybean Diseases 5th Edition. APS Press, St. Paul, MN
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Poudel, B., Ho, T., Khadgi, A. and Tzanetakis, I.E. 2015. Epidemiology of Blackberry chlorotic ringspot virus. Acta Horticulturae 1064: 311-316
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Di Bello P.L., Ho, T. and Tzanetakis, I.E. 2015. Identification of the rose rosette disease agent. Acta Horticulturae 1064: 295-298
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Tzanetakis I.E. and Martin R.R. 2014. Incidence of major strawberry viruses in North America. Acta Horticulturae 1049: 595-598.


Progress 03/07/14 to 09/30/14

Outputs
Target Audience: The berry, ornamentals and soybean industries face major problems caused by viruses. There is a need and demand from the stakeholders for educational sessions and publications that would provide knowledge and assist in the development of superior Standard Operating Procedures, minimizing the impact of viruses, maximize profitability for the growers and provide affordable, high qualitymaterial to consumers.The past year we reached all major target groups: growers, nurseries, extension personnel, scientists and consumers. More information can be found in the 'Product' section of this report. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Four graduate students (two PhD and two MS) and five postdoctoral scientists working full or part time are part of this project. There have also been several undergraduates that are participating in research associated with thisproject and two of them have expressed their interest in continuing for a graduate degree. All graduate students and postdocs have presented their work in national and regional meetings whereas three have also presented in international meetings enhancing their chances for a successful professional career. How have the results been disseminated to communities of interest? The results of this research has been presented to the stakeholders as peer-reviewed publications, articles in the popular pres andpresentation in scientific and grower meetings. The stakeholders are eager to learn and our group provides them with high quality knowledge that they use to improve their livelihoods and make better decisions, in the farm or their local nursery. What do you plan to do during the next reporting period to accomplish the goals? We will continue using the same mode of action as there are no major pitfalls identified and research is moving at an accelerated pace.

Impacts
What was accomplished under these goals? Next generation sequencing and bioinformatics: The virology program leads the efforts for the implementation of the technology for testing NCPN G1 plants. New methodologies have been developed and are used by several laboratories in the United States but also internationally. The bioinformatics pipeline (Virfind), a tool for virus detection and discovery is being used across the world. The Arkansas NCPN Center has started or anticipate to receive G1 material from Berries, Citrus, Sweet Potato and Potato for NGS and bioinformatics analyses. Work on emaraviruses has revealed additional genomic complexity but also differences in transmission vs what has previously been reported. After proving that emaraviruses are causal agents of significant diseases of ornamental crops we are working on identifying cryptic mite species that may be involved in transmission. In strawberry, the virology program works on the genomic complexity of strawberry crinkle virus, a persistent propagative virus that is becoming widespread in several growing areas. We are also exploring next generation sequencing as a tool to dissect the complexity of the virus epidemics observed over the last two years. In blackberry and elderberry we have discovered several new viruses and study their role in disease onset. The discovery of viruses that are able to incorporate in the plant genome (badnaviruses) has made this work a priority as we are evaluating a pipeline were all plants will be tested for badnaviruses before they are used as breeding material or shipped across country lines. In addition to research, Dr. Tzanetakis has led work on the development of national berry certification programs. The draft guidelines for blueberry and Rubus are completed while Dr. Tzanetakis assisted in the development of the certification guidelines for strawberry completing all major berry crops. This project has saved growers millions of dollars either directly by providing better managing strategies for disease control or indirectly by providing high qulity berry and rose propagation material which is performing better andis productive for a longer period of time in the field. In the case of soybean the knowledge on the epidemiology of soybean vein necrosis virus has minimized sprays (as the disease was previously thought to be caused by fungal pathoghens) whereas we are working on developing resistance to the virus and/or vector.

Publications

  • Type: Book Chapters Status: Accepted Year Published: 2015 Citation: MacFarlane, S., McGavin W. and Tzanetakis I.E. 201x. Virus-testing by PCR and RT-PCR amplification in berry fruit. Methods in Molecular Biology - Plant Pathology: Techniques and Protocols, in press
  • Type: Book Chapters Status: Accepted Year Published: 2015 Citation: Ho, T., Martin. R.R. and Tzanetakis, I.E. 201x. Next Generation Sequencing of Elite Berry Germplasm and Data Analysis Using a Bioinformatics Pipeline for Virus Detection and Discovery. Methods in Molecular Biology - Plant Pathology: Techniques and Protocols, in press
  • Type: Book Chapters Status: Published Year Published: 2014 Citation: Martin R.R., and Tzanetakis I.E. 2014.Pathogen-Tested Planting Material. In: Neal Van Alfen, EIC. Encyclopedia of Agriculture and Food Systems, Vol. 4, pp. 304-312.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Gergerich, R.C., Welliver, R., Gettys, S., Osterbauer, N.K., Kamenidou, S., Martin, R.R., Golino, D., Eastwell, K., Fuchs, M., Vidalakis, G. and Tzanetakis, I.E. 2015. Safeguarding fruit crops in the age of agricultural globalization. Plant Disease http://dx.doi.org/10.1094/PDIS-07-14-0762-FE
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Martin, R.R. and Tzanetakis I.E. 2014. Control of virus diseases of berry crops. Advances in Virus Research 91:44-81.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Ho, T. and Tzanetakis, I.E. 2014. Developing a virus detection and discovery pipeline using next generation sequencing. Virology 471-3:54-60
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Thekke-Veetil, T., Ho, T., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. 2014. A new ophiovirus associated with blueberry mosaic disease. Virus Research 189: 92-96.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Poudel, B., Ho, T., Laney, A., Khadgi, A. and Tzanetakis, I.E. 2014. Epidemiology of Blackberry chlorotic ringspot virus. Plant Disease 98:547-550.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Tzanetakis I.E. and Martin R.R. Incidence of major strawberry viruses in North America. Acta Horticulturae 1049: 595-598.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Poudel, B., Ho, T., Khadgi, A. and Tzanetakis, I.E. Epidemiology of Blackberry chlorotic ringspot virus. Acta Horticulturae, in press.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: Di Bello P.L., Ho, T. and Tzanetakis, I.E. Identification of the rose rosette disease agent. Acta Horticulturae, in press.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: 3) Thekke-Veetil, T., Polashock, J., Plesko, I.M., Keller, K.E., Martin, R.R., Ho, T. and Tzanetakis, I.E. Blueberry mosaic associated virus - a putative new member of the Ophioviridae. Acta Horticulturae, in press.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2015 Citation: 5) Thekke-Veetil, T., Polashock, J., Marn, M.V., Plesko, I.M., Keller, K.E., Martin, R.R., Ho, T. and Tzanetakis, I.E. Molecular characterization and population structure of Blueberry mosaic associated virus. RUcore, in press