PLANT AND FUNGAL VIRUS EPIDEMIOLOGY AND CONTROL

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017254
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
Plant Pathology

Non Technical Summary
Plant diseases account for significant losses every year estimated in billions of dollars. Control of emerging and re-emerging diseases is a challenging undertaking as knowledge of virus epidemiology is limited. The situation has become more complicated because a significant number of the diseases that have emerged in the last quarter century are caused by virus complexes rather than individual viruses.Since 2008, the plant virology program at the University of Arkansas has identified more than 100 new virus species associated with emerging and re-emerging diseases of plants or plant pathogenic fungi. Most of those viruses are associated with diseases caused by complexes.The number of new viruses in economic crops will likely increase as agriculture moves to new production areas. This practice, in addition to the predicted climate change towards warmer and drier growing conditions that favor vector movement and survival, elevate the possibility for the emergence of new diseases. In addition, the global 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 losses and destruction associated with the 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 unknown that infect the material.The proposed project will tackle emerging disease problems in Arkansas, US and global agriculture by studying virus epidemiology. It will generate knowledge on the understudied mycoviruses of plant pathogenic fungi and evaluate them as biocontrol agents. It also aims to develop or improve existing detection protocols based on molecular techniques (including high throughput sequencing) which will detect and identify known and unknown viruses; making the interception of exotic viruses possible and provide the nursery industry with a powerful tool in the quest for virus-free propagation material.

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
212	1129	1101	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1129 - Berries and cane fruits, general/other;

Field Of Science
1101 - Virology;

Keywords

virus

strawberry

blackberry

raspberry

blueberry

currant

elderberry

rose

peony

ornamentals

cranberry

mycovirus

Goals / Objectives
Identify potential new viruses in fungal pathogens, berries and ornamental crops.Study virus epidemiology in fungi, berries and ornamental crops.Develop management strategies for viruses infecting berries and ornamental crops.Develop methodologies to mainstream detection in clonally-propagated crops.Engage stakeholders and deliver results and new tools to stakeholders for implementation of best management practices.

Project Methods
PROCEDURES:Study virus epidemiology and develop control strategies for virus diseasesThere is a need to study emerging diseases and for this reason work on this area will continue using high throughput sequencing. The laboratory has successfully used total nucleic acids as template for discovery of viruses or assessment of virus population structures (Thekke-Veetil et al., 2014; Di Bello et al., 2015; Katsiani et al., 2018; Tzanetakis et al, unpublished data).In more detail, nucleic acids are depleted from ribosomal RNA, reverse transcribed and subjected to degenerate oligonucleotide-primed PCR as described (Ho et al, 2015). Amplicons are processed using Illumina or PacBio platforms. The outputs are analyzed using Virfind (Ho et al., 2014) or CLC (Qiagen). 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. Viruses have complex 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 a vast array of isolates. Detection techniques could be immunological (ELISA or immunoblots) with antibodies developed against the coat protein of the most representative isolate in the population or molecular (PCR and its variations) based on conserved genomic region. 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 the sequence analysis of the virus we can hypothesize the identity of a virus vector (e.g. an Emaravirus is expected to be transmitted by an eriophyid mite species), and initiate work testing transmission on different types of vectors as previously described (Di Bello et al, 2015; Hassan et al., 2017). If the 'best guess' is not a vector then other putative vectors will be assessed based on their presence in the field.In cases where the causal agent 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 (Zhou and Tzanetakis, 2013; Di Bello et al, 2015). In the case of virus complexes such as BYVD we will develop infectious clones of the dominant viruses and reconstruct the disease to determine the components needed for symptom development.Field monitoring to determine when viruses are 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 (Khadgi, 2015). Trap plants will be placed in fields where disease is present and left for 15, or 30 days depending on the mode of transmission of the virus (non-persistence, semi-persistent, persistent) before replaced with a subsequent set of plants throughout the growing season (Khadgi, 2015). Plants will be tested for all 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 (Powell, 1992), whereas chemical sprays may have resulted in higher disease incidence (Madden, 2000). 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 (Lightle, 2013). If the vector is moving slowly in the field such as soil-borne 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, 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 (Halgren, 2006; Lightle, 2013).Identify new mycoviruses as potential biocontrol agentsMycoviruses, or fungal viruses, are of interest because of their potential role in biological control. Rhizoctonia solani will be used as a model host organism for this project because of its wide host range and its effect on major agricultural crops including cotton, rice and soybean. The objective of this research is to identify mycoviruses of R. solani and determine relationships between species distribution, relationships among anastomosis groups (AG), and the effects of virus distribution on R. solani epidemiology with the aim to identify those that ameliorate host virulence and show potential as biocontrol agents. More than 25 new virus species have been identified by our group in different AGs obtained from rice/soybean or cotton/soybean rotation fields. One hundred points have been chosen per survey, with multiple isolates from each point used for analysis. Detection protocols will be developed and optimized. Those will be used to determine virus presence in all geotagged isolates. Spatial analysis will be performed on distributions of Rhizoctonia spp., AG, and mycovirus species and correlations will be generated for these three factors in addition to soil properties including pH and texture.The mycovirus exchange via hyphal anastomosis will be evaluated using genetically similar and somatically incompatible virus-infected where the presence or absence of mycovirus will be confirmed using RT-PCR. The effects of mycovirus infection on mycelial growth and R. solani virulence will be performed on seedlings and plant growth and symptom development will be evaluated. If there are viruses that show potential as biocontrol agents they will be evaluated under field conditions.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Producers, industry (nurseries- propagators), state and federal regulators, master gardeners, researchers working on berries; soybean andornamentals Changes/Problems:COVID-19 has affected the lab operations andhad to make tough decisions to address the situation.The laboratory has worked at 15%-33% capacity between March and August 2020. In order to achieve the goals set for the majority of objectives we eliminated the fungal virus research component. What opportunities for training and professional development has the project provided?A PhD and two 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 National and regional stakeholer (producer) meetings On-line reporting (ncpnberries.org) Conference proceedings 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? Rose rosette, caused by rose rosette virus,is the most important disease of rose costing the industry tensof millions of dollars in revenue as roses are notplantedin areas with high disease incidence. We have studied the population structure of the virus to better understand its epidemiology and implement that knowledge in disease control strategies. We employed a high-throughput sequencing strategy and sequenced the virus genomeof over 90 isolates collected across areas with high virus incidence. Analysesrevealed indelpolymorphisms with some resulting in protein length changes. Phylogenetic analysis showed little diversity that could be explained based on the collection site. Overall, the virus is homogeneous.We hypothesize that this is because ofregular movement of plants trhough the nursery trade,the recent emergence ofthe virus and/or because the virus is under purification selection to preserve itsbiological functions in both the mite vector and plant host Raspberry leaf blotch emaravirus, the causal agent of the homonymous disease, causes siginficant losses in the areas it occurs. The virus diversity has not been studied in depth, nor is thereknowledge on the systemic movement of the virus. The latter is a major knowledge gap that affects disease control as there have been reports claiming that the disesase is eliminated with leaf senescence. We studied virus diversity and the virus appears stable in the global scale, a similar finding to rose rosette virus discussed above. We were able to confirm that the virus moves systemically, reaching the roots. This findings has major implication for disease control as producers will no longer assume that the disease is eliminated after winter defoliation but rather it should be agressivelycontrolled via acareocidal sprays targeting the mite vector and by roguing infected material before the virusspreads to adjucent plants. Vein necrosis is the most widespread soybean virus disease in North America and its causal agent,Soybean vein necrosis virus (SNSV) reduces seed oil content and is seed-borne, affecting international movement of seed. We have studied the epidemiology of the virus and developed sustainable control strategies for the virus and its vectors. The results of this research: - Accessions with dense pubescence provides tolerance to thrips and growers should prefer them when there is a history of the disease in their fields - Transmission was lowered by 70% after using proteins that affect virus acquisition by its vectors. This strategy, if implemented in the field, can significantly lower the impact of the disease - The virus, confined to thrips feeding areas in soybean, becomes systemic once plants are co-infected with bean pod mottle virus. Growers need to be aware of the presence of both viruses in their fields and control beetles (vectors of bean pod mottle virus) and/or thrips (multiple species transmit SVNV) to minimize the impact of the mixed virus infections. - Kudzu is a SVNV host and may act as a virus source early in the season. Producers need to be aware of the fact as they plant the crop and avoid major thrips flights into the young field - The western flower thrips, the most important vector of the group of viruses that SNSV belongs to, is not a vector of the virus and therefore no control for this pest is needed in the field - The virus is not seed-transmissible in Arkansas breeding accessions. This fact would ease regulatory requirements and allow for the easier movement of soybean around the globe

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhou, J., Johnson, D.T. and Tzanetakis, I.E. 2020. Assessing Soybean Genotypes for Feeding Damage by Neohydatothrips variabilis (Thysanoptera: Thripidae). Crop Protection 128: 104983.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhou, J. and Tzanetakis, I.E. 2020. Soybean vein necrosis orthotospovirus can move systemically in soybean in the presence of bean pod mottle virus. Virus Genes 56: 104-107.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Zhou, J. and Tzanetakis, I.E. 2020. Transmission blockage of an orthotospovirus using synthetic peptides. Journal of General Virology 101: 112-121
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Sanfa�on, H., Dasgupta, I., Fuchs, M., Karasev, A.V., Petrzik, K., Thompson, J.R., Tzanetakis, I.E. van der Vlugt, R., Wetzel, T. and Yoshikawa, N. 2020. Proposed revision of the family Secoviridae taxonomy to create three subgenera Satsumavirus, Stramovirus and Cholivirus in the genus Sadwavirus. Archives of Virology 165: 527-533
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Thekke-Veetil, T., Ho, T., Postman, J. D., and Tzanetakis, I. E. 2020. Comparative analysis of a new blackcurrant waikavirus with other members of the genus. Eur. J. Plant Pathol. 175: 59-64
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Delic, D., Radulovic, M., Vakic, M., Sunulahpaaic, A., Villamor, D.E.V. and Tzanetakis, I.E. 2020. First Report of black currant reversion virus and gooseberry vein banding associated virus in currants in Bosnia and Herzegovina. Plant Disease 104:2036
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Katsiani, A., Stainton, D., Lamour, K. and Tzanetakis, I.E. 2020. The population structure of Rose rosette virus in the United States. Journal of General Virology 101: 676684
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Delic, D., Radulovi?, M., Vakic, M., Sunulahpaaic, A., Villamor, D.E.V. and Tzanetakis, I.E. 2020. Raspberry leaf blotch emaravirus in Bosnia and Herzegovina: population structure and systemic movement. Molecular Biology Reports 47: 48914896
• Type: Book Chapters Status: Published Year Published: 2020 Citation: Tzanetakis, I.E., Sabanadzovic S. and Valverde R. 2020. Amalgaviruses (Amalgaviridae) In Reference Module in Life Sciences 2020. https://doi.org/10.1016/B978-0-12-809633-8.21527-6
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Wait, A., Villamor, D.E.V., Martin, R. and Tzanetakis, I.E. Characterization of Two New Strawberry Viruses in the National Clonal Germplasm Repository Collection. 2020 APS meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Villamor, D.E.V., Keller, K., Martin, R. and Tzanetakis, I.E. Comparison between high throughput sequencing (HTS) and standard protocol for virus detection in berry crops (Fragaria, Rubus and Vaccinium spp.). 2020 APS meeting
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Villamor, D.E.V., Sierra, A., Keller, K., Martin, R. and Tzanetakis, I.E. Discovery of two new viruses infecting blueberry. 2020 APS meeting
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Shaffer, C., Michener, D., Vlasava, N.B. and Tzanetakis, I.E. Population structure, evolution, and detection of Lychnis mottle virus in peony. 2020 APS meeting
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Shaffer, C., Michener, D., Vlasava, N.B. and Tzanetakis, I.E. Brief report of Gentian Kobu-sho associated virus in peony. 2020 APS meeting
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Ho, T. and Tzanetakis, I.E. 2020. Using Next Generation Sequencing and VirFind Bioinformatic Tool to Discover Plant, Animal and Human Viruses. 2020 Indian Virological Society meeting.

Progress 10/01/18 to 09/30/19

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 and twoMasters 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?International, national and regional scientific meetings International, national and regional stakeholer (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 have developed a protocolbased on reverse transcription-PCR that simultaniously IDs eriophyiod mites and detects viruses in a single tube.The new nethod can ID the mite species based on a individual or egg because of andin additionto the genomic DNA, it amplifies the transcripts of targeted genes. At the same time it allows for the amplification of both RNA or DNA viruses. Because of the protocolefficiencies we can mainstream the identification of potential virus vectors. We could amplify viruses in all vectors tested whereas there was no amplification in any of the non vectors. There is alwaysgoing to be a need to perform transmission experiments to verify the in-vitro resultsbut we can eliminatespecies as potential vectors ifwe areunable to detect viruses in their bodies. Blackberry leaf mottle associated virus has been identified as a major component of yellow vein disease in the southern United States. We have studied the population structure of the virus and identified several markers that point to recombination and reassortment . Based on that data we were able to develop a sensitive quantitative reverse transcription PCR test that is able to detect all virus isolates identified to date. Lemoine's disease of peony, a disorder described more than 100 years ago remains a main interest of th lab. there are now seven viruses identified in peonies collected from around the country. Intriguingly, all sevenhave been viruses that have been described and only reported in Asia till now. It appears that the viruses made it to the Western hemisphere via the movement of peony propagation material.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Tzanetakis, I.E. Almeyda, C., Golino, D. and Martin R.R. How the Clean Plant Network safeguards the strawberry industry. 9th North American Strawberry Symposium, February 4th 2019, Orlando, Florida
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Katsiani, A., Lamour K., and Tzanetakis I.E. Studies on the population structure of rose rosette virus using high throughput sequencing. 19th Hellenic Phytopathological meeting
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Druciarek, T., Lewandowski, M. and Tzanetakis, I.E. Phyllocoptes (Acari: Eriophyoidea) from rose: how many species? 15th International Congress of Acareology
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Hassan, M., Shahid, M.S. and Tzanetakis, I.E., 2018. Molecular characterization and detection of a novel vitivirus infecting blackberry. Archives of Virology 163:28892893.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Druciarek, T., Lewandowski, M. and Tzanetakis I.E. 2019. First report of European mountain ash ringspot-associated emaravirus in Sorbus aucuparia in Poland. Plant Disease 103:166
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhou, J. and Tzanetakis, I.E. 2019. Soybean vein necrosis virus: an emerging virus in North America. Virus Genes 55: 12-21
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hassan, M., and Tzanetakis I.E. 2019. Population structure, evolution and detection of blackberry leaf mottle associated virus, an emerging Emaravirus. Plant Pathology 68: 775-782
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Shaffer, C., Gress, J.C., and Tzanetakis I.E. 2019. First Report of cycas necrotic stunt virus and lychnis mottle virus in Peony in the USA. Plant Disease 103: 1048.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Zurn, J.D., Ho, T., Li, R., Bassil, N.V., Tzanetakis, I.E., Martin, R.R. and Postman, J.D. 2019. First report of blackcurrant reversion virus in Ribes nigrum germplasm in the United States. Plant Disease, 103: 1051.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Villamor, D.E., Ho, T., Al Rwahnih, M., Martin, R.R. and Tzanetakis, I.E., 2019. High Throughput Sequencing in Plant Virus Detection and Discovery. Phytopathology 109: 716-725.
• Type: Book Chapters Status: Published Year Published: 2019 Citation: Improving plant propagation methods for fruit disease control. InIntegrated management of diseases and insect pests of tree fruit. Edited by Professor Xiangming Xu and Dr Michelle Fountain NIAB EMR, UK
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Tzanetakis, I.E. Start clean, stay clean: National Clean Plant Network APS annual meeting August 7th 2019, Cleveland, Ohio
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Tzanetakis, I.E. How bioinformatics impact plant movement across border lines. 5th Arkansas Bioinformatics Consortium meeting, February 25th 2019, Little Rock, Arkansas