VIRUS DISEASE DIAGNOSTICS IN SUPPORT OF THE NY GRAPE & NURSERY INDUSTRIES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1004285
• Project Start Date: Dec 17, 2014
• Project End Date: Sep 30, 2017
• Program Code: [(N/A)]- (N/A)

Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853

Performing Department
Plant Pathology

Non Technical Summary
Grape planting stock free of pathogens is essential for efficient production and the sustainable management of vineyards; profitable grape production depends on healthy vines to optimize yield and returns. Viruses and virus-like diseases are graft- and vector-transmissible. If present in the propagation material, these pathogens will be perpetuated and compromise the productivity of progeny vines. Addressing this problem requires three coordinated efforts: 1) effective pathogen detecting technologies and services must be in place to support a nursery industry, 2) foundation planting stocks for grapevine propagation need to be established and monitored, and 3) a certification program is required to provide quality assurance. This project addresses the first step by monitoring the prevalence of a newly emerging virus of grape and refining a state-of-the-art diagnostic technology; it addresses the last step by implementing the use of large scale sequencing for diagnostics and evaluating this technology as a more efficient alternative to traditional methods.

Animal Health Component

70%

Research Effort Categories

Basic

30%

Applied

70%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1131	1101	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1131 - Wine grapes;

Field Of Science
1101 - Virology;

Keywords

grape virus testing

grape nursery industry

Goals / Objectives
Objective 1 is to test for, monitor and educate growers about the newly emerging grapevine red blotch-associated virus in NY vineyards. We will determine the distribution of this virus in grape growing regions within the state and among the specific cultivars being grown. Educational presentations about the biology and impact of this disease in NY will be made to growers by extension staff (Fuchs, Martinson, Wise). Objective 2 is to develop diagnostic tools in support of the reactivation of a grapevine certification program in NY State. Focusing on nursery stocks and new introductions into NY, we will refine large-scale nucleic acid sequencing technologies to delineate virus, viroid, and other pathogens present in each cultivar. We will compare this technology (that provides results within weeks to months) with that of the current standard of biological indexing (that requires two to three years to obtain results).

Project Methods
For objective 1, investigators and extension cooperators will meet with vineyard and nursery managers to visually assess vine health and to collect samples from healthy and suspect vines. This approach will yield information on grapevine red blotch-associated virus (GRBaV) distribution, and in some cases, with additional sampling, prevalence. Extracts from vines will be tested for viruses using a panel of serological reagents (ELISA; protein-based) for the most common viruses of concern to producers. To test for grapevine red blotch-associated virus (GRBaV), nucleic acid-based amplification methods (PCR and rolling circle amplification) will be employed.For objective 2, newly developed methods (macroarrays, large-scale sequencing) will be compared with existing methodologies (ELISA, PCR, graft-indexing). Total nucleic acids will be extracted from grapevines and initially tested using our macroarray for the multiplexed detection of >43 viruses of grapevine. Vines of particular interest or those without viruses consistent with the observed symptoms will be processed for large-scale sequencing. DNA libraries will be prepared, sequenced and analyzed by an array of bioinformatics tools presently in use in our lab. Vines of particular interest will be subjected to graft-indexing.

Progress 12/17/14 to 09/30/17

Outputs
Target Audience:New York State nursery growers and grape growers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Over the three year period, two graduate students and seven undergraduate students trained in aspects of the diagnostics of grapevine viruses, Illumina sequencing as a diagnostic tool for plant virus diagnostics, and the biology of Grapevine red blotch virus. Additional training for growers and those in the grape and wine industry is addressed in the section below on dissemination of results to communities of interest. How have the results been disseminated to communities of interest?Over the two year period, public presentations were made at 30 grape grower and wine industry meetings reaching a total audience of 1975 participants. Additionally communications were made through newsletters (five), IPM fact sheets (two) and grower field days (two). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1 . We have determined that grapevine redblotch virus (GRBV) is established in commercial cultivars of wine grapes (Vitis vinifera) on Long Island and in the Finger Lakes wine-growing regions. We have tested for grapevine red blotch-associated virus (GRBaV) among samples from cultivated (n=194) and wild grapevines (n=31). We have determined that the distribution of GRBaV does not correlate with geographic location or variety. In surveys of wild (non-cultivated) vines in New York, GRBV has not been detected. There is no evidence of spread of the virus within New York vineyards. This contrasts with the situation in California, where the virus is both present in wild vines and is spreading within vineyards. Wild vines may serve as a reservoir, but at present there is no evidence that the virus is moving from wild into cultivated vines. There are two phylogenetic clades of GRBV and isolates from both of these genetic clades are observed in New York. No differences in disease severity is associated with different isolates of the virus. The main source of the virus in New York appears to be the nursery stocks purchased and planted by growers. The planting of certified grapevines free of GRBV should allow New York growers to avoid the damaging effects of this virus. A limiting factor for growers wishing to plant or replant vineyards is the availability of grape planting stocks that are free of GRBV and other viruses. New York State Nursery Growers are keenly aware of the importance of GRBV and this project has refined the tools necessary to allow for the detection and exclusion of the virus in propagated materials. These research findings have been communicated to nursery growers and commercial wine and grape-growers at regional and national industry meetings in all three years of this project. Objective 2. The primary diagnostic tool we have refined in support of grapevine certification is that of high throughput sequencing (HTS). Using total nucleic acid extracts from vines, libraries of small RNAs are prepared and submitted for Illumina sequencing. A bioinformatic pipeline is operational and allows the detection of all viruses present in any given vine sample. The time required for the use of HTS as a diagnostic tool for grapevines is approximatly 5 weeks (one week for sample preparation, 4 weeks for sequencing, two days for data analysis). This time requirement could be reduced to one week if an Illumina sequencng instrument were immediately accessible (e.g. in the same lab). This diagnostic technology has been applied to the testing of ~111 vine samples (Vitis sp.), including commercially cultivated plants, Vitis collections, and non-cultivated (wild) vines from within and outside of New York. We have observed four viroids and 11 virus species. Some of these agents were not previously reported in New York and are described in research publications from this project, including Australian grapevine viroid, Grapevine asteroid mosaic-associated virus, Arabis mosaic virus, and Grapevine virus E. Three viroids, Hop stunt viroid and Grapevine yellow spreckle viroid 1, and Grapevine yellow spreckle viroid 2 are common in commercial vines and hop stunt viroid can be detected in nearly all vines tested. Other viruses detecting using this diagnostic system include Grapevine red blotch virus, Grapevine syrah virus 1, Grapevine rupestris stem-pitting virus, Grapevine rupestris vein feathering virus, Grapevine red globe virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 2, and Grapevine leafroll-associated virus 3.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Cieniewicz, E. J., Pethybridge, S. J., Loeb, G., Perry, K. L., and Fuchs, M. (2018). Insights into the ecology of grapevine red blotch virus in a diseased vineyard. Phytopathology 98:94-102. Perry, K. L., McLane, H., Thompson, J. R., & Fuchs, M. (2017). A novel grablovirus from non-cultivated grapevine (Vitis sp.) in North America. Archives of Virology, 163(1), 259262. Li, R., Fuchs, M., Perry, K. L., Mekuriad, T., Zhang, S. 2017. Development of a fast AmplifyRP Acceler8 diagnostic assay for grapevine red blotch-associated virus. Journal of Plant Pathology 99:657662. Vargas-Asencio, J., Perry, K.L., Wise, A., Fuchs, M., 2017. Detection of Australian grapevine viroid in Vitis vinifera in New York. Plant Disease. 101:848. Cieniewicz, E. J., Pethybridge, S. J., Gorny, A., Madden, L. V., McLane, H., Perry, K. L., and Fuchs, M. 2017. Spatiotemporal spread of grapevine red blotch-associated virus in a California vineyard. Virus Research 241:156-162. (http://dx.doi.org/10.1016/j.virusres.2017.03.020). Vargas Asencio J., Wojciechowska K., Baskerville M., Gomez A.L., Perry K.L., Thompson J.R. 2017. The complete nucleotide sequence and genomic characterization of Grapevine asteroid mosaic associated virus. Virus Research 227:82-87.
• Type: Other Status: Published Year Published: 2015 Citation: Cieniewicz, E. and Fuchs, M. 2015. Grapevine leafroll disease. IPM FactSheet. http://www.nysipm.cornell.edu/factsheets/grapes/diseases/grape_leafroll.pdf
• Type: Other Status: Published Year Published: 2014 Citation: Cieniewicz, E. and Fuchs, M. 2014. Grapevine red blotch disease. IPM FactSheet. http://www.nysipm.cornell.edu/factsheets/grapes/diseases/gv_red_blotch.pdf

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

Outputs
Target Audience:The targeted audiences are NY grapevine nursery growers and commercial vineyard growers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Five undergraduate students and one graduate student have worked on the detection of GRBaV and other grapevine viruses. They have also contributed to the large-scale sequencing of grapevine nucleic acids as a diagnostic tool for to detect viruses in grapevine. How have the results been disseminated to communities of interest?Six webinar venue presentations were made; these are listed under other products. What do you plan to do during the next reporting period to accomplish the goals?A continuation of the same set of activities as originally described and as performed during the last reporting period.

Impacts
What was accomplished under these goals? During this reporting period, we have tested for grapevine red blotch-associated virus (GRBaV) among samples from cultivated (n=194) and wild grapevines (n=31). We have determined that the distribution of GRBaV does not correlate with geographic location or variety, and no patterns have been observed that would be suggestive of movement within the field. The presence of the virus appears to be a function of the source of the planting stocks. Wild vines may serve as a reservoir, but at present there is no evidence that the virus is moving from wild into cultivated vines. We have established the methodologies to allow for large-scale nucleic acid sequencing to delineate virus, viroid, and other pathogens present. Libraries of grapevine small RNAs have been constructed, sequenced, and the data processed bioinformatically. Viroids are present in most all cultivated grapevine samples. The most commonly observed viruses are Grapevine rupestris stem pitting virus and members of the family Tymoviridae.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Vargas Asencio J., Wojciechowska K., Baskerville M., Gomez A.L., Perry K.L., Thompson J.R. 2017. The complete nucleotide sequence and genomic characterization of Grapevine asteroid mosaic associated virus. Virus Research 227:82-87.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Perry, K. L., McLane, H., Hyder, M. Z., Dangl, G. S., Thompson, J. R., and Fuchs, M. F. 2016. Grapevine red blotch-associated virus is present in free-living Vitis sp. proximal to cultivated grapevines. Phytopathology 106:663670.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Vargas-Asencio, J. A., Al Rwahnih, M., Rowhani, A., Thompson, J. R., Fuchs, M. & Perry, K. L. (2016). Limited genetic variability among American isolates of Grapevine virus E from Vitis spp. Plant Disease. 100:159-163.

Progress 12/17/14 to 09/30/15

Outputs
Target Audience:New York State nursery growers, grape growers, and wine industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Educational presentations about the biology and impact of this disease in NY were made to growers by extension staff.? What do you plan to do during the next reporting period to accomplish the goals?We will be testing for grapevine viruses in NY State vineyards. We will continue torefinelarge-scale nucleic acid sequencing technologies to delineate virus, viroid, and other pathogens present in grapevine cultivars.

Impacts
What was accomplished under these goals? Grapevine red blotch-associated virus has been surveyed for in NY State vineyards. It was detected in vines from Long Island and the Fingerlakes region. We arerefininglarge-scale nucleic acid sequencing technologies to delineate virus, viroid, and other pathogens present in grapevine cultivars. This has involved RNA extraction methods, purification (or not) of small RNAs, purification of amplified library products, size analysis of amplified library products, analyses of large-scale sequence data sets, computational searches for andanalyses of viral sequences in the data sets.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Thompson, J., Fuchs, M., McLane, H., Toprak-Celebi, F., Fisher, K., Potter, J., and Perry, K. L., 2014. Profiling viral infections in grapevine using a randomly primed reverse transcription-polymerase chain reaction/macroarray multiplex platform. Phytopathology 104, 211-219.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Krenz, B., Thompson, J. R., McLane, H., Fuchs, M., and Perry, K. L. 2014. Grapevine red blotch-associated virus is widespread in the United States. Phytopathology 102:1232-1240.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2015 Citation: Vargas-Asencio, J. A., Al Rwahnih, M., Rowhani, A., Thompson, J. R., Fuchs, M. & Perry, K. L. (2015). Limited genetic variability among American isolates of Grapevine virus E from Vitis sp. Plant Disease (Accepted for publication) http://dx.doi.org/10.1094/PDIS-05-15-0556-RE.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Sudarshana, M. R., Perry, K. L. and Fuchs, M. F. (2015). Grapevine red blotch-associated virus, an emerging threat to the grapevine industry. Phytopathology 105, 10261032.