Update on Potato Corky Ringspot Research Gaurav Raikhy1, N.C. Gudmestad2, S.M. Gray3, and H.R. Pappu1

1Department of Plant Pathology, Washington State University, Pullman, WA 2Department of Plant Pathology, North Dakota State University, Fargo, ND

USDA-ARS, Ithaca, NY ABSTRACT

Tobacco rattle virus (TRV) (Genus: Tobravirus) is becoming increasingly important in many potato-growing areas of the world including the USA. TRV spreads from plant to plant by members of the stubby root nematode complex. The goal of this project is to understand the genetic diversity of TRV isolates from several states in the US. TRV isolates from CO, ID, MN, and ND were characterized at the molecular level. The genome was cloned and sequenced and compared to known TRV isolates from the US and other parts of the world. Knowledge gained would be useful in understanding the ecology, and epidemiology of the virus and will facilitate improved virus detection tools and technologies that could be applied to reduce the virus incidence and its impact.

Introduction •The value of potatoes produced in the US has been estimated as 3.9 billion USD (1). • The Pacific Northwestern (PNW) region (states of Idaho, Oregon, and Washington) accounts for more than one half of the country’s potato production (24 billion lbs against the national annual production of 46.7 billion lbs) (1).

• Tobacco rattle virus (TRV) (Genus: Tobravirus) has a wide host range (>600 plant species) and occurs in many different countries throughout the world including the USA (2). •.TRV causes important diseases of potato, ornamental plants, sugar beet and tobacco (3). • The virus spreads from plant to plant by root-feeding, members of the stubby root nematode complex (4). • TRV causes various symptoms on potato such as corky ringspot on potato tubers, stem mottle and transient yellow chevrons in the foliage (4).

•TRV was first reported from Florida (4). Since then it has been reported from 19 different states including Colorado, Michigan, Minnesota, Oregon, Washington and Wisconsin (Fig. 1) (5). • The complete genome of TRV is comprised of two positive sense RNAs, RNA-1 and RNA-2. RNA-2 shows variability both in length and in the number of open reading frames (ORFs). •To better understand the genetic diversity and epidemiology of TRV, molecular characterization of RNA-2 of the virus from four states was carried out. Materials and Methods •Twenty-two TRV-infected potato tissue and RNA samples were received from 4 different states of the USA from December 2014 to October 2015.

•Oligonucleotides were designed and used to amplify the conserved region in RNA-1 and complete RNA-2 of TRV (3).

• Amplicons were cloned into pGEM-T easy vector and sequenced. •Phylogenetic trees were constructed using MEGA 5 (4).

Results • Nearly complete RNA-2 was amplified by using the primer combination 158 and TR1 (Schmidt and Koeing,1999). RNA-2 from TRV isolates from CO, ID, MN, and ND were found to be 3537, 3606, 3024 and 2928 nucleotides long, respectively.

•The sequence obtained from all the four isolates had the complete coat protein (CP) and 2b protein.

•Only two isolates (from CO and ID) had the ORF for the complete 2c protein, while MN and ND isolates had truncated 2c protein. The isolates from MN and ND could be nematode non-transmissible as they encode for a truncated 2c protein. •Phylogenetic analysis based on a comparison of nucleotide and amino acid sequences of CP and 2b protein with other TRV isolates reported from other parts of the world, showed that the North American isolates cluster as a distinct group along with Asian isolates while European isolates formed a separate cluster. Significant homologies among American isolates were observed, while European isolates were found to be more diverse.

Conclusions In summary, the genome characterization and diversity studies of TRV among the US states will help in development of sensitive and rapid diagnostics and could be useful in designing various approaches for virus resistance. References 1. Crop production 2012 summary, January 2013, USDA/NASS 2. Otulak et al (2012). Canadian Journal of Plant Pathology 34:126-138. 3. Dikova (2006). Biotechnology & Biotechnological Equipment 20: 49-59. 4. Xu and Nie (2006). Canadian Journal of Plant Pathology 28:271-279. 5. EPPO Global Database (2015).

https://gd.eppo.int/taxon/TRV000/distribution 6. Crosslin et al. (2010). Archives of Virology 155: 621-625. 7. Schmidt and Koenig (1999). Archives of Virology 144: 503-511.

Figure 2. Incidence of Tobacco rattle virus in the USA

Acknowledgments: Funded by USDA NIFA Specialty Crop Research Initiative Grant # Supported by WSU Agricultural Research Center. Department of Plant Pathology, College of Agricultural, Human and Natural Resource Sciences, Hatch Project # WNPO 0545.

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Figure 3. Phylogenetic analysis of coat protein sequences of Tobacco rattle virus. The American and European isolates are grouped into two different clusters. Isolates highlighted in boxes were characterized in this study.

Figure 1. Corky ringspot symptoms by Tobacco rattle virus on potato tubers.

Biological and Economic Impacts of Emerging Potato Necrotic viruses and The Development of Comprehensive and Sustainable Management Practices

Funding Support: USDA National Institute of Food and Agriculture, USDA, ARS, Pacific Northwest Potato Commission, University of Wisconsin, Wisconsin Seed Potato Certification Program, University of Idaho, Montana State University Seed Potato Certification Program, Idaho Crop Improvement, Minnesota Department of Agriculture, North Dakota State University, North Dakota State Seed Dept., Michigan State University, Michigan Certified Seed Potatoes, University of Maine, Maine Potato Board Certified Seed Program, Cornell University, New York Seed Improvement Project, Colorado State University Potato Certification Program, Washington State University, Washington Certified Seed, Oregon State University.