1. Studies on Viruses Infecting Mint Germplasm J.D. Postman I.A. Tzanetakis R.R. Martin USDA Agricultural Research Service and Oregon State University Corvallis, Oregon

2. National Plant Germplasm System  Ft.Collins Beltsville Corvallis Davis Riverside Hilo Brownwood Mayaguez Geneva Washington D.C.  Pullman Griffin Ames  National Clonal Germplasm Repository  National Germplasm Resources Lab  National Seed Storage Lab  Regional Plant Introduction Station

3. Collect, Maintain, Evaluate and Distribute Mentha Germplasm

4. NCGR Mentha Collection  436 plant accessions in screenhouse  57 seed accessions  141 back-up plant accessions in tissue culture

5.  This variegated mint known since 1798  Heat therapy eliminates symptoms  Graft transmissible  Causal agent unknown ‘Golden Ginger Mint’ Mentha × gentilis L. 'Variegata' ( a.k.a. ‘Green & Gold’, ‘Scotch Mint’)

6. Before and After Heat Therapy

7. Identification of New Viruses (I. Tzanetakis)  dsRNA extracted from infected mint plants  dsRNA converted to dsDNA using reverse transcriptase and cloned into a vector.  Vector is sent to commercial lab to determine nucleotide sequence  BLAST program is to search public databases for sequences corresponding to the translated amino acid sequence.

8. Three Viruses Found in ‘Golden Ginger Mint’ 1.Strawberry latent ringspot virus (SLRV) (already reported to infect mint in Europe) 2.A new Potexvirus (Flexiviridae) we will call “Mint Virus X” (MVX) 3.A new Closterovirus we will call “Mint Veinbanding Associated Virus” (MVBV)

9. Strawberry latent ringspot virus in mint  European “nepo” virus*  Also recently found in strawberry in California and British Columbia  No similarities with any other virus  Available sequence only from RNA II

10.  Strawberry latent ringspot virus (SLRSV) is/was a quarantine virus in North America.  Electron microscopy revealed typical 30-32nm icosahedral particles

11. Strawberry latent ringspot virus in mint  IC-RT-PCR verified that the molecule belongs to SLRSV L GB GER Agdia TBRV RRSV block

12. SLRV from mint:  RNA II sequence shares ~80% nucleotide identity with published sequences.  ‘Golden Ginger Mint’ (MEN 454)  also carries SLRV satellite RNA  encodes a smaller protein than reported for other SLRSV isolates (33KDa vs 36KDa)  Mint SLRV movement protein also different  Nematode Transmission?

13.  Symptoms not consistent with SLRSV infection  dsRNA extractions, cloning, electron microscope revealed presence of 2 other viruses

14. Potexvirus Closterovirus 1.Strawberry Latent Ringspot Virus (SLRV) 2.“Mint Virus X” (MVX) - new Potexvirus (Flexiviridae) 3.“Mint Veinbanding Associated Virus” (MVBV) - new Closterovirus

15. Potexvirus - Mint Virus X  Host range on indicators is similar to other potexviruses.  Sequence data indicates a typical potexvirus (Genome is mostly sequenced).  Phylogenetic analysis reveals clustering with Potato Virus X and White Clover Mosaic Virus

16. Closterovirus Mint Vein Banding associated Virus Also found in another NCGR clone - Mentha canadensis (MEN 617) More than 8 kpb sequenced

17. Polymerase Crinivirus Ampelovirus Closterovirus MVBV A unique member of Closteroviridae shares homology with all three genera of the family. Ancestral member of the family? Progenitor of all three genera? aphid transmitted monopartite genome mealybug transmitted monopartite genome whitefly transmitted bipartite genome

18. A Variegated Ginger Mint that didn’t look right… This plant from an Oregon nursery had symptoms different from other ‘Golden Ginger’ mints. PCR tests for the viruses found in other clones were negative.

19. dsRNA extractions revealed multiple bands including one ~ 15Kbp

20. Another new Closterovirus (about 95% sequenced) with similarities to BYV, GLRaV 2 and CTV Crinivirus Ampelovirus Closterovirus MLV Mint Latent Virus (MLV)

21. Mint Virus A (MVA) Yet another virus! While multiple dsRNA bands are consistent with closterovirus infestation, cloning revealed the presence of another virus. This virus belongs to the Vitivirus genus, family Flexiviridae, closely related to GVA.

22. Molecular detection is available for all 5 viruses Clos Viti C- V- VB SLRSV MVX MVX- SLRSV- VB- Detection of New Viruses in Commercial Mint Stocks RNA nucleotide sequence  develop primers for PCR detection

23. Incidence of viruses in USDA mint germplasm:  MVBV in at least two plants, SLRV only in ‘Golden Ginger’.  MVX in several plants (MYV and MVA not yet tested)  tobacco ringspot virus detected by ELISA in several plants exhibiting mild vein-banding Incidence of viruses in commercial mint fields:  The two closteroviruses (MVBV and MLV) were found in samples from several production fields in Oregon.  SLRV and MVX not found in commercial stocks (not yet tested for MVA)

24. Incidence of new viruses in mint from retail and mail-order nurseries:  All ‘Golden Ginger Mint’ from nurseries in Maryland, Michigan, Nebraska, Ohio, and Oregon tested positive for the potexvirus (MVX) – many also tested positive for MVBV and SLRV.  SLRV was only found in ‘Golden Ginger’ Mint  MVA (vitivirus) was only found in variegated mint from one Oregon Nursery (most other sources not yet tested)  MVBV and MLV (second closterovirus) – found in some plants from many sources.

25. What is the significance to mint production? ?

26. We will not look for any new mint viruses! 2005 Plans

27.  Produce antiserum against the coat proteins of the newly discovered potexvirus and two closteroviruses to allow rapid detection by ELISA

28. 2005 Plans  Use heat therapy and meristem culture to eliminate new viruses from infected clones

29. 2005 Plans  complete sequences for new viruses  determine which virus combination is responsible for symptoms  insect transmission tests

30. Acknowledgements  Mint Industry Research Council  Bob Martin – USDA/ARS  Yannis Tzanetakis – Oregon State University  Rose Gergerich lab in Arkansas (beetle transmission of SLRSV)

31. Mentha ‘Green & Gold’ Golden Ginger Mint Variegated Peppermint Variegated Melissa Melissa All Gold Oregon Ginger Mint