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. National Clonal Germplasm Repository = National Germplasm Resources Lab O National Seed Storage Lab
Studies on Viruses Infecting Mint Germplasm J.D. Postman I.A. Tzanetakis R.R. Martin USDA Agricultural Research Service and Oregon State UniversityCorvallis, Oregon
National Clonal Germplasm Repository = National Germplasm Resources Lab O National Seed Storage Lab n Regional Plant Introduction Station National Plant Germplasm System Pullman n Geneva Corvallis n Ames n O Ft.Collins = Davis Beltsville Washington D.C. n Griffin Riverside Hilo Brownwood Mayaguez
Collect, Maintain, Evaluate and Distribute Mentha Germplasm
NCGR Mentha Collection • 436 plant accessions in screenhouse • 57 seed accessions • 141 back-up plantaccessions in tissue culture
‘Golden Ginger Mint’Mentha × gentilis L. 'Variegata' (a.k.a. ‘Green & Gold’, ‘Scotch Mint’) • This variegated mint known since 1798 • Heat therapy eliminates symptoms • Graft transmissible • Causal agent unknown
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.
Three Viruses Found in ‘Golden Ginger Mint’ • Strawberry latent ringspot virus (SLRV)(already reported to infect mint in Europe) • A new Potexvirus (Flexiviridae) we will call “Mint Virus X” (MVX) • A new Closterovirus we will call “Mint Veinbanding Associated Virus” (MVBV)
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
Strawberry latent ringspot virus (SLRSV) is/was a quarantine virus in North America. • Electron microscopy revealed typical 30-32nm icosahedral particles
L GB GER Agdia TBRV RRSV block Strawberry latent ringspot virus in mint • IC-RT-PCR verified that the molecule belongs to SLRSV
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?
Symptoms not consistent with SLRSV infection • dsRNA extractions, cloning, electron microscope revealed presence of 2 other viruses
Closterovirus Potexvirus • Strawberry Latent Ringspot Virus (SLRV) • “Mint Virus X” (MVX) - new Potexvirus (Flexiviridae) • “Mint Veinbanding Associated Virus” (MVBV) - new Closterovirus
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 andWhite Clover Mosaic Virus
ClosterovirusMint Vein Banding associated Virus Also found in another NCGR clone - Mentha canadensis (MEN 617) More than 8 kpb sequenced
Polymerase Closterovirus Ampelovirus Crinivirus MVBV A unique memberof Closteroviridae shares homology with all three genera of the family. Ancestral member of the family? Progenitor of all three genera? aphid transmittedmonopartite genome mealybug transmittedmonopartite genome whitefly transmitted bipartite genome
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.
Closterovirus MLV Ampelovirus Crinivirus Mint Latent Virus (MLV) Another new Closterovirus (about 95% sequenced) with similarities to BYV, GLRaV 2 and CTV
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.
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 Molecular detection is available for all 5 viruses
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)
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.
2005 Plans We will not look for any new mint viruses!
2005 Plans • Produce antiserum against the coat proteins of the newly discovered potexvirus and two closteroviruses to allow rapid detection by ELISA
2005 Plans • Use heat therapy and meristem culture to eliminate new viruses from infected clones
2005 Plans • complete sequences for new viruses • determine which virus combination is responsible for symptoms • insect transmission tests
Acknowledgements • Mint Industry Research Council • Bob Martin – USDA/ARS • Yannis Tzanetakis – Oregon State University • Rose Gergerich lab in Arkansas (beetle transmission of SLRSV)
Mentha ‘Green & Gold’ Golden Ginger Mint Variegated Melissa Oregon Ginger Mint Variegated Peppermint Melissa All Gold