Studies on Viruses Studies on Viruses Infecting Mint GermplasmInfecting Mint Germplasm
J.D. Postman I.A. Tzanetakis R.R. MartinJ.D. Postman I.A. Tzanetakis R.R. Martin
USDA Agricultural Research ServiceUSDA Agricultural Research Serviceand Oregon State Universityand Oregon State University
Corvallis, OregonCorvallis, Oregon
National Plant Germplasm System
Ft.Collins Beltsville
Corvallis
Davis
RiversideHilo
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
Collect, Maintain, Evaluate andDistribute Mentha Germplasm
NCGR Mentha Collection
436 plant accessions in screenhouse
57 seed accessions
141 back-up plantaccessions in tissue culture
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’)
Before and After Heat Therapy
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’
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)
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
Strawberry latent ringspot virus in mint
IC-RT-PCR verified that the molecule belongs to SLRSV
L GB GER Agdia TBRV RRSV block
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
Potexvirus Closterovirus
1. Strawberry Latent Ringspot Virus (SLRV)
2. “Mint Virus X” (MVX) - new Potexvirus (Flexiviridae)
3. “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 and White Clover Mosaic Virus
ClosterovirusMint Vein Banding associated
VirusAAlso found in another lso found in another
NCGR clone - NCGR clone - Mentha Mentha canadensis (canadensis (MEN 617)MEN 617)
MMore than 8 kpb ore than 8 kpb sequencedsequenced
Polymerase
Crinivirus
Ampelovirus
Closterovirus MVBVA 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 transmittedbipartite 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.
dsRNA extractions revealed multiple bands including one ~ 15Kbp
Another new Closterovirus (about 95% sequenced) with similarities to BYV, GLRaV 2 and CTV
Crinivirus
Ampelovirus
Closterovirus
MLVMint Latent Virus(MLV)
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.
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
Incidence of viruses in USDA mint germplasm: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: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 Incidence of new viruses in mint from retail and mail-order nurseries: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.
What is the significance to mint production?
??
We will not look We will not look for any new mint viruses!for any new mint viruses!
2005 Plans
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’Mentha ‘Green & Gold’Golden Ginger MintGolden Ginger Mint
VariegatedVariegated Peppermint Peppermint
Variegated MelissaVariegated Melissa
Melissa All GoldMelissa All Gold
Oregon Ginger Mint Oregon Ginger Mint