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 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 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

MLV

Mint 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