Virus Research Needs for the Floral Industry John Hammond; USDA-ARS R. Jordan, H-T. Hsu; USDA-ARS A. Dodds, D. Mathews, U.CA Riverside Melodie Putnam,

Virus Research Needs Virus Research Needs for the Floral Industryfor the Floral Industry

John Hammond; USDA-ARSJohn Hammond; USDA-ARSR. Jordan, H-T. Hsu; USDA-ARSR. Jordan, H-T. Hsu; USDA-ARS

A. Dodds, D. Mathews, U.CA A. Dodds, D. Mathews, U.CA RiversideRiverside

Melodie Putnam, Oregon State Melodie Putnam, Oregon State

S. Nameth, Ohio StateS. Nameth, Ohio State

Necessity for clean Necessity for clean propagation stockpropagation stock

• Viruses in certain crops are Viruses in certain crops are difficult to detect reliably – difficult to detect reliably – biggest problem in vegetatively biggest problem in vegetatively propagated cropspropagated crops– What cultural conditions are optimal for virus What cultural conditions are optimal for virus

replication, to increase detection?replication, to increase detection?– What tissues to sample for reliable detection?What tissues to sample for reliable detection?– Can tissue culture plants be sampled directly?Can tissue culture plants be sampled directly?– If not, how long after TC, and what conditions?If not, how long after TC, and what conditions?

Crops under Crops under examinationexamination

• Argyranthemum (Jordan)Argyranthemum (Jordan)• Bacopa (Hsu)Bacopa (Hsu)• Diascia (Dodds/Mathews)Diascia (Dodds/Mathews)• Lobelia (Dodds/Mathews)Lobelia (Dodds/Mathews)• Petunia (Nameth; Hammond)Petunia (Nameth; Hammond)• Scaevola (Hammond)Scaevola (Hammond)• Verbena (Putnam/Jordan; Verbena (Putnam/Jordan;

Dodds/Mathews)Dodds/Mathews)– Osteospermum, New Guinea Impatiens, othersOsteospermum, New Guinea Impatiens, others

Discussion meeting, Discussion meeting, Ecke Ranch, January Ecke Ranch, January

20032003

Source of cuttingsSource of cuttings

Rooting cuttingsRooting cuttings

Flats of ScaevolaFlats of Scaevola

Scaevola - 1Scaevola - 1

• Putative potyvirusPutative potyvirus– Electron microscopy – few flexuous particles Electron microscopy – few flexuous particles

when plants first obtainedwhen plants first obtained– No clear serological reactionNo clear serological reaction– No alternate host identified so farNo alternate host identified so far– No clear dsRNANo clear dsRNA– No particles found by electron microscopy in No particles found by electron microscopy in

summer monthssummer months

Scaevola - 2Scaevola - 2

• Current approachesCurrent approaches– Fresh plant material, maintain vegetativeFresh plant material, maintain vegetative– Grow under different conditionsGrow under different conditions– Repeat electron microscopy, serology Repeat electron microscopy, serology – Expand search for alternate hostExpand search for alternate host– PCR with group-specific primersPCR with group-specific primers

BacopaBacopa

• Presumed to be Presumed to be Broad bean wilt virusBroad bean wilt virus type I; general chlorosistype I; general chlorosis– Electron microscopy – angular isometric particles Electron microscopy – angular isometric particles

consistent with BBWVconsistent with BBWV– Serology (ELISA) negative with available BBWV antiseraSerology (ELISA) negative with available BBWV antisera– Transmission to Transmission to N. tabacumN. tabacum, , C. quinoaC. quinoa, but lost after 3, but lost after 3rdrd

transfertransfer– Loss of detectable infectivity from Bacopa following Loss of detectable infectivity from Bacopa following

summer temperatures; so far not recovered infectivity summer temperatures; so far not recovered infectivity after transfer to cooler growing conditionsafter transfer to cooler growing conditions

– Obtain fresh material and repeatObtain fresh material and repeat

ArgyranthemumArgyranthemum

• Electron microscopy inconclusiveElectron microscopy inconclusive• dsRNA inconclusivedsRNA inconclusive• ELISA detects ELISA detects Chrysanthemum Chrysanthemum

virus Bvirus B in many plants, but not all in many plants, but not all• No symptoms on any potential No symptoms on any potential

alternate hosts tested to datealternate hosts tested to date• Current continuing dsRNA analysis, Current continuing dsRNA analysis,

and RT-PCR for and RT-PCR for Chrysanthemum Chrysanthemum stunt viroid stunt viroid andand Chrysanthemum Chrysanthemum chlorotic mottle viroidchlorotic mottle viroid

Detection of TMV in Petunia

S. Nameth and A. Grincewicz

Ohio State University

E L I S A R e s u l t s o f T M V i n o c u l a t i o n i n P e t u n i a

Plant 1.1 Plant 1.2 Plant 2.1 Plant 2.2Control 0.057 0.069Jan. 15 0.059 0.18 0.075 0.117Jan. 17 0.178 0.4 0.085 0.309Jan. 19 0.068 0.145 0.07 0.311Jan. 21 0.086 3.022 0.955 0.276Jan. 23 2.467 0.317 3 0.592Jan. 25 3 0.177 0.61 0.626

Average ELISA Results

• Positive result x>=0.126 (Two times control average)

• Positive results are bolded

Ave.PL 1 Ave PL 2Control 0.063Jan. 15 0.1195 0.096Jan. 17 0.289 0.197Jan. 19 0.1065 0.1905Jan. 21 1.554 0.6155Jan. 23 1.392 1.796Jan. 25 1.5885 0.618

Graph of ELISA results

TMV Titer

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

Jan. 15 Jan. 17 Jan. 19 Jan. 21 Jan. 23 Jan. 25

Sample Date

OD

40

5n

m Ave.PL 1

Ave PL 2

VerbenaVerbena

Melodie PutnamMelodie Putnam

Oregon State UniversityOregon State University

Verbena potyvirusVerbena potyvirus

• Identified as closely related to Identified as closely related to Pea Pea mosaic virusmosaic virus and and Bean yellow mosaic Bean yellow mosaic virusvirus-CS (with Ramon Jordan)-CS (with Ramon Jordan)

• Readily detectable in tissue culture by Readily detectable in tissue culture by both bioassay and ELISA (PTY 1), both bioassay and ELISA (PTY 1), but –but –

• Titer can disappear rapidly (both Titer can disappear rapidly (both bioassay and ELISA) over three weeks bioassay and ELISA) over three weeks from a strong titer to +/- undetectablefrom a strong titer to +/- undetectable

• Titer varies widely with environmentTiter varies widely with environment– Light appears to have a bigger effect than temperatureLight appears to have a bigger effect than temperature

Detection of viruses in Verbena at all stages

of its propagation cycle

D.M. Mathews, J.A. Heick, and J.A. Dodds

Dept. of Plant PathologyUC Riverside

USDA/ARS Floral and Nursery Crop Research Initiative

• Five different host species: Verbena ‘Temari Bright Pink’ Verbena ‘Ron Deal’ Diascia ‘Red Ace’ Diascia ‘Hannah Rose’ Lobelia ‘Tioga Blue’

Objectives

• Identify viruses present through the use of:-dsRNA analysis-host range reactions-virus purification -coat protein

-viral RNA -electron microscopy -antibody production

• Determine best tissue sources for detection (age, type, etc).

Verbena ‘Temari Bright Pink’

• No visible symptoms in plants provided, low titre of several dsRNAs found in 2 individual plants.• Host range analysis: C. quinoa-red LL, becoming systemic leading to plant death.

` G. globosa-red systemic lesions. N. clevelandii- very mild systemic mosaic.

• Same dsRNA pattern found in each alternate host. Titre very low.

•Attempts at virus particle purification not successful to date. Infected tissue preserved by drying for later activation in new plants and further study.

T+S V-TBP N.c.

DsRNAs extracted from Verbena ‘Temari Bright Pink’(V-TBP) and Nicotiana clevelandii inoculated with an extract from V-TBP (N.c.), with the dsRNAs of tobaccomosaic virus (TMV) and satellite TMV as controls (T+S).

6,400 nt

1,000 nt

Verbena ‘Ron Deal’• Strong mosaic in plants provided. Two or three major dsRNAs isolated.• Host range analysis led to LL and systemic infection in N. clevelandii. DsRNAs recovered match V-RD pattern.

• Identified as infected by ScrMV by outside testing lab.• No reaction in Datura stramonium, diagnostic host for ScrMV. No dsRNA found in inoculated D. stramonium plants.

V-RD N.c.

DsRNAs extracted fromVerbena ‘Ron Deal’ (V-RD)and Nicotiana clevelandii(N.c.) inoculated with an extract from V-RD.

Mosaicsymptomson Verbena ‘Ron Deal’

• Virus purification: 3 peaks on SDGC. 2 peaks similar to tymovirus group.

• Purified virus moves toward anode in electrophoresis, opposite of that reported for ScrMV. SsRNAs correct size(s) for tymovirus.• Possible candidate: Ononis yellow mosaic tymovirus.

Verbena ‘Ron Deal’ (cont’)

T1

T2Scan of purified virus from V-RD centrifuged through a sucrose densitygradient. T1=proposed empty capsidpeak for typical tymovirus; T2=peakof typical intact tymovirus particles;X1=possible additional virus, very low titre.

X1

TMV T1 X1 T2

Single stranded RNAs isolated from virus particles purified using SDGC above. T1=possible subgenomic RNA for cp found in empty capsids of tymovirus; T2=expected size for tymovirus genomic RNA; X2=2 or 3 RNAs of unknown origin with some T1 and T2 RNAs also present; TMV ssRNA provided as control.

6,400 nt

Diascia ‘Red Ace’• No visible symptoms on provided plants. Identified as positive for ScrMV by outside testing lab. Several dsRNAs found.• Chlorotic LL and severe systemic mosaic in N. clevelandii, most dsRNAs retained.

• No reaction in D. stramonium, N. benthamiana., N. tabacum ‘Xanthi nc’ or C. quinoa.• Virus purification: 2 peaks on SDGC, tymovirus like. Coat protein and ssRNA match tymovirus group. Purified virus causes same symptoms in N. clevelandii. Virions move to anode as in Verb.’R. Deal’.

RA N.c. N.c.-PV RA N.c. N.c.-PV

DsRNAs isolated from Diascia ‘Red Ace’ (RA); Nicotianaclevelandii inoculated with an extract from D-RA (N.c.);and N. clevelandii inoculated with purified virus from D-RA (N.c.-PV). Left hand photo is normal exposure, right hand photo is reduced exposure of same gel to resolvehigh titre bands. Note that host range plants have higher titre of dsRNA than original D-RA plant.

c.a. 6,500 nt

c.a. 850 nt

Diascia ‘Hannah Rose’• No visible symptoms, multiple dsRNAs found-some same as D-RA and V-RD.

•No reaction on D. stramonium, further host range pending.• Virus purification: 3+ peaks on SDGC, 2 same as V-RD and D-RA, third further down gradient. Tymovirus likely, others not yet determined.

CTV STMV DHR DRA VRD TMV

6,400 nt19,250 nt

1,050 nt

DsRNAs extracted from citrus tristeza virus(CTV), TMV and STMV, Diascia ‘HannahRose’ (DHR), Diascia ‘Red Ace’ (DRA), andVerbena ‘Ron Deal’ (VRD).

Lobelia ‘Tioga Blue’• Ringspots found in isolated leaves. 4 dsRNAs isolated, different pattern than others found in Verbena and Diascia spp.

• Host range: C. quinoa, N. glutinosa, N. tabacum ‘Xanthi and ‘Xanthi nc’, N. sylvestris- necrotic LL; N. benthamiana- mild mosaic, tip epinasty, leading to plant death. DsRNAs not recovered from host range plants, but limited tissue available.• Virus purification pending.

TMVSTMV LOB

DsRNAs extracted fromLobelia ‘Tioga Blue’(LOB) with those from TMV and STMV ascontrols.

6,400 nt

1,050 nt

Tip epinasty 2-3 wks dpi Plant death 4-5 wks

Symptoms of N. benthamiana plants inoculated with extract from Lobelia ‘Tioga Blue’

Summary – Dodds/Mathews

• Viruses abound in ornamentals tested.• Tymovirus group predominant in these, but probably not typical ScrMV, possibly OYMV?• At least 2-3 other viruses present.• Additional host range data needed.• Different separation methods needed before virus preparations can be used for Ab production.• Electron microscopy to confirm particle types, sizes.

ConclusionsConclusions

• Some of the viruses are indeed difficult Some of the viruses are indeed difficult to detect and identifyto detect and identify

• Some appear to be significantly Some appear to be significantly affected by environmental conditionsaffected by environmental conditions

• Progress is being made to develop Progress is being made to develop diagnostic methods, and to diagnostic methods, and to understanding environmental factors understanding environmental factors influencing detectioninfluencing detection

• Partnership works; communication is Partnership works; communication is importantimportant

• On-going work is neededOn-going work is needed