# Presentation: 688029

## 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, Oregon State **
**S. Nameth, Ohio State**

## Necessity for clean propagation stock

**Viruses in certain crops are difficult to detect reliably – biggest problem in vegetatively propagated crops**
- What cultural conditions are optimal for virus replication, to increase detection?
- What tissues to sample for reliable detection?
- Can tissue culture plants be sampled directly?
- If not, how long after TC, and what conditions?

## Crops under examination

**Argyranthemum (Jordan)**
**Bacopa (Hsu)**
**Diascia (Dodds/Mathews)**
**Lobelia (Dodds/Mathews)**
**Petunia (Nameth; Hammond)**
**Scaevola (Hammond)**
**Verbena (Putnam/Jordan; Dodds/Mathews)**
- Osteospermum, New Guinea Impatiens, others

## Discussion meeting, Ecke Ranch, January 2003

## Source of cuttings

## Rooting cuttings

## Flats of Scaevola

## Scaevola - 1

**Putative potyvirus**
- Electron microscopy – few flexuous particles when plants first obtained
- No clear serological reaction
- No alternate host identified so far
- No clear dsRNA
- No particles found by electron microscopy in summer months

## Scaevola - 2

**Current approaches**
- Fresh plant material, maintain vegetative
- Grow under different conditions
- Repeat electron microscopy, serology
- Expand search for alternate host
- PCR with group-specific primers

## Bacopa

**Presumed to be *****Broad bean wilt virus***** type I; general chlorosis**
- Electron microscopy – angular isometric particles consistent with BBWV
- Serology (ELISA) negative with available BBWV antisera
- Transmission to *N. tabacum*, *C. quinoa*, but lost after 3rd transfer
- Loss of detectable infectivity from Bacopa following summer temperatures; so far not recovered infectivity after transfer to cooler growing conditions
- Obtain fresh material and repeat

## Argyranthemum

**Electron microscopy inconclusive**
**dsRNA inconclusive**
**ELISA detects *****Chrysanthemum virus B***** in many plants, but not all**
**No symptoms on any potential alternate hosts tested to date**
**Current continuing dsRNA analysis, and RT-PCR for *****Chrysanthemum stunt viroid *****and***** Chrysanthemum chlorotic mottle viroid***

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## Verbena

**Melodie Putnam**
**Oregon State University**

## Verbena potyvirus

**Identified as closely related to *****Pea mosaic virus***** and *****Bean yellow mosaic virus*****-CS (with Ramon Jordan)**
**Readily detectable in tissue culture by both bioassay and ELISA (PTY 1), ****but –**
**Titer can disappear rapidly (both bioassay and ELISA) over three weeks from a strong titer to +/- undetectable**
**Titer varies widely with environment**
- Light appears to have a bigger effect than temperature

## Detection of viruses in Verbena at all stages

**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 Pathology**
**UC Riverside**
**USDA/ARS Floral and Nursery Crop Research Initiative**

## Objectives

**	****	****	****  ****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 tobacco**
**mosaic 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 from**
**Verbena ‘Ron Deal’ (V-RD)**
**and *****Nicotiana clevelandii***
**(N.c.) inoculated with an **
**extract from V-RD.**

**Mosaic**
**symptoms**
**on Verbena **
**‘****Ron Deal’**

## Verbena ‘Ron Deal’ (cont’)

** **
** **
** ****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**

**T2**

**Scan of purified virus from V-RD **
**centrifuged through a sucrose density**
**gradient.  T1=proposed empty capsid**
**peak for typical tymovirus; T2=peak**
**of 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’.**

## 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.**

**DsRNAs extracted from citrus tristeza virus**
**(CTV), TMV and STMV, Diascia ‘Hannah**
**Rose’ (DHR), Diascia ‘Red Ace’ (DRA), and**
**Verbena ‘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.**

**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.**

## Conclusions

**Some of the viruses are indeed difficult to detect and identify**
**Some appear to be significantly affected by environmental conditions**
**Progress is being made to develop diagnostic methods, and to understanding environmental factors influencing detection**
**Partnership works; communication is important**
**On-going work is needed**