Virus Research Needs for the Floral Industry

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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

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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?

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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

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Discussion meeting, Ecke Ranch, January 2003
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Source of cuttings
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Rooting cuttings
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Flats of Scaevola
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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

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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

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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

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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

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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

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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
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Objectives

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

• 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).

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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.

TS V-TBP N.c.
6,400 nt
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 (TS).
1,000 nt
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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.
Mosaic symptoms on Verbena Ron Deal
DsRNAs extracted from Verbena Ron Deal
(V-RD) and Nicotiana clevelandii (N.c.)
inoculated with an extract from V-RD.
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Verbena Ron Deal (cont)

• 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.

Scan of purified virus from V-RD centrifuged
through a sucrose density gradient. T1proposed
empty capsid peak for typical tymovirus
T2peak of typical intact tymovirus
particles X1possible additional virus, very low
titre.
T2
T1
X1
TMV T1 X1 T2
Single stranded RNAs isolated from virus
particles purified using SDGC above. T1possible
subgenomic RNA for cp found in empty capsids of
tymovirus T2expected size for tymovirus genomic
RNA X22 or 3 RNAs of unknown origin with some
T1 and T2 RNAs also present TMV ssRNA provided
as control.
6,400 nt
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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.

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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

DsRNAs extracted from citrus tristeza
virus (CTV), TMV and STMV, Diascia Hannah Rose
(DHR), Diascia Red Ace (DRA), and Verbena Ron
Deal (VRD).
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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
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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.

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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