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Virus Research Needs for the Floral Industry

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.

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Virus Research Needs for the Floral Industry

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  1. 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

  2. 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?

  3. 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

  4. Discussion meeting, Ecke Ranch, January 2003

  5. Source of cuttings

  6. Rooting cuttings

  7. Flats of Scaevola

  8. 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

  9. 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

  10. 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

  11. 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

  12. Verbena Melodie Putnam Oregon State University

  13. 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

  14. 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

  15. 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).

  16. 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. 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 (T+S). 1,000 nt

  17. 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.

  18. 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. T1=proposed empty capsid peak for typical tymovirus; T2=peak of typical intact tymovirus particles; X1=possible additional virus, very low titre. T2 T1 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

  19. RA N.c. N.c.-PV RA N.c. N.c.-PV c.a. 6,500 nt DsRNAs isolated from Diascia ‘Red Ace’ (RA); Nicotiana clevelandii 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 resolve high titre bands. Note that host range plants have higher titre of dsRNA than original D-RA plant. c.a. 850 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’.

  20. TMV CTV STMV DHR DRA VRD 19,250 nt 6,400 nt 1,050 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. DsRNAs extracted from citrus tristeza virus (CTV), TMV and STMV, Diascia ‘Hannah Rose’ (DHR), Diascia ‘Red Ace’ (DRA), and Verbena ‘Ron Deal’ (VRD).

  21. TMV STMV LOB Tip epinasty 2-3 wks dpi Plant death 4-5 wks 6,400 nt DsRNAs extracted from Lobelia ‘Tioga Blue’ (LOB) with those from TMV and STMV as controls. 1,050 nt 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’

  22. 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.

  23. 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

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