2008 Annual Report 1a.Objectives (from AD-416) Developing sensitive, reliable, detection methods for viruses infecting specific ornamental crops. Identification and characterization of these viruses, and determination of the environmental factors that affect virus titer and detectability. Determine how the presence of multiple viruses affects detection. 1b.Approach (from AD-416) Isolate and perform basic characterization of the causal viruses. Develop reagents, sampling procedures and protocols for the sensitive and reliable detection of the viruses. Examine the influence of environmental conditions (lighting, daylength, temperature, effects of tissue culture) on virus titer and distribution within the ornamental host plant. 3.Progress Report Virus detection following tissue culture for virus elimination. Tissue culture is often used for attempted virus elimination, as well as for rapid propagation of many ornamental plants; however in some instances tissue culture suppresses viral infections below the sensitivity of detection, and viral titer and symptoms re-emerge after material is re-established in the greenhouse. Verbena plants grown from seed were initially used. These were inoculated with Nemesia ring necrosis virus (NeRNV), then meristem shoot tips (0.3mm) were removed from systemically infected plants and grown in vitro on previously optimized plant growth media. While approximately 90% of the resulting plantlets tested negative for NeRNV at the first screening at 30 days post isolation, by the end of 6 weeks of weekly testing, 67% of the plants tested positive indicating that the tissue culture was not sufficient in most cases for true virus exclusion even though virus titer was apparently suppressed, initially preventing accurate detection. It was discovered that some of the commercially obtained Verbena seed harbored infections of NeRNV or another similar virus that cross-reacts in ELISA. Subsequent trials utilized Verbena plants obtained from rooted cuttings of a commercial hybrid variety that were extensively tested prior to use to ensure that existing infections were not present. Currently, plantlets are being analyzed over time using ELISA, RT-PCR and host inoculation. Similar experiments using Verbena plants infected with Angelonia flower break virus (AnFBV) and mixed infections of NeRNV and AnFBV are also underway. Detection of multiple viruses in mixed infections. Ornamental plants are frequently infected with more than one virus, and in such cases detection of one virus may be less successful than in a single infection. Nemesia plants were inoculated with single or mixed infections of Ribgrass mosaic virus (RMV), NeRNV, or AnFBV. Plants were tested 3 weeks after inoculation by ELISA and RT-PCR. All plants inoculated with RMV (from a Nemesia source plant) became infected while those inoculated with NeRNV and AnFBV (both from Verbena sources) became infected at rates of only 33% and 0% respectively (12 plants each). After additional attempts to establish similar infections, it was apparent that inoculum prepared from Verbena plants contained an inhibitor which prevented successful infections of NeRNV and AnFBV from being established in Nemesia. Extracts of NeRNV from Verbena and AnFBV from Angelonia plants were inoculated onto Nicotiana benthamiana plants (six each). After 3 weeks, 5 of 6 plants were infected with high levels of NeRNV and 3 of 6 plants were infected with low levels of AnFBV. Each infected plant was transferred to additional N. benthamiana plants, and 100% infection was achieved. These infections from N. benthamiana were used to infect a new mixed infection trial in two varieties of Nemesia. Plants are currently being analyzed by ELISA, RT-PCR and dsRNA analysis to assess the interaction of the viruses. Monitoring activities for this project included e-mails, conference calls and discussions at the annual society meeting.