Location: Subtropical Plant Pathology Research2011 Annual Report
1a. Objectives (from AD-416)
1) Finish multi-year research on detection of viruses in single and mixed infections after removal and tissue culture of virus infected verbena shoot tip meristems; 2) Survey ornamental crops in California for newly reported plant viruses, e.g. Phlox virus M and Alternanthera mosaic virus; and 3) Determine the efficiency of slow sand filtration in eliminating infectious plant viruses from recycled irrigation water.
1b. Approach (from AD-416)
1) Shoot tip meristems from verbena plants naturally and experimentally infected with viruses will be removed and grown in tissue culture. Resulting plants will be regularly tested over time for the presence of the viruses to assess the conditions and timing that are required for sensitive and accurate detection; 2) Known hosts of newly reported viruses not previously detected in California will be collected from retail, wholesale and landscape locations in multiple counties and tested by ELISA and/or RT-PCR. If these viruses are found to be present, data will be provided to the California Department of Food and Agriculture for assessment. This work should prevent the establishment of quarantines on crops that harbor new, but non-agriculturally threatening viruses; and 3) Irrigation water from ornamental plants will be collected and artificially contaminated with mechanically transmissible plant viruses. The water will then be applied to a slow sand filtration system. Aliquots of water will be taken from multiple levels within the system, inoculated to indicator hosts and used for virus detection by RT-PCR.
3. Progress Report
This project is related to inhouse objective 3: Develop or improve comprehensive integrated disease management strategies. Experiments were conducted to determine the timing and sensitivity of virus detection in verbena plants infected with Angelonia flower break virus (AnFBV) alone, Impatiens necrotic spot virus (INSV) alone, and mixed infections of Nemesia ring necrosis virus (NeRNV) with AnFBV or INSV after removal of shoot tip meristems and tissue culture. After inoculation of each virus to verbena and subsequent testing of individual shoots over time, some shoots would be negative for both viruses, some would be NeRNV positive, and only a few would contain both viruses. Some shoots that initially tested positive for both viruses, would later test negative for AnFBV. These results indicate that AnFBV is not able to compete effectively in mixed infections. Thirty meristems were removed for each of the four different sets of plants and tested every two weeks once tissue was available, usually 3-5 weeks after meristem removal. Plants are still being analyzed for long term results, but initial results for single infections of AnFBV and INSV were very similar to those with NeRNV; testing needs to continue for a minimum of 8 weeks in order to detect all the positive plants. For plants with mixed infections of AnFBV and NeRNV, positive results for the presence of NeRNV were still being obtained at week 10, while all plants were negative for AnFBV at every harvest date in these mixed infections in contrast to single infections of AnFBV where several positives were found throughout the trial. This seems to correspond to the above results that indicate that AnFBV is suppressed in mixed infections. For the NeRNV/INSV mixed infection plants, it was possible to detect both viruses or either virus alone in individual regenerated plantlets. For the majority of plants, enzyme linked immunosorbent assay (ELISA)was sufficient for detection, polymerase chain reaction (PCR) in a few cases detected virus one week before ELISA. The presence of more than one virus in shoot tip meristems seems to further delay their detection when plants are regenerated. This result means that the ornamental plant industry needs to be especially vigilant in the testing and selection of crops that are susceptible to multiple viruses. We are still in a preparatory mode for the study of virus exclusion using slow sand filtration. Most of the mechanical devices are on site and awaiting final assembly at the University of California South Coast Research and Extension Center in Irvine, CA. Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) are purified and INSV infected N. benthamiana plants are available for application to the filters and subsequent testing.