Location: Subtropical Plant Pathology Research2012 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 research related to inhouse project objective: 1. Characterize ecology, biology, epidemiology, molecular genetics, and vector and host (crop and weed) interactions of domestic, exotic, newly emerging, and re-emerging pathogens, and 3. Develop or improve comprehensive integrated disease management strategies. This year two new projects were started involving elimination of plant viruses via slow sand filtration and a survey and characterization of viruses in ferns. Plant viruses are known to be present in water runoff from infected plants and can cause new infections in healthy plants. Slow sand filters (SSF) have been used with success to eliminate human viruses from drinking water, but little work has been published using SSF for eliminating plant viruses. In collaboration with the University of California we set up a pilot system in Irvine, CA using tobacco mosaic virus (TMV) to test the efficiency of SSF in eliminating this common plant virus in water. Purified TMV was applied to a column containing a 1 m bed of sand every day for the first 10 days, then M-F each week for 12 weeks. Water samples were collected prior to the first virus application, at 24 hrs post TMV, then once every week thereafter from the top of the water chamber before and after the daily virus application, from a valve in the middle of the column just above the sand bed, and at the bottom, below the sand filter. Aliquots of water from each of the four weekly samples were mechanically inoculated onto Nicotiana glutinosa, Chenopodium quinoa (both local lesions hosts of TMV), N. tabacum ‘Turkish’, and N. benthamiana (both systemic hosts), as well as being tested for the presence of TMV by enzyme-linked immunosorbent assay (ELISA). TMV was readily detected by all methods in the upper chamber after adding virus each week establishing the viability of the virus diluted in water. After 24hrs TMV was also detected in the upper chamber before the addition of new virus and the middle port, but not the bottom port. After 1 week TMV was detected in water from the bottom port indicating no viral suppression. However by week 5, a noticeable decrease in the amount of local lesions and ELISA values occurred in water from the bottom port although systemic infections continued. By week 10, systemic infections were delayed or eliminated from bottom port water. Therefore the SSF was able to eliminate or substantially reduce the level of TMV in the water within 3 months and the study is continuing. We extracted dsRNAs from several genera of ferns (Polypodium, Dicksonia, Cibotium, Lastreopsis, Rumohra) and found multiple virus-like patterns. Using polymerase chain reaction (PCR) primers to a newly reported virus found in Japanese holly fern, we confirmed the presence of that virus in 3 of the California ferns, but all dsRNAs found cannot be accounted for by this virus. It appears that there are multiple unknown viruses in ferns and we are cloning and sequencing some of them for comparison.