Location: National Germplasm Resources Laboratory2013 Annual Report
1a. Objectives (from AD-416):
The objectives of this project are to 1) Characterize unknown and poorly described pathogens (primarily viruses and viroids) and diseases highly significant to the USDA plant germplasm quarantine program; 2) Develop and transfer sensitive, reliable, and cost-effective methods to clientele for the rapid detection of virus and virus-like pathogens of quarantine significance; and 3) Develop and transfer methods to clientele for the therapeutic elimination of virus and virus-like pathogens from infected plant genetic resources. The objectives focus on characterizing quarantine pathogens and determining the causal agents responsible for diseases that could threaten U.S. agriculture and ecosystems, and developing tools to effectively detect and eliminate them. The research areas are developed in consultation with USDA-APHIS and the data, protocols, and tools developed by this project are shared with them. Because problems that require immediate investigation can arise rapidly in the plant quarantine system, this project will remain flexible to allocate resources to new or emerging quarantine problems as warranted.
1b. Approach (from AD-416):
Conduct laboratory and greenhouse research to identify new methodologies and protocols for diagnostic testing of quarantined plant germplasm, with emphasis on highly sensitive molecular techniques that can shorten the duration time that material is held in quarantine and increase the reliability of indexing programs. Determine the etiology of poorly described quarantine diseases using a wide range of greenhouse and laboratory techniques. Conduct molecular characterization studies of quarantine pathogens and investigate their genetic diversity in order to refine and optimize testing methods. Develop protocols for the in vitro cultivation of prohibited genera germplasm and the therapeutic elimination of quarantine pathogens, thereby ensuring that valuable and sometimes unique and endangered germplasm is available for safeguarding and utilization. Transfer research data, protocols, and products to USDA-APHIS for incorporation into testing programs and to support science-based regulatory decisions.
3. Progress Report:
Gooseberry vein banding associated (GVBaV) and Rubus yellow net viruses infect small fruits such as gooseberries, blackberries, and raspberries. Some viruses in the same family as these two occur as integrated sequences in their host’s genome and do not typically cause diseases, whereas virus particles are detrimental. Research is continuing to investigate whether these two viruses exist as part of their host genome. Their coat protein genes were cloned and expressed in a bacterium. Antiserum against GVBaV has been obtained, and will be tested to see if it can detect virus proteins in Rubus spp. Epiphyllum spp. is an important genus of ornamental plants. A diseased Epiphyllum plant was determined to be virus infected using electron microscopy by a scientist at the California Department of Food and Agriculture (CDFA). To identify the virus, a project has been initiated at the request of CDFA. A partial sequence of 2.2 kb was obtained and data to date suggests this may be a newly described virus. Research is ongoing to determine complete genomic sequence and host range of the virus. Many viruses infect and cause diseases in yam (Dioscorea spp.), an important tuber food crop. Diseased plants of Chinese yam were identified as infected with a novel strain of Japanese yam mosaic virus by scientists from Yunnan Agricultural University. A project has been initiated in collaboration with this group to determine the complete genomic sequence of this virus strain. Sequence information obtained from this study will be used to improve the detection method for this virus in the USDA quarantine system. Many viruses infect and cause diseases in sweet potato. A project has been initiated to develop a multiplex molecular assay to detect simultaneously three viruses infecting sweet potato. The assay appears to be very sensitive and reliable, and will be further validated using field samples. The multiplex assay is also simple and cost-effective, and will improve the efficiency of the sweet potato plant quarantine program. Various Prunus species are imported to the U.S. for improving crops such as peaches, plums, nectarines, almonds, etc. Tissue culture can be used to eliminate viruses from such valuable germplasm. A project was initiated to develop a universal culture medium in which to grow several Prunus species. Different carbohydrate sources, one of most important nutrients for good cultivation, were tested at the culture initiation stage. Research is ongoing to optimize nutrient formulations at other stages, such as the multiplication stage and for long term maintenance in vitro (at least 1-2 years). A universal culture medium will allow continuous in vitro culture of multiple Prunus spp., which is very beneficial for plant quarantine and certification programs.
1. First report of taro virus in Hawaii. Many viruses infect and cause diseases in taro (Colocasia esculenta), an important tuber crop and ornamental plant. One virus, Taro bacilliform virus (TaBV), causes a lethal disease called Alomae when it occurs as a mixed infection with a second virus. TaBV, which had only been previously reported from some Pacific Island countries and South America, was detected in Hawaii in a recent survey. To confirm presence of the virus, a project with scientists from University of Hawaii and USDA-APHIS was initiated at the request of APHIS. Two viruses, Dasheen mosaic virus and TaBV were detected from the diseased plants. The complete genomic sequence of TaBV was determined, which proved this is a new strain of the virus. This is also the first report of TaBV in the United States. This information will help regulatory officials decide on the best approach to address this problem in Hawaii.
2. Improved detection of sweet potato viruses. Sweet potato latent virus (SPLV) is one of six sweet potato viruses associated with Sweet Potato Viral Disease (SPVD), the most devastating viral disease in sweet potato. The virus is an important target in our USDA Plant Germplasm Quarantine Program because it has not been reported in the U.S. The complete genomic sequence of SPLV was determined in collaboration with scientists from APHIS at Beltsville, MD and the International Potato Center at Lima, Peru. Results show that the SPLV is different genetically from a related virus – the sweet potato feathery mottle virus lineage. The information helps refine the detection methods of SPVD and strengthens our ability to keep SPLV out of the U.S. Sweet potato virus G (SPVG) is another virus associated with SPVD. The genetic variation among this virus is high, and this increases the risk of false-negatives in the plant quarantine testing. The complete sequence of SPVG has been determined and used to develop an improved broad spectrum detection technique for this virus.
Cheong, E.J., Mock, R.G., Li, R. 2012. Elimination of five sugarcane viruses from sugarcane using in vitro culture of axillary bud and apical meristem. Plant Cell Tissue And Organ Culture. 109:439-445.