Location: National Germplasm Resources Laboratory2012 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:
A molecular detection assay is being developed for three viruses collectively called Asian Prunus Viruses (designated APV 1-3), Plum Bark Necrosis Stem Pitting Associated Virus (PBNSPaV), and Peach Latent Mosaic Viroid (PLMVd). The assay (real-time RT-PCR) is highly sensitive and amenable to an automated detection technique that avoids the use of agarose gels to visualize the reaction products. Furthermore, the multiplex component of the assay means that all five pathogens can be detected in a single reaction tube, thereby saving resources and reducing the opportunities for contamination when separate individual tests are performed. Preliminary results suggest the method is from 10-100 times more sensitive than the more conventional RT-PCR assay in detecting positive reactions for the corresponding pathogen. This research will be applicable to quarantine and certification programs that test numerous samples for these pathogens. Many viruses in family Betaflexiviride infect tree fruits and are targets of quarantine programs. To improve detection of flexiviruses, a nucleic acid based (RT-PCR) detection assay using degenerate primers is being developed to simplify the current three-step assay. The assay is being validated using positive control and field samples. A group detection assay has the advantage of detecting most flexiviruses in a single reaction, thereby reducing operational costs and the opportunities for cross-contamination that are present in a multiple-step assay.