2008 Annual Report 1a.Objectives (from AD-416) Investigate pathogens and diseases of quarantine significance that occur in clonal plant germplasm that must enter the U.S. through federal quarantine programs. Characterize poorly described pathogens and diseases of quarantined crops that limit entry and distribution of plant germplasm. Enhance testing of quarantine crops for known pathogens by developing detection methods that represent improvements in sensitivity, speed, and reliability when compared to currently used procedures. Develop tools to effectively eliminate pathogens of quarantine significance to facilitate the availability of pathogen-free germplasm to stakeholders in a safe and timely fashion. Transfer research data, methods, and protocols to USDA-APHIS to support testing programs and science-based regulatory decisions. 1b.Approach (from AD-416) Conduct research to identify new methodologies and protocols for diagnostic testing of quarantined plant germplasm, with an emphasis on molecular techniques, to shorten the duration 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 testing methods. Develop protocols for the in vitro cultivation of prohibited genera germplasm and the therapeutic elimination of quarantine pathogens, thereby salvaging valuable and sometimes unique and endangered germplasm. Transfer research data, protocols, and products to USDA-APHIS for incorporation into testing and regulatory programs. 3.Progress Report An existing hoophouse was upgraded to a screenhouse that meets the requirements for performing research on regulated quarantine pathogens. The screenhouse passed all inspections and was occupied in March 2008. This is an essential facility component of the project’s research with infected deciduous tree and small fruits that require winter dormancy. The complete sequence of an isolate of Lolium latent virus (LoLV) from England has been obtained. Sequence analyses indicate it is a distinct virus in the family Flexiviridae, a result consistent with those obtained from our serological study. The data supports the hypothesis that LoLV is a novel virus in a new genus. The information will have an impact on the indexing protocols for grasses at the USDA quarantine program, and is also important to viral taxonomy. Molecular characterization of sugarcane streak mosaic virus, a virus occurring in Southwest Asia, has been initiated in collaboration with South China Agricultural University. Approximately half of the genomic sequence has been obtained and the results suggest this is a novel virus in the family Potyviridae. Work will continue to obtain the complete viral genome sequence. Two cherry necrotic rusty mottle virus isolates obtained from Washington State were identified as, at least, distinct strains based on the partial sequences and host plant reactions after rub inoculations. Research will continue to obtain their complete sequences. This research will have an impact not only on the USDA quarantine programs, but also on the taxonomy of this unassigned virus. A survey, in collaboration with ARS curators, has been initiated to screen cherry germplasm repository samples for the presence of eight viruses, including the recently characterized cherry virus A. The results will have an impact on the indexing protocols for this crop at the USDA quarantine program and the management of cherry germplasm at the USDA repositories. An optimized medium has been developed for growth of several Saccharum species. Cultures infected with sugarcane pathogens were established in vitro and have been subjected to therapeutic conditions to attempt pathogen elimination. Similarly, samples of Prunus species have been established both in vivo and as cultures in vitro. Work on growth media optimization is underway and the development of therapeutic strategies for elimination of Prunus quarantine pathogens is being investigated. Several species of Ribes and Rubus infected with important viral pathogens have been received and are being maintained in the greenhouse. Many isolates have been established as in vitro cultures and research into the therapeutic elimination of those pathogens has begun. The emphasis will be on developing optimized media for in vitro cultivation of a wide range of small fruit species, as well as developing procedures for therapeutic elimination of quarantine pathogens. The progress from this research supports Problem Statement 1B (Detection, Identification, Characterization, and Classification of Pathogens) of Component I (Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens)NP 301. 4.Accomplishments 1. A reliable and inexpensive method of nucleic acid extraction for the detection of plant pathogens by PCR or RT-PCR An extraction method was adapted that works well for the preparation of either DNA or RNA for the PCR-based detection of plant pathogens. It uses a mechanical device to grind samples. Thus, large numbers of samples can be processed while reducing potential contamination. The supplies and chemicals needed are inexpensive and readily available. The procedure performed as well as and, in some cases, better than more expensive commercially available kits in preparation of DNA and RNA samples suitable for PCR-based pathogen detection. This protocol has proven reliable in detecting a wide range of plant pathogens including viruses, viroids, phytoplasmas, and bacteria from diverse woody and herbaceous crops. This information will be useful to quarantine and certification programs that need to test valuable plant material for pathogens. These research accomplishments support Problem Statement 1B (Detection, Identification, Characterization, and Classification of Pathogens) of Component I (Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens) of National Program 303. This research also provides collateral support to Problem Statement 1A (Efficiently and Effectively Manage Plant and Microbial Genetic Resources) of the Plant and Microbial Genetic Resource Management component of National Program 301. 2. Detection of Fiji disease virus by RT-PCR Fiji disease virus (FDV), a serious pathogen of Saccharum species, has not been reported in the U.S. The method currently used to detect FDV in the USDA quarantine program is visual examination only of germplasm for symptoms. Research was conducted to develop a sensitive, rapid, and reliable molecular detection method to supplement the existing quarantine protocol. A newly selected pair of primers was tested after comparisons with previously reported primers, and tissue throughout the plant was assayed for presence of the virus. The nucleic acid extraction and RT-PCR protocols that were developed in our lab reliably detected FDV from tissues throughout the plant. This protocol should be used to supplement the existing quarantine procedure to ensure that imported Saccharum germplasm does not enter the U.S. with FDV, an exotic pathogen that could threaten the domestic sugarcane industry. These research accomplishments support Problem Statement 1B (Detection, Identification, Characterization, and Classification of Pathogens) of Component I (Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens) of National Program 303. This research also provides collateral support to Problem Statement 1A (Efficiently and Effectively Manage Plant and Microbial Genetic Resources) of the Plant and Microbial Genetic Resource Management component of National Program 301. 3. Detection of yellow dwarf viruses by RT-PCR Infection of yellow dwarf viruses (YDVs) such as Barley yellow dwarf virus-MAV and Cereal yellow dwarf virus is common in grasses. A published multiplex RT-PCR assay was evaluated for detection of three YDVs. The result indicated that method is rapid and reliable for detection of theses viruses. The procedures and results were documented and transferred to APHIS. The information will have an impact on the indexing protocols for cereal grasses at the USDA quarantine program. These research accomplishments support Problem Statement 1B (Detection, Identification, Characterization, and Classification of Pathogens) of Component I (Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens) of National Program 303. This research also provides collateral support to Problem Statement 1A (Efficiently and Effectively Manage Plant and Microbial Genetic Resources) of the Plant and Microbial Genetic Resource Management component of National Program 301. 4. First report of cherry virus A in sweet cherry trees in China Cherry virus A (CVA) is a recently described virus infecting sweet cherry and has been found in Europe, North America and Japan. The virus was detected in China for the first time in collaboration with researchers in China using an RT-PCR assay developed in the NGRL. Sequence analysis shows that the isolates found in China are very similar to that of the type species CVA-German isolate. The study demonstrated that the virus is probably distributed worldwide due to inadequate detection methods for stone fruits in the quarantine and certification programs. The information will have an impact on the indexing protocols for stone fruits at the USDA quarantine program. These research accomplishments support Problem Statement 1B (Detection, Identification, Characterization, and Classification of Pathogens) of Component I (Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens) of National Program 303. This research also provides collateral support to Problem Statement 1A (Efficiently and Effectively Manage Plant and Microbial Genetic Resources) of the Plant and Microbial Genetic Resource Management component of National Program 301. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of New Commercial Licenses Executed 3 Number of Other Technology Transfer 3 Review Publications Li, R. and Hartung, J.S. 2007. Reverse Transcription-Polymerase Chain Reaction-Based Detection of Cherry green ring mottle virus and Cherry necrotic rusty mottle virus in Prunus spp. UNIT 16C.1 In: Current Protocols in Microbiology. New Jersey: John Wiley and Sons. p.1-9. Li, R., Maroon Lango, C.J., Mock, R.G., Hammond, J. 2008. Lolium latent virus. In: Rao, G.P., Kharana, S.M.P., Lenardon, S.L., eds. Molecular Diagnosis of Plant Viruses. Houston, Studium Press LLC. pp. 215-220. Li, R., Mock, R.G. 2008. Characterization of a flowering cherry strain of cherry necrotic rusty motttle virus. Archives of Virology 153(5):973-978. usty. Archives of Virology.