2004 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Floral crop sales comprise one of the fastest growing segments of U.S. agriculture, forming a $5.1 billion industry in 2003. International trade in ornamentals plays an important role in this industry, but may also serve as a conduit for introduction of diseases that may threaten agronomic crops as well as horticultural crops. Bacterial wilt is one such example. Ralstonia solanacearum (Smith) causes bacterial wilt, a soilborne vascular disease that is distributed worldwide in tropical and warm temperate regions. The disease attacks over 450 plant species including ornamentals such as geranium, dahlia and impatiens, and is the major constraint on production of many economically important crops. Control of bacterial wilt depends mainly on the use of resistant cultivars, when available, and proper rotation or fallow. Such control, however, has not been very successful due to the pathogen¿s wide host range, broad distribution, great variability and ability to survive in soil and water. R. solanacearum is a complex species with considerable diversity. It is generally classified into 5 races based on host range, and five biovars according to the ability to utilize or oxidize three disaccharides and three hexose alcohols. The five races and biovars do not correspond except that in general, race 3 is equivalent to biovar 2. Race 3, biovar2 (R3bv2) causes brown rot, a very destructive disease of potatoes that has resulted in significant losses in potato production in Europe since 1976 following several outbreaks of the disease and subsequent quarantine restrictions. Compared to race 1, race 3 is more adapted to cooler temperatures found in temperate climates and at higher elevations and latitudes in the tropics. R3bv2 is reported as having a narrow host range limited mainly to potato, tomato, eggplant and solanaceous weeds like bittersweet. Recently, however, it was found in stinging nettle and a number of latently infected non-solanaceous plants. R3bv2 had never been reported in the US until 1999 when it was first introduced to the US in geranium cuttings imported from Guatemala. Since R3bv2 is considered a potential threat to the potato industry if the bacterium is established and spread into major potato producing areas in the US, R. solanacearum R3bv2 was listed as one of the 10 plant pathogens in USDA's Agricultural Bioterrorism Protection Act of 2002, and is subject to the strictest eradication requirements and the most stringent security regulations. As a result, the recent introduction of R. solanacearum R3bv2 in the spring of 2003 on imported geranium and its subsequent outbreak in 127 nurseries in 27 states in the US had forced APHIS to destroy over 2 million geranium and non-geranium plants at an estimated $5 million losses to the geranium industry. Since very little is known about R3bv2 of R. solanacearum in geranium, our research is directed at studies on host range, epidemiology, genetic comparison, and control of the geranium race of R. solanacearum. These studies are important steps towards a better understanding of the pathogen and will result in better quarantine measures to control and prevent the disease it causes from becoming established in the United States. This will provide a direct benefit to the $300 million per year geranium industry, and help safeguard the $1.2 billion per year US potato industry. This research is carried out under National Program 303, Plant Diseases, and contributes to Components I (Identification and Classification of Pathogens), III (Cultural Control), and IV (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors). 2.List the milestones (indicators of progress) from your Project Plan. Since this project is a Specific Cooperative Agreement, there is no formal project plan and no specific milestones. Milestones for the associated in-house research are described in the report for CRIS 1230-22000-012-00D. The followings are specific goals of this project: -Determine varietal differences in the responses of geranium to infection by R. solanacearum, and correlation with host plant genotype based on Amplified Fragment Length Polymorphism (AFLP). -Compare R. solanacearum isolates from different geographic origins and from different outbreaks using Repetitive Extragenic Palindromic-Polymerase Chain Reaction (Rep-PCR) and Fatty Acid Methyl Ester (FAME) analysis. -Examine freeze survival of R. solanacearum under continuous cold or varying freeze-thaw cycles. -Examine the host range of R. solanacearum R3bv2 among ornamental crops. -Determine inoculation threshold and the effect of temperature on virulence of R3bv2. -Examine chemical control of R. solanacearum using copper compounds, phosphoric acid, chelated silver, and an inducer of systemic acquired resistance. 3.Milestones: The following goal has been substantially met: -Examine chemical control of R. solanacearum using copper compounds, phosphoric acid, chelated silver, and an inducer of systemic acquired resistance. Preliminary research has been conducted to achieve the following goals: -Determine varietal differences in the responses of geranium to infection by R. solanacearum, and correlation with host plant genotype based on AFLP. -Compare R. solanacearum isolates from different geographic origins and from different outbreaks using Rep-PCR and FAME analysis. -Examine freeze survival of R. solanacearum under continuous cold or varying freeze-thaw cycles. B. List the milestones (from the list in Question #2) that you expect to address over the next 3 years (FY 2005, 2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The following studies will be continued: -Determine varietal differences in the responses of geranium to infection by R. solanacearum, and correlation with host plant genotype based on AFLP. -Compare R. solanacearum isolates from different geographic origins and from different outbreaks using Rep-PCR and FAME analysis. -Examine freeze survival of R. solanacearum under continuous cold or varying freeze-thaw cycles. -Examine chemical control of R. solanacearum using copper compounds, phosphoric acid, chelated silver, and an inducer of systemic acquired resistance. The following studies will be conducted: -Examine the host range of R. solanacearum R3bv2 among ornamental crops. -Determine inoculation threshold and the effect of temperature on virulence of R3bv2. 4.What were the most significant accomplishments this past year? D. This report documents research carried out by the University of Florida under Specific Cooperative Agreement 58-1230-3-170, CRIS Project 1230-22000-012-09S. Additional details can be found under the annual report of the parent project, 1230-22000-012-00D. Using Ralstonia solanacearum Race 1 as a model system, we were able to screen bactericides for their ability to protect geranium plants from infection. We found that plants can be protected from bacterial wilt infection by applying compounds containing phosphorous acid (H3PO3). It is thought that the phosphorous acid is protecting the plants from infection by acting as a Systemic Acquired Resistance Compound. Similar compounds such as H3PO4 or P2O5 do not provide disease protection. In order to be effective, phosphorous acid must be applied as a drench before disease onset. Even though strict sanitation procedures have been implemented in geranium production facilities, a low cost effective protectant would greatly benefit the industry. Phosphorous acid would be low cost, easy to apply, and would provide phosphorous needed for plant growth. This research finding will provide a direct benefit to the $300 million/year geranium industry and will help safeguard the $1.2 billion/year US potato industry. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was initiated in September 2003, so the Progress Report above (4D) reflects the accomplishments to date. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Participated in the R. solanacearum race 3 working group meetings on March 16-17, 2004 at USDA-APHIS Headquarters in Riverdale, MD. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. Presented invited talks entitled 'R. solanacearum race 3 in Geranium: An Overview and Research Plans', and 'Host range studies on Race 3 and control measures' at the R. solanacearum race 3 working group meeting on March 16 in Riverdale, Maryland. Attended the American Phytopathological Society annual meeting and presented a paper entitled: "Screening of chemicals for the control of bacterial wilt of geranium".