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. Southern wilt of geranium (Pelargonium hortorum) is caused by Ralstonia solanacearum (Smith), a soilborne bacterium that typically invades plants through the roots and colonizes host xylem vessels. Symptoms include leaf yellowing, wilting and necrosis, as well as vascular browning; though infected geranium plants may die, they can also sustain moderate levels of disease or remain latently infected for weeks or months. R. solanacearum is best known as the cause of bacterial wilt, a disease that affects diverse plants ranging from solanaceous crops like tomatoes and tobacco to peanuts, bananas, ginger and eucalyptus. The pathogen is widely distributed in tropical and warm-temperate regions, where it is responsible for large crop losses. R. solanacearum is a species complex, a large heterogeneous group of related strains. It has been subdivided into races based on host range, and biovars based on carbohydrate utilization. A phylogenetically coherent cluster known as race 3, biovar 2 (R3bv2) contains cool-tolerant strains that originated in the Andes. R3bv2 strains cause potato brown rot, which ranks among the most destructive diseases of potato in Africa, Asia, and Central and South America. The pathogen commonly forms latent (asymptomatic) infections in the cool tropical highlands, but when infected seed tubers are planted in warmer lowland fields, the resulting plants quickly wilt and die. R3bv2 was introduced into northern Europe in the late 1980's and caused considerable economic and political havoc when it appeared on potatoes in 1995. Despite ongoing eradication efforts, the bacterium is still present in many fields and waterways of northern Europe, although it has caused only minor direct crop losses. Recent ecological studies found that a R3bv2 strain could survive at least 12 months in temperate field soil and for at least 110 days in surface water at 12ºC. Although the host range of R3bv2 strains was once thought to be limited to potatoes, eggplant, and tomato, it is now known that these strains can infect many ornamental and weed species as well. Solanum dulcamara, a common semi-aquatic weed, is an important survival refuge and source of inoculum in Europe. R3bv2 is not established in North America. Geraniums sold in Europe and North America are commonly grown from cuttings produced in the highland tropics of Africa and Central America, where R3bv2 is endemic. Southern wilt was reported on U.S. geraniums grown from imported cuttings as early as 1981. The volume of geranium cuttings imported to the United States has grown dramatically, increasing from an estimated 10 million cuttings per year in 1985 to an estimated 100 million in 2003. Perhaps not coincidentally, R. solanacearum has been repeatedly introduced to Europe and North America in geraniums in recent years. Most geranium isolates of R. solanacearum that have been classified belong to R3bv2. Southern wilt is a minor disease of geranium, but any introduction of R3bv2 to North America raises concerns because the presence of R3bv2 on potato could cause quarantine and trade-related losses to the potato industry. R3bv2 was among 10 plant pathogens listed in the Agricultural Bioterrorism Protection Act of 2002, so any finding of this organism in the U.S. is now subject to the strictest eradication requirements and the most stringent security regulations. In February 2003, R. solanacearum R3bv2 was detected in the U.S. in geraniums imported from Kenya; later that season the pathogen was also found in geraniums from Guatemala and Costa Rica, and additional infected geraniums originating from Guatemala were found in December, 2003. The resulting response cost growers and regulators an estimated $7 million and involved the destruction of over 2 million plants. To address this outbreak, APHIS prepared a Ralstonia Action Plan specifying measures that growers and regulators must take when Race 3-infected plants are detected. However, these measures were based on best guesses and reasonable extrapolations from other hosts such as potato, because very little research has been done on the geranium-Ralstonia interaction. The regulatory problem is complicated by the ability of the bacterium to form latent infections, which are difficult to detect but can generate inoculum that leads to disease spread. Understanding R3bv2 pathogenesis and latency in geranium plants is key to designing rapid, accurate diagnostic methods and rational control responses. We initiated this research project to address some of the many unanswered questions about this plant-pathogen interaction. Our goal was to discover baseline information about the interactions between R. solanacearum and this ornamental host, with the larger aim of developing improved diagnostic methods and data-based regulatory responses to accidental introductions. The beneficiaries of this research are the geranium production industry, the hundreds of US greenhouse ornamental growers that grow and sell geraniums, and USDA-APHIS-PPQ, the regulatory agency responsible for developing policies that exclude and if necessary, eradicate this Select Agent pathogen. 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 following are specific goals of this project: -Determine conditions conductive to formation of latent infection on geranium. -Whether holding plants at a particular temperature can force symptom development. -Whether R3bv2 can be transmitted by contact with wounded vegetation. -Determine the amount of bacteria occurring in run-off water from symptomatic or latently-infected plants. -Determine R. solanacearum distribution in geranium. -Examine the host range of R3bv2 among ornamental crops. -Determine inoculation threshold and the effect of temperature on virulence of R3bv2. -Determine genetic diversity among and between geranium and potato isolates of R3bv2. -Determine direct effects of environmental factors such as temperature, plant growth stage, and nutritional status on latent infection of geranium by R3bv2. -Characterize the natural population structure of R. solanacearum in Guatemala at the genetic and phenotypic level, and developing tools for rapid specific diagnosis. -Use functional genomics of R. solanacearum R3bv2 to identify new diagnostic targets and explore the biology of latency in geranium plants. 3.Milestones: The following goals for FY2004 have been substantially met: -Determine conditions conductive to formation of latent infection on geranium. -Whether holding plants at a particular temperature can force symptom development. -Whether R3bv2 can be transmitted by contact with wounded vegetation. -Determine the amount of bacteria occurring in run-off water from symptomatic or latently-infected plants. -Determine R. solanacearum distribution in geranium. 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 conducted: -Examine the host range of R3bv2 among ornamental crops. -Determine inoculation threshold and the effect of temperature on virulence of R3bv2. -Determine genetic diversity among and between geranium and potato isolates of R3bv2. -Determine direct effects of environmental factors such as temperature, plant growth stage, and nutritional status on latent infection of geranium by R3bv2. -Characterize the natural population structure of R. solanacearum in Guatemala at the genetic and phenotypic level, and developing tools for rapid specific diagnosis. -Use functional genomics of R. solanacearum R3bv2 to identify new diagnostic targets and explore the biology of latency in geranium plants. 4.What were the most significant accomplishments this past year? D. This report documents research carried out by the University of Wisconsin-Madison under Specific Cooperative Agreement 58-1230-3-174, CRIS Project 1230-22000-012-08S. Additional details can be found under the annual report of the parent project, 1230-22000-012-00D. Using UW551, a virulent R3bv2 geranium isolate from Kenya, we characterized development of Southern wilt disease and R3bv2 latent infection on geranium plants. Following soil inoculation, between 12 and 26% of plants became latently infected, carrying average bacterial populations of 4.8x108 CFU/g crown tissue in the absence of visible symptoms. Such latently infected plants shed an average of 1.3x105 CFU/ml in soil run-off water, suggesting a non-destructive means of testing pools of asymptomatic plants. Similarly, symptomatic plants shed 2x106 CFU/ml run-off water. A few hundred R. solanacearum cells introduced directly into geranium stems resulted in death of almost all inoculated plants. However, no disease transmission was detected after contact between wounded leaves, meaning that all plants shipped with an infected plant need not be destroyed. Increasing temperatures to 28ºC for two weeks did not convert all latently infected plants to active disease, although disease development was temperature dependent. Holding plants at 4ºC for 48 hours, a routine practice during geranium cutting shipment, did not increase frequency of latent infections. R. solanacearum cells were distributed unevenly in the stems and leaves of both symptomatic and latently infected plants, meaning that random leaf sampling is an unreliable testing method. Our single most significant finding was the discovery that symptomless plants release easily detectable numbers of bacteria from their roots; we are working to develop this discovery into a rapid field screening method. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Southern wilt of geraniums caused by the soilborne bacterium R. solanacearum R3bv2 has inflicted significant economic losses when geranium cuttings latently infected with this quarantine pest were imported into the United States and Europe. Our research has begun to better understand the interactions between geranium plants and the Southern wilt pathogen, R. solanacearum R3bv2, and to develop improved data-based regulatory responses. The information and tools from this research will greatly help regulatory scientists and geranium producers to develop detection methods and offshore production standards that will reliably prevent further introductions of this quarantine pathogen on imported geranium cuttings. 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 'Studies of pathogenesis and latent infection of geraniums by R. solanacearum Race 3' at the R. solanacearum race 3 working group meeting on March 16 in Riverdale, Maryland.