Location: Floral and Nursery Plants Research2011 Annual Report
1a. Objectives (from AD-416)
To better understand the biological interactions between the plant pathogenic bacterium Ralstonia solanacearum Race 3 and its ornamental host plants, particularly geraniums. More specifically, we propose to study the environmental and genetic variables affecting disease onset, latent infection, disease transmission, and pathogen survival. The long-range goal of this project is to improve our knowledge base for detection, identification, epidemiological prediction, and strategies for eradication of this exotic plant pest.
1b. Approach (from AD-416)
To develop improved diagnostic methods, we will screen existing genomes of Race 3 biovar 2 (R3bv2) strain UW551 and bv3 strain GMI1000 to identify novel R3bv2 diagnostic targets. Explore immunological and oligonucleotide-based methods diagnose R3bv2-infected plants using these targets; apply new methodologies such as nanodetection and magnetic capture/multiplex PCR combinations to move these diagnostic tools into practical use. Our lab will thoroughly test and validate diagnostic specificity and sensitivity, working with both geraniums and other commercial host plants. Note: This work will be accomplished in collaboration with colleagues at other universities in synergy with an ongoing diagnostic tools development project. To understand R3bv2 cold tolerance, we will use a transcriptomic approach to compare total pathogen gene expression during infection of plants at 20°C (cool, permissive for R3bv2) and 28°C (warm, permissive for both R3bv2 and bv3 strains). We will use two complementary microarray chips custom-designed to represent the complete genomes of R3bv2 strain UW551 and bv3 strain GMI1000. Using a 4-way experimental design, we will compare gene expression of tropical strain GMI1000 and cool-temperate strain UW551 during plant infection under cool and warm conditions. This comparative analysis will identify candidate cold tolerance genes that can be tested for function using site-directed mutagenesis followed by virulence and survival assays at the two temperatures. Microarray data will also reveal larger patterns of pathway expression or suites of genes that are likely to play roles in this complex trait; the contribution of such patterns to cool-temperate bacterial wilt disease will be tested using rational mutagenesis designed to abrogate the pathway or trait in question. To determine the biological basis of R3bv2 latent infection, we will draw on the custom-designed UW551 microarray chips developed above, we will compare R3bv2 pathogen gene expression during latent and active (symptomatic) infection of host plants. Identifying conditions that predictably produce latently infected plants has been a challenge, but cooler temperatures and lower inoculum are promising. A comparative analysis of total gene expression during the two kinds of infection will test the hypothesis that the pathogen exists in a different physiological and defensive state during latent infection. The practical purpose of these experiments is to identify chemical or environmental triggers that would either prevent or reverse latent infections, allowing offshore growers to block or expose latently infected plants before they could be accidentally introduced to the U.S.
3. Progress Report
The R3bv2 subgroup of the plant pathogen Ralstonia solanacearum is a quarantine pest and a Select Agent pathogen. Accidental introductions of R3bv2 to the U.S. in geranium cuttings have disrupted ornamental production and inflicted large losses. Our goals are to improve methods for detection and exclusion of this bacterium and to better understand the biology of its spread, infection, and persistence. During the past year, we measured survival of native U.S. and R3bv2 strains of this pathogen in water and in infected potato, tomato, and geranium tissue. Both strains survived >6 months at constant low temperature (-20°C). However, bacteria rapidly became undetectable after moderate temperature fluctuations (48h at 5°C followed by 48h at -10°C). This suggests that R3bv2 is no more likely to become established in the northern U.S. than the native southeastern R. solanacearum strains, which have failed to persist in the north despite repeated introductions. We presented these data in a comment supporting removal of R3bv2 from the Select Agent list. In collaboration with colleagues in Georgia and Hawaii, we used R. solanacearum genomes to develop R3bv2-specific diagnostic tools. The resulting primers have been incorporated into two DNA-based detection methods, Loop-activated isothermal AMPlification (LAMP) and Magnetic Capture Hybridization (MCH). We are now directly comparing six diverse R3bv2 detection methods for sensitivity, accuracy, speed, ease of use, and cost. To explore the biological traits of R3bv2, we did experiments to identify traits uniquely expressed by this organism when in contact with plants at cool temperatures. We measured the virulence of mutant strains lacking these traits and found that a fucose-mannose-binding lectin, a stress-related DNA-protective protein, and two conserved genes of unknown function are differentially reduced in virulence at cool temperatures. Communications to monitor progress were carried out by e-mail, written reports, and by a meeting during the Annual meeting of the American Phytopathological Society.