2010 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.
The race 3, biovar 2 (R3bv2) subgroup of the bacterial plant pathogen Ralstonia solanacearum is a quarantine pest and a listed Federal Select Agent. Accidental introductions of R3bv2 to the U.S. in infected geranium cuttings 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. R3bv2 is considered a grave threat partly because it causes disease in cool temperate zones. However, R3bv2 will not become established if it cannot survive temperate winters. We found that in water at 4 degrees C, R3bv2 does not survive as long as native U.S. strains, but R3bv2 remains viable longer than U.S. strains in potato tubers at 4 C. We assessed the ability of R3bv2 and a native U.S. strain to survive typical temperate winter temperature cycles of two days at 5 C followed by two days at -10 C. We measured pathogen survival in infected tomato and geranium plants, in infected potato tubers, and in sterile water. Population sizes of both strains declined rapidly under these conditions in all three plant hosts and in sterile water, and no culturable R. solanacearum cells were detected after five to seven temperature cycles in plant tissue. The fluctuations played a critical role in loss of bacterial viability, since at a constant temperature of -20 C, both strains could survive in infected geranium tissue for at least six months. These results suggest that even when sheltered in infected plant tissue, R3bv2 is unlikely to survive the temperature fluctuations typical of a northern temperate winter. To further explore this trait, we compared total transcriptomes of R3bv2 and a tropical R. solanacearum strain under four conditions: in tomato plants at 20 C and 28 C, and in culture at 20 C and 28 C. Excitingly, this analysis identified a cluster of R3bv2 genes that contribute to differential virulence at 20 C. One of these encodes a lectin, and studies are underway to determine how this protein helps R3bv2 cause disease at cooler temperatures. Finally, field tests in two Race 3 hot-spots (Guatemala and Reunion Is., France) have given us informative feedback to optimize our immuno-magnetic separation and Polymerase chain reaction (PCR) diagnostic method to specifically and sensitively detect R3bv2 in plants and water.
Research activities under this agreement were monitored by e-mails and written reports.