2012 Annual Report
1a.Objectives (from AD-416):
To determine the susceptibility of commercial geranium (Pelargonium) cultivars to bacterial wilt caused by Ralsonia solanacearum, and whether certain plants may act as asymptomatic hosts; to determine whether susceptibility is correlated with genetic relatedness of the host cultivar; to determine the susceptibility of other greenhouse-grown ornamental crops to Ralstonia solanacearum; to compare isolates of the bacterium from different outbreaks to correlate with geographic origin; to determine freeze survival of the bacterium in soil; to investigate chemical control measures; and to identify genes associated with the survival and pathogenicity at different temperatures, evaluate pathogen populations in relation to their ability to cause pathogenicity to agricultural crops at various temperatures and determine potential environmental fate and movement in the United States.
1b.Approach (from AD-416):
An empirical approach will be used to examine the genetic and physiological capability of exotic strains of bacterial wilt to access their ability to become established and spread within U.S. agriculture. Once introduced Ralstonia solanacearum is know historically to be spread via river systems. The project will further address the survival of distinct genetic populations in various water types and assess the ability to infect and survive under different temperature regimes.
Problem addressed: Ralstonia solanacearum is distributed in tropical and subtropical areas of the world. R. solanacearum race 3 biovar 2 (R3B2) strains are found in the coldest areas of their range and are thought to be pathogenic at low temperatures. Growth and twitching motility characterization of selected strains of Ralstonia were completed in order to identify potential phenotypes associated with pathogenicity at low temperature. These results were published recently. Cellular and secreted proteins differentially expressed at warm and cool temperatures were identified by proteomics experiments. Analysis of these results resulted in a list of potential virulence factors at low temperatures which are being analyzed currently.
Results: Among the set of proteins differentially expressed in our proteomics study, proteins with functions associated with oxidative-stress responses, known virulence factors, and two hypothetical proteins stood out due to their pattern of expression. qRT-PCR on RNA extracted from bacterial populations of strains R3B2 UW551 and R1B1 GMI1000 in contact with the tomato rhizosphere at 30°C and 18°C confirmed that one secreted and five cellular proteins selected from the list of differentially expressed proteins are indeed differentially expressed at the two temperatures. Virulence studies at the two temperatures listed above suggest that R3B2 and R1B1 deletion mutants of PilQ, a gene which codes for a porin necessary for twitching motility, have delayed and reduced virulence at 18°C. We are repeating the virulence experiments and we are producing mutant strains for two more candidate virulence factors at low temperatures. We are currently in the final stages of preparing a manuscript describing the results of our proteomics experiment.
Potential impact: Because of historical evidence of its ability to survive under cool climate conditions and kill solanaceous crops such as potato, R3B2 is designated as a “Select Agent” under the Agricultural Bioterrorism Protection Act of 2002. In this work bacterial wilt strains currently present in the United States that have the potential to survive and infect tomato and potato plants at low temperatures have been identified. It is important to determine the potential environmental fate and movement of these populations within the United States and to identify their virulence determinants at low temperature. If specific genes involved in establishment under temperate conditions are identified for R3B2 and other races of Ralstonia, new methods of control can be developed to safeguard US tomato and potato production.