Progress 10/01/06 to 09/30/11
Outputs
OUTPUTS: Our research group conducted experiments to test the specific hypotheses about bacterial wilt disease of crop plants caused by the soilborne bacterium Ralstonia solanacearum. Much of our experimentation has focused on hypotheses generated by a large gene expression microarray analysis that compared the total transcriptomes of two different strains growing either in artificial culture medium or within infected plants during pathogenesis. Specifically, we measured the virulence of site-directed bacterial mutant strains lacking one or more functions identified as strongly upregulated during tomato pathogenesis. These functions include sucrose metabolism, the assimilatory and dissimilatory nitrate pathways, a high-affinity oxygen uptake system, and oxidative stress tolerance. We also participated in annotation and analysis of genome sequences of three phylogenetically distant members of the R. solanacearum species complex that all cause bacterial wilt of tomato, and in a collaborative study of the quantitative virulence factor PopW, a Type 3 effector protein produced by R. solanacearum. PARTICIPANTS: Professor Caitilyn Allen; Dr. Jenniifer M. Colburn-Clifforn (former PhD student); Jonathan Jacobs (current PhD student); Dr. Annett Milling (postdoc); Jacob M. Scherf (former MS student). TARGET AUDIENCES: The target audience for these changes in knowledge is the international community of plant pathology scientists, as well as scientists working to understand microbial pathogenesis of eukaryotes in general. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our research this year generated the following changes in knowledge: 1. R. solanacearum requires the ability to metabolize sucrose for full virulence. Mutant strains lacking the scrRABY operon were significantly reduced in virulence on both wilt-susceptible and partially wilt-resistant tomato varieties. This finding was somewhat surprising since R. solanacearum infects and multiplies in the plant xylem tissue, which contains only small amounts of sucrose, at least in healthy plants. 2. Nitrate metabolism is important for fitness of R. solanacearum during tomato pathogenesis. Mutant strains lacking the NasA protein (and thus the assimilatory nitrate pathway) had lower wilt virulence than their wild-type parent strains. 3. The CBB-3 type cytochrome C oxidase, a high-affinity oxygen uptake system in R. solanaceatum, is important both for the ability to grow in hypo-oxyic environments like the host plant xylem vessel and for bacterial wilt virulence. 4. A homolog of DPS, a DNA-binding protein induced by stress in E. coli, is upregulated during bacterial growth in tomato rhizospheres and is required for full virulence. This suggests that R. solanacearum encounters significant stress during bacterial wilt pathogenesis, possibly as a result of host plant defenses responses.
Publications
- Colburn-Clifford, J. M., J. M. Scherf, and C. Allen. 2010. Ralstonia solanacearum Dps contributes to oxidative stress tolerance, colonization, and virulence on tomato plants. Applied and Environmental Microbiology 76:7392-7399.
- Remenant, B., B. Coupat-Goutaland, A. Guidot, G. Cellier, E. Wicker, C. Allen, M. Fegan, O. Pruvost, M. Elbaz, A. Calteau, G. Salvignol, D. Mornico, S. Mangenot, V. Barbe, C. Medigue and P. Prior. 2010. Genomes of three diverse tomato pathogens within the Ralstonia solanacearum species complex reveal evolution in action. BMC Genomics 11:379.
- Colburn-Clifford, J. M., and C. Allen. 2010. A cbb3-type cytochrome c oxidase contributes to Ralstonia solanacearum R3bv2 growth in microaerobic environments and to bacterial wilt disease development in tomato. Molecular Plant-Microbe Interactions 23:1042-1052.
- Li, J.-G., H.-X. Liu, J. Cao, L.-F. Chen, C. Gu, C. Allen, and J-H. Guo. 2010. PopW of Ralstonia solanacearum, a new two-domain harpin targeting the plant cell wall. Molecular Plant Pathology 11: 371-381.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Activities: Conducted experiments to better understand the interactions of the plant pathogenic bacterium Ralstonia solanacearum with its plant hosts. We study several strains of the bacterial wilt pathogen in my lab: a typical Asian tropical strain, a native strain from the southeastern US, and a Race 3 biovar 2 strain that originated in the Andes and can cause brown rot of potatoes at much cooler temperatures than other R. solanacearum strains. Race 3 biovar 2 strains are US Select Agent quarantine pests and a source of losses and concern both for subsistence farmers in the tropical highlands and for US ornamental producers who fear importing the pathogen from offshore production sites. This year, we monitored total pathogen transcriptional activity in order to identify the bacterial genes that are expressed during plant infection and at different temperatures. Bacterial RNA from cells growing under various conditions were hybridized to custom-designed microarray chips containing the complete genomes of both a tropical and a Race 3 biovar 2 strain. Analysis of the resulting gene expression data revealed that about 20% of the total pathogen transcriptome is differentially expressed (DE) during plant pathogenesis, relative to growth in culture. The diverse metabolic pathways upregulated in planta portray the physiological state of R. solanacearum during its life in the host xylem. These include genes involved in uptake and metabolism of nitrate and sucrose. Preliminary experiments indicate that ability to use sucrose is essential for full virulence on tomato, even though the xylem of healthy tomato plants is not reported to contain sucrose. A different approach identified bacterial genes expressed in response to host root exudates. These included a high-affinity cytochrome c oxidase needed for both growth in hypoxic conditions and for full virulence on tomato plants. A second root exudate-induced gene encodes Dps, a non-specific DNA-binding protein that helps bacteria survive oxidative and other stress. It too is necessary for full virulence. Continuing our studies of the differential epidemiological potential of diverse R. solanacearum strains, we measured population sizes of R3bv2 and tropical strains in infected geranium and tomato stems at various temperatures. Both strains survived over 6 months in host stems at -20C, but populations of both strains dropped to undetectable following a more biologically realistic series of six 2-day cycles of +5C/-10C. Dissemination: In addition to the formal peer-reviewed journal articles described below, we also presented data at two international meetings and one national scientific meeting attended by both academic researchers and representatives of the ornamental industry and regulatory agency scientists from APHIS concerned with developing and modifying quarantine regulation. PARTICIPANTS: Jennifer Clifford, PhD student, Dept of Plant Pathology, University of Wisconsin-Madison; Jacob Scherf, MS student,Dept of Plant Pathology, University of Wisconsin-Madison; Dr Annett Milling, Postdoctoral Fellow,Dept of Plant Pathology, University of Wisconsin-Madison TARGET AUDIENCES: Our target audiences are: 1) The community of plant pathology researchers working to control crop losses caused by plant diseases; 2) Growers affected by bacterial wilt, especially U.S. ornamental cutting producers and small-scale cash and subsistence farmers in the developing tropics; 3) Federal and state plant health regulators responsible for developing policy to prevent introduction of potentially destructive quarantine plant pests to the U.S. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our finding that Race 3 biovar 2 strains of Ralstonia solanacearum do not survive repeated freeze-thaw cycles even when sheltered in plant tissue may impact regulatory policy regarding the Select Agent quarantine pest, paricularly in association with policy affecting the imported ornamental industry, and eventually the decision on whether R3bv2 should continue to be considered a Select Agent pathogen. In collaboration with colleagues in France and Cameroon, we characterized the diverse R. solanacearum strains present in West Africa, as part of an ongoing effort to breed regionally-appropriate wilt-resistant tomato plants.
Publications
- Toukam, G. M. S., G. Cellier, E. Wicker, C. Guilbaud, R. Kahane, C. Allen, and P. Prior. 2009. Broad diversity of Ralstonia solanacearum strains in Cameroon. Plant Disease: 93:1123-1130.
- Allen, C., A. F. Bent, and A. O. Charkowski. 2009. Underexplored niches in research on plant pathogenic bacteria. Plant Physiology 150:1631-37.
- Flores-Cruz, Z. and C. Allen. 2009. Ralstonia solanacearum encounters an oxidative environment during tomato infection. Molecular Plant-Microbe Interactions: 22:773-782.
- Mejia, L., B.E. Garcia, A.C. Fulladolsa, E.R. Ewert, J.-F. Wang, J.W. Scott, C Allen, and D.P. Maxwell. 2009. Evaluation of recombinant inbred lines for resistance to Ralstonia solanacearum in Guatemala and preliminary data on PCR-based tagging of introgressions associated with bacterial wilt-resistant line, Hawaii 7996. Tomato Genetics Cooperative Report 59:32-41.
- Milling, A., F. Meng, T. P. Denny, and C. Allen. 2009. Interactions with hosts at cool temperatures, not cold tolerance, explain the unique epidemiology of Ralstonia solanacearum Race 3 biovar 2. Phytopathology 99:1127-1134.
- Champoiseau, P., J. Jones, and C. Allen. 2009. Ralstonia solanacearum Race 3 biovar 2 causes tropical losses and temperate anxieties. Plant Health Progress doi:10.1094/PHP-2009-0313-01-RV.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Activities: Conducted experiments to better understand the interactions of the plant pathogenic bacterium Ralstonia solanacearum with its plant hosts. Several strains of the bacterial wilt pathogen are studied in my lab: a typical Asian tropical strain, a native strain from the southeastern US, and a Race 3 biovar 2 strain that originated in the Andes and can cause brown rot of potatoes at much cooler temperatures than other R. solanacearum strains. Race 3 biovar 2 strains are US Select Agent quarantine pests and a source of losses and concern both for subsistence farmers in the tropical highlands and US ornamental producers who fear importing the pathogen from offshore production sites. This year we developed methods to extract bacterial RNA from infected plants in order to identify the bacterial genes that are expressed during plant infection. We designed microarrays chips containing the complete genomes of both a tropical and a Race 3 biovar 2 strain that will allow us to compare the pathogen genes expressed during plant infection at tropical and cool temperatures in the two different strains. We further determined that tropical and Race 3 biovar 2 strains survive similarly in water and soil, at warm & cool temperatures, but that Race 3 biovar 2 strains can survive much longer than tropical strains at cool temperatures if they are in plant tissues, like potato tubers or tomato stems. We determined that the ability to overcome oxidative stress, such as that produced by plant defensive reactive oxygen species, is critical for pathogen success. Further, a mutant bacterial strain lacking a high-affinity oxygen uptake system was reduced in virulence and in the ability to grow in low-oxygen environments resembling the host plant xylem vessel, which is microaerobic. From the perspective of the plant host, we used quantitative RT-PCR to identify plant defense pathways that are activated in resistant and susceptible tomato plants in respone to infection by either tropical or Race 3 biovar 2 strains. To our surprise, we found that resistant tomatoes can recognize and respond to purified bacterial extrapolysaccharide from a Race 3 biovar 2 strain, a susceptible tomato cultivar cannot do so. Reciprocal experiments with extracellular polysaccharide-deficient mutants of R. solanacearum confirm that this pathogen's EPS appears to specifically trigger expression of plant defense pathways. Dissemination: Results were disseminated in two ways in addition to the formal peer-reviewed journal articles described below. First, we presented data at two national scientific meetings attended by both academic researchers and representatives of the ornamental industry and regulatory agency scientists from APHIS concerned with developing and modifying quarantine regulation. Second, I co-authored a review article intended for a non-academic office, which will be published as the primary feature article on the American Phytopathological Society's website in January 2009. PARTICIPANTS: Postdocs trained: Dr. Annett Schoenwalder Milling and Dr. Lavanya Babujee. Graduate students trained: Zomary Flores-Cruz, Jennifer Clifford, Fanhong Meng, Jonathan Jacobs, Jacob Scherf. Undergraduate students Ryan Leung, Eric Flanagan, Brad Lang, and Nikki Levin did independent research in the lab during 2008. Collaborative partners include: Professor Luis Mejia (University of San Carlos de Guatemala); Professor Jeff Jones and Timur Momol (University of Florida-Gainesville); Professors Mark Schell and Timothy Denny (University of Georgia); Professor Anne Alvarez (University of Hawaii); Professor Dirk Hoffmeister (University of Minnesota); Dr. Philippe Prior , Dr. Philippe Rott, and Dr. Monique Royer (CIRAD, France); Dr. Mike Klopmeyer (Ball FloraPlant, Inc., West Chicago, IL). TARGET AUDIENCES: Target audiences for this projects are: 1) Fellow academic researchers in plant pathology, environmental microbiology, and microbial pathogenesis; 2) Government agency scientists responsible for developing regulatory policy concerning quarantine and Select Agent plant pathogens; 3) Ornamental industry growers who are eager to effectively exclude and detect ornamental cuttings infected with R. solanacearum Race 3 biovar 2; 4) Commercial producers of detection technologies who may wish to commercialize rapid and sensitive diagnostic and detection systems for R. solanacearum based on our research results. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Our finding that Race 3 biovar 2 strains of Ralstonia solanacearum express their characteristic cold tolerance only in the presence of plant host tissue will likely impact regulatory policy regarding the Select Agent quarantine pest. These data suggest that this pathogen is no more likely to survive in soil or water in the northern U.S. than native Race 1 strains of R. solanacearum, which have been present since before European settlement but have not become established in the northern US. Results of ongoing experiments on Race 3 biovar 2 survival in infected geranium roots under simulated winter conditions will be directly applicable to regulatory policy affecting the imported ornamental industry, and eventually the decision on whether Race 3 biovar 2 should continue to be considered a Select Agent pathogen. As part on an ongoing collaboration with plant breeders at the University of San Carlos in Guatemala, we determined the level of bacterial wilt resistance of a set of candidate wilt-resistant tomato breeding lines. It was necessary to screen and evaluate these lines under controlled conditions in Madison because the field selections for wilt resistance in Guatemala are complicated by the presence of several destructive tomato virus diseases in our test-plots. These results will inform selection of parents for additional breeding crosses to incorporate this complex disease resistance trait into tomato cultivars that have agronomic traits desired in Guatemalan markets. These cultivars will eventually be marketed by a locally-owned seed business in Antigua, Guatemala.
Publications
- Nakaho, K. and C. Allen. 2008. A pectinase-deficient Ralstonia solanacearum strain induces reduced and delayed structural defenses in tomato xylem. Journal of Phytopathology: doi:10.1111/j.1439-0434.2008.01467.x
- Hong, J.C., T. Momol, J. Jones, P. Ji, S. Olson, C. Allen, A. Sanchez-Perez, P. Pradhanang, K. Guven. 2008. Detection of Ralstonia solanacearum in irrigation ponds and aquatic weeds associated with ponds in North Florida. Plant Disease: 92:1674-82.
- Young,J. C., C. Allen, T. Coutinho, T. Denny, J. Elphinstone, M. Fegan, M. Gillings, T. R. Gottwald, J. H. Graham, J. D. Janse, M. M. Lopez, C. Morris, N. Parkinson, J. Rodrigues Neto, M. Scortichini, and Y. Takikawa. 2008. Plant pathogenic bacteria as bioterror weapons: A real threat Phytopathology 98:1060-1065.
- Sanchez-Perez, A., L. Mejia, M. Fegan, and C. Allen. 2008. Diversity and distribution of Ralstonia solanacearum strains in Guatemala and rare occurance of tomato fruit infection. Plant Pathology 57:320-331.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: My lab has two main research thrusts. First, we work to understand the basic biology of the interactions between the bacterial wilt pathogen, Ralstonia solanacearum, and its diverse plant hosts (we use tomato as a model host). These studies currently focus on genes that the pathogen needs to find, invade, and colonize the plant. A genetic screen for pathogen genes upregulated during growth in the host suggested that genes involved in motility and in tolerance of stresses were important for R. solanacearum success in tomato. We therefore constructed mutant bacterial strains lacking either various motility and chemotaxis functions and mutant strains lacking functions likely involved in tolerance of host-imposed stresses like plant antimicrobial compounds. (described in the report on Hatch project WIS04765.) A second set of mutants affected in tolerance of plant reactive oxygen species (ROS) include one lacking Bcp (a peroxidase needed for hydrogen peroxide and superoxide
tolerance) and another lacking OxyR, the global regulator of oxidative stress responses. The Bcp mutant is fully virulent, which is unsurprising since there are several redundant peroxidase genes in the pathogen genome. However, the OxyR mutant grows poorly in the plant and is reduced in virulence, suggesting this trait is important in pathogen fitness. To identify genes important in the earliest stages of bacterial wilt disease development, we conducted a second screen to find pathogen genes that are upregulated following exposure to tomato root exudates (rex genes). This screen identified, among other genes, a cytochrome C oxidase (cco) biosynthetic gene cluster that we hypothesize is important for growth under the low oxygen tension conditions in the plant rhizosphere and in plant xylem vessels. A second, more applied line of research seeks tools to more effectively diagnose, detect, and prevent bacterial wilt both in the US, where one strain is a highly regulated quarantine pest,
and in the developing tropics, where bacterial wilt is a source of serious and increasing crop losses. We have an ongoing collaborative project with the groups of Jeff Jones and Tim Momol at the University of Florida on epidemiology and diagnosis of R. solanacearum in Florida. We found that a group of recently-introduced Race 1 biovar 1 strains give a false positive on a commonly used PCR assay for Race 3 biovar 2. Further, we found that R. solanacearum is present in ponds and weedy plants in South Florida, suggesting a potential source of outbreaks, In separate work with colleagues at the University of San Carlos in Guatemala, we found that while Race 3 biovar 2 can cause disease on tomatoes in the Guatemala highlands, neither Race 1 nor Race 3 bacteria are usually present in fruit from infected plants. Two additional publications offer a feminist analysis of molecular genetic research on sex determination in humans, and on the social effects on women working in the Guatemalan
ornamental industry of the regulatory response to R. solanacearum Race 3 biovar 2 bacterial wilt on geraniums (Allen 2007a and b).
PARTICIPANTS: Caitilyn Allen, Professor of Plant Pathology Jill Swanson, Research Specialist UW-Madison Amilcar Sanchez-Perez, Graduate Research Assistant in UW-Madison Dept of Plant Pathology and currently faculty member in the Faculty of Agronomy, University of San Carlos of Guatemala. Zomary Flores, Graduate Research Assistant, UW-Madison, PhD candidate MDTP.
TARGET AUDIENCES: Biology researchers focusing on the molecular basis of plant-bacterial interactions. Regulators concerned with detection, diagnosis, eradication, and control of bacterial wilt disease and especially its quarantine / Select Agent pathogen Ralstonia solanacearum Race 3 biovar 2. Vegetable and ornamental growers in the US and the developing tropics who are trying to avoid or reduce crop losses to bacterial wilt disease.
Impacts
Our basic biology work affects primarily other researchers working to understand the molecular basis of interactions between plant-associated bacteria and their hosts; we disseminate this work at professional meetings and in peer-reviewed journals. Our collaborative work with colleagues in Florida (Ji et al 2007) has changed the standard operating procedures used by the USDA-APHIS national plant disease diagnostic lab in Beltsville Maryland to diagnose R. solanacerarum Race 3 biovar 2. They now routinely conduct a biovar test in addition to the PCR test that we showed could give false positives. Our collaborative work with colleagues in Florida and in Guatemala (Swanson et al 2007; Hong et al, in press) will serve state and Federal regulatory official determining optimal procedures for surveying and detection of R. solanacearum Race biovar 2. USDA-APHIS regulators will use our findings on the rareness of tomato fruit infection with R. solanacearum Race 3 biovar 2
(Sanchez-Perez et al 2008) as part of their science-based decision making process regarding permitting import of fresh tomato fruits from Guatemala to the United States.
Publications
- Allen, C. 2007a. It's a Boy! Gender expectations intrude on the study of sex determination. DNA and Cell Biology. 26(10): 699-705.
- Allen, C. 2007b. Bacteria, Bioterrorism, and the Geranium Ladies of Guatemala. pp.169-177 in: Wages of Empire: Neoliberal policies, repression, and women's poverty. (A. L. Cabezas, E. Reese, and M. Waller, editors) Paradigm Press, Boulder, Colorado.
- Hong, J.C., T. Momol, J. Jones, P. Ji, S. Olson, C. Allen, A. Sanchez-Perez, P. Pradhanang, K. Guven. 2008. Detection of Ralstonia solanacearum in irrigation ponds and aquatic weeds associated with ponds in North Florida. Plant Disease: accepted pending revision.
- Sanchez-Perez, A., L. Mejia, M. Fegan, and C. Allen. 2008. Diversity and distribution of Ralstonia solanacearum strains in Guatemala and rare occurance of tomato fruit infection. Plant Pathology: 57: e-print before publication doi: 10.1111/j.1365-3059.2007.01769.x
- Swanson, J. K., L. Montes, L. Mejia and C. Allen. 2007. Detection of latent infections of Ralstonia solanacearum Race 3 biovar 2 in geraniums. Plant Disease 91:828-834.
- Ji, P., C. Allen, A. Sanchez-Perez,, J. Yao, J G. Elphinstone, J. Jones, and T. Momol. 2007. New diversity and diagnostic challenges associated with Ralstonia solanacearum strains in Florida. Plant Disease 91:195-203.
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