Progress 01/15/07 to 01/14/12
Outputs
OUTPUTS: Activities: This collaborative integrated plant biosecurity project had three objectives: 1. Develop rapid, robust, and reliable diagnostic assays for Ralstonia solanacearum race 3 biovar 2 (R3bv2); 2: Develop a package of optimized training modules on R3bv2 for students and stakeholders; and 3. Identify Ralstonia solanacearum R3bv2 traits involved in cold adaptation and growth in plant hosts. We have addressed Objective 1 with four approaches tailored to the differing needs of growers, who need a simple, moderately sensitive method that is robust, and regulators, who need a highly sensitive method and have the technical expertise to use complex demanding approaches. We field tested detection methods in potato fields in the Guatemalan highlands, where R3bv2 is endemic. LAMP, a sensitive but technically demanding method, was compared to magnetic capture hybridization plus PCR, FTA card plus PCR, and immunomagnetic separation plus PCR. A broadly comparative analysis is in preparation for publication. We addressed Objective 2 with the extension and outreach activities described under Outcomes/Impacts below. To address Objective 3 we characterized the role in cool virulence of a cluster of 5 genes found to be differentially upregulated under cool conditions in R3bv2 compared to a tropical R. solanacearum strain. The 5 genes encode a lectin, two related but cryptic proteins, and a quorum sensing system. Mutants lacking any of these genes are affected in cool virulence, confirming the role of their products in the epidemiologically important pathogen trait. A paper describing these results is currently in review. PARTICIPANTS: Individuals: Co-PIs were: Prof. Caitilyn Allen, University of Wisconsin-Madison (lead PI); Prof. Timothy P. Denny, University of Georgia; Prof. Mark Schell, University of Georgia; Prof. Jeffrey B. Jones, University of Florida; Prof. Anne Alvarez, University of Hawaii. Postdocs working on this project: Youngsil Ha (U. Georgia); Benoit Remenant (U. Wisconsin-Madison); Annett Milling (U. Wisconsin-Madison); and Lavanya Babujee (U. Wisconsin-Madison). Graduate students trained on this project: Jonathan M. Jacobs, Alejandra Huerta, and Fanhong Meng. Partner organizations: Society of American Florists; Ball FloraPlant, Inc.; USDA-APHIS PPQ (Russ Bullock); NPDN. TARGET AUDIENCES: The target audiences for this project are: potato, tomato, and geranium growers in the US and abroad whose productivity is threatened by bacterial caused by Ralstonia solanacearum; regulatory officials (e.g. USDA-APHIS, EPPO, and similar, as well as state plant health/quarantine officers; plant disease diagnosticians both private and public, as well as the NPDN; academic and industrial researchers and students interested in the applied and fundamental aspects of bacterial wilt disease and the pathogen that causes it. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The impact of the extension and education components of the project to plant biosecurity is to disseminate reliable information about the pathogen, the disease, regulatory standards, recent research findings, and effective diagnostic tools for the pathogen. The goal is to ensure effective preparedness and training of official regulators, diagnosticians and all individuals responsible for first identification and response to a possible R3b2 discovery in the US. Growers and regulators concerned with R3bv2 are served by our project website (http://plantpath.ifas.ufl.edu/rsol/, online since September 12, 2008), which contains modules on the pathogen, the disease, and disease management in English and in Spanish, as well as alerts, a current bibliography, distribution maps, and images. The website also offers E-learning modules available on the National Plant Disease Diagnostic Network (NPDN) Training and Education site. Those who pass the associated post-test receive an NPDN Certificate of Completion. To disseminate the new detection methods developed in this project, we organized a workshop at the 2011 APS national meetings, entitled "DNA-based Pathogen Detection Methods: Ralstonia solanacearum, a case study." This 8-h workshop, co-sponsored by the APS Bacteriology Committee and the APS Diagnostics Committee, filled to capacity of 18 people with a 10-person waitlist. Attendees were from industry, regulatory agencies, and plant pathology graduate programs.
Publications
- Flores-Cruz, Z., and C. Allen. 2011. Necessity of OxyR for the hydrogen peroxide stress response and full virulence in Ralstonia solanacearum. Applied and Environmental Microbiology 77: 6426-6432.
- Kubota, R., M. A. Schell, G. D. Peckham, J. Rue, A. M. Alvarez, C. Allen, and D. M. Jenkins. 2011. In silico genomic subtraction
 guides development of highly accurate, DNA-based diagnostics for
 Ralstonia solanacearum Race 3 biovar 2 and Blood Disease Bacterium. Journal of General Plant Pathology 77:182-193.
- Meng, F., J. Yao, and C. Allen. 2011. A hypermotile motN mutant of Ralstonia solanacearum is reduced in virulence. Journal of Bacteriology 193:2477-2486.
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Progress 01/15/10 to 01/14/11
Outputs
OUTPUTS: Goal 1 (Diagnostics) - We have identified a set of five non-redundant PCR primer pairs that together detect 100% of tested Race 3 biovar 2 (R3bv2) strains of R. solanacearum, but do not detect any tested related strains. We have developed two different low-tech rapid methods for application of this diagnostic method and will test them in the field next year. Goal 2 (Outreach and Education) - We have constructed an accessible content-dense online resource for growers, regulators, and scientists working with R. solanacearum, including targeted educational modules, protocols, and a reference database of publications and images. Site traffic reports demonstrate a significant global impact. In addition, project personnel have delivered numerous public presentations to growers, regulators, and scientists. Goal 3 (Biological Characterization of R3bv2 Cold Tolerance) - We published a thorough analysis of cold tolerance and cold virulence in diverse R. solanacearum strains as a precursor to gene expression analysis. We performed bacterial transcriptome analysis of relative gene expression in tropical ("Race 1") and temperate R3bv2 strains under tropical (28C) and temperate (20C) conditions; the results are clear, consistent, and interpretable. Excitingly, a 5-gene cluster upregulated in R3bv2 at 20C (vs. 28C) encodes three proteins differentially required for full virulence under cold conditions. PARTICIPANTS: Professor Mark A. Schell (U. Georgia); Professor Timothy P. Denny (U. Georgia); Dr. Youngsil Ha (postdoc, U. Georgia); Professor Anne Alvarez (U. Hawaii); Dr. Gabriel Peckham (postdoc, U. Hawaii); Professor Jeffery B. Jones (U. Florida); Dr. Patrice Champoiseau (postdoc, U. Florida); Professor Caitilyn Allen (U. Wisconsin); Dr. Annett S. Milling (Postdoc, U. Wisconsin); Alejandra Huerta (PhD student, U. Wisconsin); Fanhong Meng (PhD student, U. Wisconsin). TARGET AUDIENCES: The target audiences for this work are: -The international community of research biologists studying the mechanisms of plant-bacterial interactions and working to develop methods to manage or prevent bacterial wilt disease of diverse crop plants. -The USDA-APHIS-PPQ, the federal agency charged with protecting the nation's agriculture from exotic pests. Some of our research results are directly applicable to regulatory policy and decisions about the select agent plant pathogen Ralstonia solanacearum Race 3 biovar 2. - Plant disease diagnosticians (for example, the NPPDN, the National Plant Disease Diagnostic Network) responsible for detecting and identifying potential outbreaks of bacterial wilt or brown rot caused by R. solanacearum Race 3 biovar 2. -Ornamental plant producers, especially geranium growers who import cuttings from the highland tropics to North America and Europe. Ornamental cuttings latently infected with R. solanacearum Race 3 biovar 2 have been a source of accidental introductions of this select agent pathogen. Our improved diagnostic techniques are intended to reduce the risk of such future introductions. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project generated the following changes in knowledge: We determined that tomato plants can specifically recognize the extracellular polysaccharides produced by all strains of R. solanacearum, and that in response to infection by the pathogen or to purified cell-free EPS plants launch defense responses via the ethylene and salycylic acid signaling pathways. We now know that R3bv2 strains cannot survive the moderate freeze-thaw cycles typically occur in a temperate winter. This result suggests that this pathogen is unlikely to survive and become established in the commercial potato-growing regions of the United States and Canada. Field testing of the immuno-magnetic separation (IMS) detection system for R3bv2 in Guatemala and Reunion Island France helped us optimize the parameters for this assay and suggested that because of its extreme sensitivity the LAMP assay needs substantial modification before it is appropriate for field use.
Publications
- Scherf, J. M., A. Milling, and C. Allen. 2010. Moderate temperature fluctuations rapidly reduce viability of Ralstonia solanacearum Race 3 biovar 2 in infected geranium, tomato, and potato. Applied and Environmental Microbiology 76:7061-7067.
- Milling, A., L. Babujee, and C. Allen 2011. Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants. PloS One 6(1): e15853
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Progress 01/15/09 to 01/14/10
Outputs
OUTPUTS: Objective 1: Develop rapid, robust, and reliable diagnostic assays for Ralstonia solanacearum race 3 biovar 2. We coupled a species-specific antibody to a magnetic bead capture system that enables concentration of low Ralstonia populations from large volumes of aqueous plant or soil extractions (this is called IMS, or immunomagnetic separation). The "captured" bacteria can then be subjected to a R3bv2-specific real-time or conventional PCR. Limits of detection are currently being evaluated to optimize the assay. Objective 2: Develop a package of optimized training modules on R3bv2 for students and stakeholders. This includes i) Educational modules at different levels for diverse target audiences, ii) Delivery of educational materials using diverse formats such as fact sheets, oral presentations, workshops, project website, and online course modules to serve as many audiences as possible, and iii) Evaluation of program impact and effectiveness. * Dedicated project website (http://plantpath.ifas.ufl.edu/rsol/index.html) designed to continuously educate and connect with our target audiences. After noting many site users from Spanish-speaking countries, we translated core website content into Spanish; this resource is among our most frequently-downloaded. (http://plantpath.ifas.ufl.edu/rsol/RalstoniaPublications_PDF/Ralston ia_solanacearum_ES_PDF.pdf). * Educational Modules. This year we launched four online e-learning modules: A) R. solanacearum race 3 biovar 2, B) brown rot of potato, C) Southern wilt of geranium, and D) bacterial wilt of tomato. These use a Content Management System for e-learning applications, and are accessible through the NPDN Training and Educational Site at http://cbc.at.ufl.edu/. Objective 3: Identify Ralstonia solanacearum race 3 biovar 2 traits involved in cold adaptation and growth in plant hosts. Most strains of R. solanacearum are tropical, but R3bv2 strains can attack plants in temperate zones and tropical highlands. Can R. solanacearum strains survive typical environmental conditions of a North American winter Strains in water or in naturally infected tomato, potato, and geranium plants were subjected to cycles of 2 days at +5C followed by 2 days at -10C. US strain K60 and R3bv2 strain UW551 dropped below detectability after 12 cycles. But in infected plant tissue these strains were killed by only 5-7 cycles. Thus, even when sheltered in plant tissue, R3bv2 is unlikely to survive the temperature fluctuations typical of a northern temperate winter. We used microarray chips to monitor gene expression of both R3bv2 and tropical strain GMI1000 during growth in culture and in infected plants at 20C (temperate) and 28C (tropical conditions). This identified candidate genes specifically expressed in R3bv2 at the cooler temperature. We created mutants lacking each gene. Virulence assays at 20C and 28C revealed that all three genes are required for full R3bv2 virulence, and most importantly, mutants lacking the lectin are differentially reduced in virulence at cooler temperatures. PARTICIPANTS: Individuals: Patrice Champoiseau - Postdoc, University of Florida Gainesville (outreach); Youngsil Ha -Postdoc, University of Georgia (diagnostics); Gabriel Peckham -Postdoc, University of Hawaii (diagnostics); Jonathan Jacobs -Doctoral student, University of Wisconsin (research); Partner Organizations: National Plant Disease Diagnostics Network (NPDN) TARGET AUDIENCES: Our target audience includes students, educators, growers, and government agency personnel charged with monitoring and managing this plant pathogen. We work to disseminate reliable information to these audiences about the pathogen, the disease, regulatory standards, recent research findings, and effective diagnostic tools for the pathogen. The goal is to ensure effective preparedness and training of official regulators, diagnosticians and all individuals responsible for first identification and response to a possible R3b2 discovery in the US. We have therefore developed an integrated education and outreach program. This 3-step program involves development of educational and training content by a team of experts, delivery of educational materials to target audiences by diverse means including current web-based technologies, as well as use of various evaluation tools to assess program effectiveness. Monitored access to our Ralstonia/bacterial wilt-dedicated website shows that stakeholders from diverse organizations both within and outside the US regularly use this resource to obtain updated and accurate information on R. solanacearum R3bv2 and bacterial wilt disease management. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Objective 1: (Diagnostics) Laboratory phytopathology course. To ensure that diagnostic reagents and protocols work as expected in novice hands, and to familiarize future scientists with R3b2-specific diagnostic tools, we tested these methods on students in a U. Florida Bacterial Plant Pathogens Laboratory course taught by co-PI Jeff Jones. A total of 16 students in the Plant Medicine and Plant Pathology Programs learned background on bacterial wilt and performed detection protocols. Objective 2: (Outreach) Outreach Meetings and Conferences Attended in 2009: to directly disseminate information and to establish channels with end users of the educational modules. - December 6-10, 2009. 2nd National Meeting of the National Plant Diagnostic Network, Miami, Florida. Oral communication title: "Ralstonia solanacearum race 3, biovar 2: Description and current diagnosis methods for best management of a Select Agent pathogen". Number of attendees: 200 researchers, diagnosticians, and government representatives - November 3-5, 2009. 24th Tomato Disease Workshop, State College, Pennsylvania."Description and strategies for best management of Ralstonia solanacearum race 3 biovar 2 as a potential incitant of bacterial wilt of tomato". - September 9, 2009. Florida Tomato Institute Meeting, Naples, Florida. "Ralstonia solanacearum race 3 biovar 2: Description and strategies for best management of a select agent pathogen as a potential incitant of bacterial wilt of tomato". - May 16-19, 2009. Joint meeting of the Florida Phytopathological Society and the American Phytopathological Society Caribbean Division, Orlando, Florida. "Developing an effective international education program for management of Ralstonia solanacearum race 3 biovar 2". - March 9-14 2009. DRCAFTA-USAID training program, Guatemala, To 20 lab leaders involved in Sanitary/Phytosanitary testing from Guatemala, Costa Rica, Dominican Republic, Honduras, and El Salvador.
Publications
- Allen, C., A. F. Bent, and A. O. Charkowski. 2009. Underexplored niches in research on plant pathogenic bacteria. Plant Physiology 150:1631-37.
- Champoiseau, P., Jones, J. B. and Allen, C. 2009. Ralstonia solanacearum race 3 biovar 2 causes tropical losses and temperate anxieties. Plant Health Progress. doi:10.1094/PHP-2009-0313-01-RV. Published 13 March 2009.
- Milling, A., Meng, F., Denny, T.P., and Allen, C. 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.
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Progress 01/15/08 to 01/14/09
Outputs
OUTPUTS: Aim 1: Develop rapid, robust, and reliable diagnostic assays for Ralstonia solanacearum race 3 biovar 2, using both immunological and DNA-based approaches. This year, we identified a set of R3bv2-specific primer sets and tested them against a broad array of diverse R. solanacearum strains as well as a group of common soil and rhizosphere bacteria. Some of these probes are being optimized for use in a LAMP (loop-mediated isothermal amplification) detection assay (see publication by Kubota et al 2008). We are further investigating using a related but proprietary detection technology, called NEAR, in collaboration with a company licensed to develop and market NEAR for agricultural uses. Screening for R. solanacearum R3bv2-specific antibodies continues but to date has been less rewarding. Aim 2: Identify Ralstonia solanacearum race 3 biovar 2 genes involved in cold adaptation and growth in plant hosts, using a microarray-based post-genomic approach. We have found that interactions with hosts at cool temperatures, not cold tolerance, explain the unique epidemiology of Ralstonia solanacearum Race 3 biovar 2. Most strains of R. solanacearum are tropical, but R3bv2 strains can attack plants in temperate zones and tropical highlands. The basis of this distinctive ecological trait is not understood. We compared the survival of tropical, R3bv2, and warm-temperate North American strains of R. solanacearum under different conditions. In water at 4C, North American strains remained culturable the longest (up to 90 days) whereas tropical strains remained culturable for the shortest time (about 40 days). However, live/dead staining indicated that cells of representative strains remained viable for over 160 days. In contrast, inside potato tubers R3bv2 strain UW551 survived more than 4 months at 4C, while North American strain K60 and tropical strain GMI1000 were undetectable after less than 70 days in tubers. GMI1000 and UW551 grew similarly in minimal medium at 20C and 28C, and while both strains wilted tomato plants rapidly at 28C, UW551 was much more virulent at 20C. Thus, any differences among the strains in the absence of a plant host were not predictive of their behavior in planta at cooler temperatures, where R3bv2 survived longer in potato tubers and was more virulent on tomato plants. These data indicate that interaction with plants is required for expression of the temperate epidemiological trait of R3bv2. To better understand the biology behind this finding, we used genomic microarrays for UW551 and GMI1000 to compare total transcriptional profiles of both strains grown in culture at 20C and 28C. These analyses identified several candidate traits that may contribute to differential survival and virulence at cool temperatures. We are currently testing mutants lacking the relevant genes. In parallel, we are also examining gene expression of both strains during natural pathogenesis of tomato plants and the two temperatures. Aim 3: Develop a package of optimized education and management training modules that will educate stakeholders to control this pathogen. PARTICIPANTS: Caitilyn Allen, Dept of Plant Pathology, University of Wisconsin-Madison (lead PI); Fanhong Meng, Dept of Plant Pathology, University of Wisconsin-Madison (predoctoral trainee); Jonathan M. Jacobs, Dept of Plant Pathology, University of Wisconsin-Madison (predoctoral trainee); Annett Milling, Dept of Plant Pathology, University of Wisconsin-Madison (postdoctoral trainee); Weihong Tan, University of Florida (co-I, leaving project in January 2009); Jeffery B. Jones, Dept of Plant Pathology, University of Florida (co-I); Patrice Champoiseau, University of Florida (Postdoctoral trainee); Mark A. Schell, Dept of Plant Pathology & Microbiology, University of Georgia (co-I); Timothy P. Denny, Dept of Plant Pathology, University of Georgia (co-I); Anne Alvarez, Dept Plant Pathology, University of Hawaii (co-I); Gabriel Peckham, Dept Plant Pathology University of Hawaii (postdoctoral trainee); Ryo Kubota,Dept Plant Pathology, University of Hawaii (predoctoral trainee) TARGET AUDIENCES: Our target audience for research publications is fellow academic researchers both domestic and international, grower group research scientists, and federal and state regulatory scientists. Our target audience for the Race 3 biovar 2 extension/outreach efforts such as the website and the APS Net online Feature Article includes: growers, plant disease diagnosticians, federal and state regulatory scientists in the US as well as regulators in other countries. Another potential target audience that we are in the process of developing is potential industrial partners who could commercialize diagnostic tools developed in the project. PROJECT MODIFICATIONS: A major change for this period was the departure of Dr Weihong Tan (U. Florida) as a co-Investigator on the project. Dr. Tan, who was responsible for developing the nanodetection methods to link with R3bv2-specific antibodies or nucleic acid-based detection methods, did not have sufficient time to devote to this project and the graduate student who was working on the project had to leave the country because of illness. Mark Schell (U. Georgia) has taken on Dr. Tan's area of responsibility for the detection method aim of the project. The funds previously allocated to Dr. Tan's lab will be transferred to Dr Schell's lab and to the lab of Caitilyn Allen (U. Wisconsin), where they will be used to support gene expression studies to identify temperature-responsive genes in R3bv2.
Impacts
A major outreach/extension accomplishment in the past year was the September 12, 2008 launching of the R. solanacearum/Bacterial Wilt Website (http://plantpath.ifas.ufl.edu/rsol/index.html), a comprehensive resource for growers, agency scientists and regulators, and researchers. This site contains expert peer-reviewed Learning Modules and in-depth articles on detection, diagnosis, identification, and management of bacterial wilt disease, with specialized modules on geraniums, potato, and tomato. In addition, the site offers an updated list with links to publications on R. solanacearum and bacterial wilt/potato brown rot, recent alerts and first reports of disease outbreaks around the world, links to the NPDN and USDA-APHIS Management Guidelines and Pest Alerts, and a description of this project. Preliminary assessment indicates that at 6 months post-launch there had been 2,003 first-time page visits in the site, corresponding to over 700 unique individuals visiting the site overall. The number of returning visitors has consistently increased over the life of the site with 102 returning visitors in the 6th month, a good indication that visitors find the site useful. About 26% of visitors spend more than 5 minutes on the site, another indicator of its usefulness. Within the site, the most popular resource is the Learning Modules, with the Race 3 Biovar 2 Learning Module accounting for 23.5% of page visits. Almost 40% of total visited website pages were visited from the United States (34 different states). However, the site has a large international impact, as more than 60% of total visited website pages were visited from 69 different countries around the world. However, the geographical origin of logs in the US revealed poor access to the website from potato-producing states where R. solanacearum race 3 biovar 2 is considered a serious threat. We will increase efforts to deliver our educational website content in these target states using our networks and mailing lists. The significant number of accessions from hispanic and francophone counties suggest that it would be useful to translate at least the core Learning Modules into French and Spanish; this is underway. A second outreach/extension accomplishment was presenting a detailed, heavily illustrated article on R. solanacearum Race 3 biovar 2 and the diseases it causes as an APS Net Feature Article, where it is freely available to the public. This invited article, which was written for an educated lay audience, was Feature of the Month in January 2009.
Publications
- 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.
- Kutin RK, Alvarez A, Jenkins DM. 2009. Detection of Ralstonia solanacearum in natural substrates using phage amplification integrated with real-time PCR assay. Journal of Microbiology Methods 76 (3): 241-246.
- J. M. Jacobs, J. M., F. Meng, and C. Allen. 2008. Identifying gene expression differences between race 1 and race 3 strains of Ralstonia solanacearum during bacterial wilt disease development at warm and cool temperatures. Phytopathology. Vol. 98 (6 July Supplement), page S73.
- Meng, F., A. Milling, and C. Allen. 2008. Interactions with hosts at cool temperature, not cold tolerance, explain the unique epidemiology of Ralstonia solanacearum race 3 biovar 2. Phytopathology. Vol. 98 (6 July Supplement), page S104.
- J. M. Young, 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.
- Kubota R, Vine BG, Alvarez AM, Jenkins, DM. 2008. Detection of Ralstonia solanacearum by loop-mediated isothermal amplification. Phytopathology 98:1045-1051.
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Progress 01/15/07 to 01/14/08
Outputs
OUTPUTS: This project has three major specific objectives: (1) Develop rapid, robust, and reliable diagnostic tools for Ralstonia solanacearum R3b2; (2) Identify R3b2 genes involved in cold adaptation and growth in plant hosts, (3) Develop a package of optimized training modules that will educate stakeholders to control this pathogen, primarily by exclusion. Because all of these are relatively complex and long-term goals, in the first year of the project we have made substantial progress on all three fronts but do not yet have product outputs. Activities: the co-PIs and relevant additional personnel have held four conference calls to plan specific research and outreach projects and to update each other on progress. Co-PI Tim Denny visited the Allen lab to plan cold tolerance research experiments. A one-day project meeting held in July 2007 was attended by the four lead PIs plus six additional personnel (other involved faculty,students, and postdocs) and three interested
representatives of industry and APHIS-PPQ. Each objective group gave formal presentations on progress to date and on proposed acticites for the following year. Results of initial experiments on ecological traits of R3bv2 strains were presented at the 2007 Americal Phytopathological Society annual meetings. The lead PI (Allen) attended the required NRI Plant Biosecurity Program Preoject Directors Meeting in July 2007.
PARTICIPANTS: Anne Alvarez, Professor, University of Hawaii (PI) Gabriel D. Peckham, postdoctoral researcher, University of Hawaii Tim Momol, Professor, University of Florida; Jeffrey B. Jones, Professor, University of Florida (P.I.); Weihong Tan, Professor, University of Florida; Jospeh Philipps, postdoctoral researcher, University of Florida; Jilin Yan, postdoctoral researcher, University of Florida; Patrice Champoiseau, postdoctoral researcher, University of Florida; Timothy P. Denny, Professor, University of Georgia (P.I.); Mark A. Schell, Professor, University of Georgia; Joanne Rue, postdoctoral researcher, University of Georgia; Caitilyn Allen, Professor, University of Wisconsin-Madison (lead P.I.); Annett Milling, postdoctoral researcher, University of Wisconsin-Madison; Fanhong Meng, graduate student, University of Wisconsin-Madison. To date, six postdoctoral researchers and one graduate student have received training as part of this project.
TARGET AUDIENCES: The target audiences for this integrated project are: 1. Practitioners in the ornamental industry, growing species that can be infected by Ralstonia solanacearum Race 3 biovar 2 (R3bv2) (i.e., geraniums), especially those producing ornamental cuttings offshore. This group includes both industrial plant pathologists and the field production managers. 2. Growers of solanaceaous plants that are potential hosts of R3bv2 in the United States, such as potatoes, tomatoes, tobacco, peppers, etc. 3. Plant diagnosticians in state, university, and federal facilities and APHIS inspectors who may need to recognize potential R3bv2 infected plant material. This includes NPDN members and scientists at the APHIS-PPQ national diagnostic lab who would be eventual users of improved diagnostic tools developed in this project. 4. Government regulators charged with developing biologically appropriate regulations for exclusion of R3bv2 from the US and response to finds of the pathogen in
the US. 5. Plant pathology graduate students who will use the learning modules on bacterial wilt, Select Agent diagnostics, and related phytosanitation. 6. Professional academic and agency plant pathologists and bacteriologists, both US and international, who work with R. solanacearum and bacterial wilt or with related pathogens or diseases, and who will draw on new basic and applied knowledge created in this project.
PROJECT MODIFICATIONS: Professor Timor Momol is no longer the lead P. I. for the University of Florida team, although he is still active in the project. Professor Jeffrey B. Jones is now the University of Florida lead P.I.
Impacts
We report the following changes in knowledge, actions, and conditions relating to each of our three project objectives. Objective (1): Develop rapid, robust, and reliable diagnostic tools for Ralstonia solanacearum R3b2. The group of Tan (U. FL) are working to link R. solanacearum-specific PS1 antibodies generated by the Alvarez group (U. HA) to dye-doped silica nanoparticles, but this method is still under optimization. The linking methods will ultimately be used to connect R3bv2-specific antibodies or oligonucleotides to nanoparticles for ultra-sensitive detection. The Alvarez group (U. HA) have done multiple mouse inoculations with whole cells or purified outer membrane proteins and screened resulting antibodies for R3bv2-specificity. To date antibodies either lacked specificity or bound weakly. An EPS-LPS R3bv2 mutant developed in collaboration with Schell (U. GA) and Allen (U. WI) was toxic. Combinatorial antibodies may offer R3bv2-specificity. A second diagnostic
method was developed and tested in the lab of Schell (U. GA). He identified candidate R3bv2-specific PCR primers from genome sequences of three R. solanacearum strains. A set of five primer sets were highly accurate and specific for R3bv2 and have diagnostic promise. These studies revealed that R3bv2 strains outside of S. America appear to be essentially clonal, with very limited genetic variation; this simplifies development of diagnostic tools. Objective (2): Identify R3b2 genes involved in cold adaptation and growth in plant hosts. The research groups of Denny (U. GA) and Allen (U. WI) measured the ability of diverse R. solanacearum strains to survive at 4 degrees C in water. Surprisingly, we found little difference in this trait between native North American Race 1 strains and the "cold-tolerant" Select Agent R3bv2 strain. In contrast, R3bv2 strains did survive significantly longer in potato tubers at 4C than US Race 1 strains. Race 1 and R3bv2 strains grew equally well in minimal
medium at 20C and 29C. However when these strains were inoculated onto tomato plants at either 20C or 29C, R3bv2 caused severe disease at both temperatures but the Race 1 strains were highly virulent at 29C but barely caused symptoms at 20C. Thus it appears that the cold tolerance of R3bv2 strains is only expressed in interaction with plants. We are using custom microarrays to measure expression of genes in Race 1 and R3bv2 strains during pathogenesis at 20C and 29C. (3) Develop a package of optimized training modules that will educate stakeholders to control this pathogen, primarily by exclusion. Dr Patrice Champoiuseau was hired as (U. FL) to lead and develop the outreach component of this project. He is assembling a web-accessible bibliography and a library of bacterial wilt images and presentations. He developed survey instruments to ascertain the state of baseline knowledge among academics and practitioners working with bacterial wilt; results of this survey are pending. He has
visited ornamental production facilities to better understand the realities and needs of this industry.
Publications
- 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
- 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)
- J. M. Young, 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 (accepted pending revision)
- Kubota, R., Alvarez, A.M., Vine, B.G., and Jenkins, D. M. 2007. Development of a loop-mediated isothermal amplification method (LAMP) for detection of the bacterial wilt pathogen, Ralstonia solanacearum. Phytopathology 97:S. 60 (abstract)
- Denny, T.P., A. Milling, V. Bhakta, and C. Allen. Ralstonia solanacearum race 3 biovar 2 strains are not uniquely cold tolerant in vitro. 2007. Phytopathology 97: S28 (Abstract)
- Milling, A., J. Swanson, and C. Allen. Tomato upregulates defense genes in response to infection by Ralstonia solanacearum. 2007. Phytopathology 97: S77 (Abstract)
- Allen, C. 2007. Strategies for managing bacterial wilt diseases of tomato, potato, and export ornamentals. Phytopathology 97: S148 (Abstract; Oral Symposium Presentation)
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