Source: UNIVERSITY OF FLORIDA submitted to NRP
BIOLOGY, EPIDEMIOLOGY AND DEVELOPMENT OF METHODS FOR DETECTION AND SUPPRESSION OF CITRUS CANKER
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
0193477
Grant No.
2002-34446-12292
Cumulative Award Amt.
(N/A)
Proposal No.
2002-06054
Multistate No.
(N/A)
Project Start Date
Aug 1, 2002
Project End Date
Jul 31, 2003
Grant Year
2002
Program Code
[RA]- (N/A)
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
CITRUS RESEARCH & EDUCATION CENTER, LAKE ALFRED
Non Technical Summary
The exotic disease, citrus canker, periodically invades U.S. citrus and threatens to disrupt production and marketing of this important fruit crop. Laboratory, greenhouse and field research objectives are proposed to better understand the biology and spread of the disease and to develop practical and effective suppression tools to limit its impact on the U.S.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2010999108020%
2120999104030%
2120999110040%
4040999202010%
Goals / Objectives
1. Characterize disease spread; 2. Detection and characterization of Xac strains; 3. Bacterial survival and disinfection of plant and non-plant surfaces; 4. Survey and detection of canker by remote sensing; 5. Improved chemical control; 6. Genetic improvement of citrus; 7. Development of delivery of canker education to diverse audiences.
Project Methods
Quarantine, laboratory, greenhouse and field research will be conducted across an array of specific experiments, and the location of the research will be dependent upon the needs and the ability to work under existing quarantine regulations and access to canker organisms. Biology and epidemiology experiments will be established in quarantine facilities where the Asian strain of Xanthomonas is permitted for growth in artificial media or on plant material. Limited epidemiological and suppression experimentation will be conducted within the mobile containment laboratory occupied by UF, IFAS and USDA, ARS scientists within the containment area of infection in Southeastern Florida. Conventional laboratory and greenhouse facilities will be used to conduct relevant experiments on surrogate Xanthomonas strains which are non-pathogenic to citrus and thus exempt from the severe regulations associated with the pathogenic strains. Field research in Florida and the U.S. will be restricted to observational studies within the infected and regulated regions, and to a lesser extent, applied field experimentation on naturally infected trees. Development of methods for early detection and tracking of canker infections can likewise be accomplished under Florida field conditions. Research being conducted with other funding is evaluating citrus canker suppression or infection prevention under field conditions in natural field infection conditions in Southeastern Brazil, with on-site collaborators. This field program allows full-scale evaluation of materials that have advanced through preliminary trials and need to be tested under field pressure. Finally, studies relating to genetic mechanisms of resistance to citrus canker will involve lab, greenhouse, and field experimentation in conjunction with a well-established plant improvement program conducted by University of Florida and USDA, ARS researchers. Additional research details are provided within the discussion of ongoing research below, and more specific details of individual research projects will be provided in the IFAS solicitation and evaluation of specific sub-projects under this program. Educational activities will be developed in concert with the University of Florida, IFAS Extension Program, which has physical presence of faculty in all 67 of Florida's counties. These agents, specialists, and directors will team with the research community to evaluate the needs for publicizing research results, developing public education tools to assist in general understanding of canker as an invasive exotic disease and the need-for sanitation, containment and eradication. Florida Agricultural Extension State Major Programs (FL-108 Citrus Management, for example) will provide the focal point for these planning, developmental and delivery efforts. The IFAS External Relations organization also will lend assistance to planning efforts to reach important audiences with timely and science-based information. Advisory organizations that interface between IFAS and client groups will be engaged in needs assessment and development of appropriate delivery plans.

Progress 10/01/03 to 09/30/04

Outputs
Characterization of strains of Xanthomonas axonopodis pv. citri, using DNA fingerprints generated from primers for ERIC repetitive elements, led to the discovery of differential sequences for a leucine responsive regulatory protein (lrp) gene in two subgroups of strains with different host range on Citrus spp. DNA hybridization and PCR-based assays using different sets of primers were designed to detect the core as well as obtain the entire sequence of the lrp gene for several Xanthomonas species and pathovars. Higher variability was observed at the nucleotide sequence level than at the amino acid level among the different species and pathovars, revealing a selection pressure on the lrp gene presumably due to an essential role of the gene in bacterial metabolism. Moderate variability in the 3' and 5' domains was used to study relationships among different species within the genus Xanthomonas. Species of this genus isolated from citrus as well as other pathovars of X. axonopodis showed highly similar lrp sequences whereas other Xanthomonas spp., especially X. campestris, had sequences more dissimilar to X. axonopodis. Thus, the lrp gene sequence is useful to distinguish X. axonopodis pv. citri groups and promising for polyphasic taxonomic analysis of the genus Xanthomonas. Data from analysis of the lrp gene sequences supports the current concepts for classification of xanthomonads based on other approaches.

Impacts
Accurate and rapid diagnosis of citrus canker by polymerase chain reaction (PCR) requires appropriate DNA primers for the detection and identification of Xac. Existing sets of primers were inadequate for detection and identification of certain canker strains. New primers that detect all canker strains have been designed. Another important aspect for eradication of citrus canker is genotyping the strains from each canker outbreak to make inferences about their origin and spread in Florida. A novel protocol utilizing rep PCR elements was applied to identify the genotypes present in every outbreak of canker in Florida and for worldwide strains. Genotype identification of Florida strains has been forwarded to the Division of Plant Industry (DPI) and routinely applied for tracking and risk assessment of the strains in existing and new outbreaks of canker. Three genotypes were discovered indicating separate introductions of the exotic disease have occurred over the last 20 years and that most of the new outbreaks of canker are due to spread of the Miami strain. PCR tests are now routinely utilized by Florida's Division of Plant Industry in the diagnostic program for citrus canker

Publications

  • Graham,J. H., T. R. Gottwald, J. Cubero,1 and D. S. Achor. 2004. Xanthomonas axonopodis pv. citri: factors affecting successful eradication of citrus canker. Molecular Plant Path. 5:1-15.
  • Cubero, J. and Graham, J.H. 2004. The leucine responsive regulatory protein (lrp) gene for characterization of the relationship among Xanthomonas spp. Intern. J. Syst. Evol. Micro. 54:429-437.


Progress 10/01/02 to 10/01/03

Outputs
Although wind and rain splash/spray are known to be important factors in spread of citrus canker, the relative effects of different wind speeds, durations and rain types have not been quantified with respect to dispersal or infection by the bacterium. In Florida , a wind tunnel has been constructed to study the effect of wind and rain splash on dispersal of the citrus canker bacterium. Where possible these aspects of the epidemiology of the disease in the field the following will be assessed within the epidemic zone in South Florida:1) Duration of dispersal of the canker pathogen in both simulated and natural storm conditions; 2) Wind speeds (0-120 kph) that cause increased infection in the presence of inoculum under different rain splash (droplet sizes/intensities) conditions; 3) The phenomenon of leaf soaking in facilitating infection under varying wind/rain splash events; 4) Distance of dispersal from a source of inoculum in relation to droplet size containing bacteria; 5) Effect of damage (insect and physical) on infection under varying wind/rain splash events.

Impacts
A thorough knowledge of the factors that allow infection and dispersal of the pathogen will contribute to developing a fundamental understanding of the disease epidemiology and will pave the way to a better informed and rational approach to its management and eventual eradication in Florida.

Publications

  • No publications reported this period


Progress 08/01/02 to 07/31/03

Outputs
OUTPUTS: Dr. Browning is no longer a faculty member at the University of Florida. Therefore a Final report canoot be completed for this project. Please terminate this project. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Dr. Browning is no longer a faculty member at the University of Florida. Therefore a Final report canoot be completed for this project. Please terminate this project.

Publications

  • No publications reported this period