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United States Department of Agriculture

Agricultural Research Service



2008 Annual Report

1a. Objectives (from AD-416)
Objective 1: Define the distribution patterns of Xylella fastidiosa and Candidatus Liberibacter spp. in planta. Objective 2: Characterize host/pathogen interactions and potential control methods for Candidatus Liberibacter spp. and Citrus. Objective 3: Characterize antagonistic or synergistic interactions between strains of Xylella fastidiosa from sweet orange and grapevine. Objective 4: Establish the etiology of citrus chlorotic dwarf disease. Objective 5: Characterize, using microassay hybridizations and other approaches, host plant gene expression pattern in response to infections by 'Candidatus Liberbacter' spp. (citrus greening pathogens).

1b. Approach (from AD-416)
We will characterize novel or exotic pathogens of citrus that threaten the U.S. citrus industry. This work will include the determination of the etiology of novel diseases, characterization of the diversity present in a pathogenic taxon, the development of diagnostic tests for the pathogen, and the developent of information about disease processes and knowledge that may lead to control of the diseases. This procect will also maintain and develop an extensive collection of exotic pathogens of citrus for use by this project and collaborators from the U.S. and around the world. Detection methods will e generally PCR-based, including real-time quantitative PCR. Antibodies will also be developed. The etiology of novel diseases will be established by plant inocolulations both in the greenhouse in Beltsville and by field experiments in the countries where the disease exists. We will characterize responses of diseased plants to infections by citrus greening pathogens through the use of microarray analyses and other nucleic acid-based approaches and methodologies to identify genes whose expression is altered in infected plants compared to disease-free plants.

3. Progress Report
A quantitative real time PCR assay for the citrus greening pathogen was validated and used in both diagnostic and research activities. This work furthered objective 1 of NP303: Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens by making an essential tool available to the research public. The assay was also applied to quantify the pathogen in diseased citrus tissues taken from both the greenhouse, field and other experimental samples. Three hundred and thirty seeds from symptomatic fruit were germinated and assayed three times by quantitative PCR for the presence of the pathogen and symptom development was monitored. It appears that the citrus greening pathogen can not be transmitted vertically through true seed. Numerous attempts were also made toward the goal of determining whether or not the citrus relative Murraya paniculata is also a host for the greening organism. This is an important question because M. paniculata is the preferred host of the insect vector of the pathogen and is very widely produced, sold and planted as an ornamental plant in subtropical regions like Florida. If it is a host of the pathogen, control of the citrus greening disease will be much more difficult. Numerous grafts from infected citrus to M. paniculata have been made as well as several transmission attempts from diseased citrus using parasitic dodder plants as the vector. These experiments continue with the next phase being the assay of inoculated plants with the quantitative PCR assay for the pathogen. The application of our quantitative assay for Ca. Liberibacter asiaticus furthered objective 2 of NP303: Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors by providing basic biological data on the host pathogen interaction. Research on the population structure of X. fastidiosa in Costa Rica was completed. In this work strains of the pathogen from sweet orange, coffee and grapevine were expensively compared with reference strains from the same hosts isolated in Brazil and the United States. The results show that the strains found in Costa Rica are generally more closely related to strains from the United States, and are likely examples of independent adaptation and selection of indigenous strains for growth in introduced horticultural crops. This work furthered objective 1 of NP303: Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens by characterizing the biological diversity present in an important pathogen of citrus. Research to characterize the cause of citrus chlorotic dwarf continued. Results indicate that the agent is a virus, though the agent is apparently not stable during purification attempts. Experiments have continued to transmit the presumed virus to a different plant host. If successful, this would greatly facilitate further research on the virus. This work furthered objective 1 of NP303: Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens by characterizing an unknown pathogen of citrus.

4. Accomplishments
1. Genetic diversity of citrus canker pathogens preserved in herbarium specimens. Citrus bacterial canker (CBC) caused by Xanthomonas axonopodis pv. citri (Xac) was first documented in India and Java in the mid 19th century. Since that time the known distribution of the disease has steadily increased. Concurrent with the dispersion of the pathogen, the diversity of described strains continues to increase, with novel strains appearing in Saudi Arabia, Iran and Florida in the last decade. Herbarium specimens of infected plants provide an historical record documenting both the geographic distribution and genetic diversity of the pathogen in the past. However no method was available to assess the genetic diversity within these herbarium samples. We have developed a novel method and applied the method to characterize the diversity present within CBC populations documented as herbarium specimens over the past century. IES is based on the specific amplification of junction fragments that define insertion events. The potential for IES in current forensic applications is demonstrated by finding an exact match of pathogen genotypes preserved in herbarium specimens from Japan and Florida, demonstrating the source of the original outbreak of citrus canker in Florida in 1911. IES is a very sensitive technique for differentiating bacterial strains and can be applied to any of the several hundred bacteria for which full genomic sequence data is available. This research was performed under The Plant Diseases National Program (NP 303) component 1: Disease diagnosis: Detection, identification and characterization of plant pathogens.

5. Significant Activities that Support Special Target Populations

Review Publications
Li, W., Li, D., Tweig, E., Hartung, J.S., Levy, L. 2008. Optimized Quantification of Unculturable Candidatus Liberibacter Species Causing Citrus Huanglongbing in Host Plants by Real-Time PCR. Plant Disease. 92(6):854-861.

Montero-Astua, M., Saborio-R, G., Chacon-Diaz, C., Villalobos, W., Moreira, L., Rivera, C., Hartung, J.S. 2008. First report of Xylella fastidiosa in Avocado. Plant Disease. 92(1):175.

Li, R. and Hartung, J.S. 2007. Reverse Transcription-Polymerase Chain Reaction-Based Detection of Cherry green ring mottle virus and Cherry necrotic rusty mottle virus in Prunus spp. UNIT 16C.1 In: Current Protocols in Microbiology. New Jersey: John Wiley and Sons. p.1-9.

Turechek, W., Hartung, J.S., Mccallister, J.E. 2008. Development and Optimization of a Real Time Detection Assay for Xanthomonas fragariae in Strawberry Crown Tissue with Receiver Operating Characteristic (ROC) Curve Analysis. Phytopathology. 98(3):359-368.

Montero-Astus, M., Hartung, J.S., Aguilar, E., Chacon, C., Li, W., Albertazzi, F., Rivera, C. 2008. Genetic diversity of xylella fastidiosa isolates from Costa Rica, Sao Paulo and the United States of America. Phytopathology. 97(10):1338-1347.

Lacava, P.T., Li, W., Araujo, W., Azevedo, J., Hartung, J.S. 2007. The endophyte curtobacterium flaccumfaciens reduces symptoms caused by xylella fastidiosa in catharanthus roseusan endophytic bacterium from citrus. Journal of Microbiology. 45(5):388-393.

Li, W., Qijian, S., Brlansky, R., Hartung, J.S. 2007. Genetic diversity of citrus bacterial canker pathogens preserved in herbarium specimens. Proceedings of the National Academy of Sciences. 104(47):18427-18432.

Last Modified: 2/23/2016
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