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United States Department of Agriculture

Agricultural Research Service

Research Project: EXOTIC PATHOGENS OF CITRUS
2007 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.


3.Progress Report
A quantitative real time PCR assay for the citrus greening pathogen was validated for use in both diagnostic and research activities. The assay was also applied to quantify the pathogen in diseased citrus tissues in both the greenhouse and field. Two species of the greening pathogen were transmitted into periwinkle, a more suitable experimental plant than sweet orange. This will facilitate future research objectives. Extensive progress was made to determine whether the citrus greening pathogen can be transmitted vertically through true seed. Three hundred and thirty seeds from symptomatic fruit were germinated and assayed two times by quantitative PCR for the presence of the pathogen and symptom development was monitored. Progress was 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.

Laboratory work was completed to determine if synergistic interactions between the grapevine and the sweet orange subspecies of Xylella fastidiosa could be identified. No in vitro synergisms, as measured by population growth, were observed. Novel quantitative real time PCR assays were developed for the purpose of simultaneous quantification of the grape and citrus subspecies of X. fastidiosa in co-inoculated experimental plants. These assays were applied in initial stages of in planta research, where limited synergism between the strains was observed in experimentally inoculated periwinkle plants.

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.

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 and has not produced detectable levels of dsRNA in repeated assays. The only experimental host for this presumptive virus is various citrus trees. We have been attempting to transmit the virus to a species of tobacco by grafting. If successful, this would greatly facilitate further research on the virus.


4.Accomplishments
National Program 303 Plant Diseases Research Component:Identification and Classification of Pathogens

Evaluation of methods for the detection of Ca. Liberibacter spp. the causal agents of citrus greening disease.

Citrus greening or Huanglongbing disease, is the most serious threat to citriculture in the world today. It is caused by a gram-negative bacterium that has not been cultured. DNA-based detection methods have been reported, but they have not been systematically compared and shown to be effective and efficient for the three species of the pathogen. We compared seven different DNA-based methods. All were effective but some had different host specificities, and we were able to improve the sensitivities of these assays as compared to their original publications. Current quantitative PCR assays were 10-100 times more sensitive than the first generation PCR assays. One of these, developed by our team, can be used to detect all three species of the pathogen. Researchers, industry and regulatory officials must have reliable and well characterized assays for this pathogen in order to diagnose infected trees and certify young trees for planting. These assays will be necessary components of any recovery plan for the Florida citrus industry and will assist current efforts to limit the further spread of the disease in the United States.


5.Significant Activities that Support Special Target Populations
None


6.Technology Transfer

Number of patent applications filed4
Number of new commercial licenses granted1
Number of non-peer reviewed presentations and proceedings2

Review Publications
Li, W., Hartung, J.S., Levy, L. 2006. Evaluation of dna amplification methods for improved detection of candidatus liberibacter species associated with citrus huanglongbing. Plant Disease. 91:51-58.

Last Modified: 9/10/2014
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