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United States Department of Agriculture

Agricultural Research Service

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Location: Molecular Plant Pathology Laboratory

2013 Annual Report

1a. Objectives (from AD-416):
Objective 1: Develop and use assays to characterize the interactions of exotic citrus pathogens with their hosts or vectors. Objective 2: Determine the etiology of exotic diseases of citrus and the diversity of exotic citrus pathogens.

1b. Approach (from AD-416):
This research project is focused on huanglongbing (HLB) and the extremely invasive pathogen Ca. Liberibacter asiaticus and another potentially invasive but uncharacterized pathogen, a presumed virus that causes citrus chlorotic dwarf (CCD) in Turkey. The pathogen has not been described, but it is transmitted by a whitefly already widespread in the United States and thus has very high potential for invasiveness. We will conduct basic and applied research which will lead to improved understanding of the biology and etiology of these diseases and provide the basis for improved diagnostic and research methods. Microarray studies of sweet orange trees infected with Ca. Liberibacter asiaticus and other phloem-limited pathogens will lead to the identification of novel host genes associated with general disease responses triggered by phloem-limited pathogens and specific disease responses triggered by Ca. Liberibacter asiaticus. Recombinant antibodies developed by bacteriophage display will be used to develop diagnostic procedures for Ca. Liberibacter asiaticus and as research tools. We will analyze data obtained from deep sequencing technology to characterize the virus causing citrus chlorotic dwarf. This information will be used to develop PCR and serologically based assays for this pathogen. The products of this research project will improve both scientific knowledge of invasive pathogens of citrus and provide novel assays useful or their detection and management.

3. Progress Report:
The goal of this project ’Invasive Citrus Pathogens’ is to prevent the introduction or spread within the citrus industry of a number of graft-transmissable and invasive pathogens of citrus. Due to quarantine considerations this work is carried out at Beltsville, Maryland. We have developed and used assays to characterize the interactions of exotic citrus pathogens with their hosts or vectors. In the first, we have continued previous work with a library of antibodies against the critically important pathogen that causes citrus greening disease, ‘Ca. Liberibacter asiaticus’. We have purified about 30 of these antibodies and have used them in ‘tissue print’ assays to reveal the presence of the pathogen in infected citrus tissues. This has never been done before with this pathogen, and allows us to both detect the pathogen and study its distribution in infected citrus trees. This work is made possible by our unique library of antibodies against this pathogen. We have also improved the production process for the antibodies to make it more productive and efficient. We have continued our study of how several of the diseases of interest effect the expression of genes in infected citrus, and have identified families of genes that are differentially expressed in sweet orange trees infected by Citrus tristeza virus and ‘Ca. Liberibacter asiaticus’. We have continued with ‘deep sequencing’ of RNA in trees infected with Citrus tristeza virus and ‘Ca. Liberibacter asiaticus’. We are presently analyzing the data which will give a more precise understanding of how gene expression varies in trees infected by these two important citrus pathogens. Because these genes are plant genes, expression is likely to be more uniform and the resulting assays to reveal the pattern of plant gene expression in response to the pathogen may be more reliable. We also have worked on projects to identify novel pathogens of citrus and establish the causes of diseases of citrus. We have worked with citrus chlorotic dwarf, which turns out to be a novel virus. We discovered this by studying very small RNA molecules, which include the remains of the virus that was attacking the plant after they fight the pathogen. Our results differ in important ways from those of another group working with the same pathogen, and we are presently working to resolve the discrepancies. In cooperation with researchers at the University of Florida and at Ft. Detrick, MD we have characterized two viral pathogens that cause a disease called citrus leprosis. Although this disease is very important in South America it is not present in the United States. In the course of this research we have identified an entirely new virus associated with citrus leprosis in Columbia and determined the complete sequence of its genome. This virus is related to another virus characterized by researchers in Brazil. We have also determined the complete sequence of the genome of a third, completely different virus that produces identical symptoms of leprosis in infected citrus. Because of this work we are able for the first time to distinguish among these three viruses which formerly could not be distinguished.

4. Accomplishments
1. Recombinant antibodies aide fight against citrus greening. Ca. Liberibacter asiaticus,’ causes citrus greening disease which threatens the citrus industry in the United States. Antibodies against the pathogen, which would be uniquely useful for both diagnostic and research purposes, have not been available. ARS researchers at Beltsville, Maryland, have for the first time created a library of antibodies against this pathogen through the use of recombinant DNA technology. Some of these antibodies have been used to detect and visualize the pathogen in cross sections of leaves from infected trees, using a process called tissue printing. The technology makes it possible to detect and visualize the pathogen in infected plant tissue, and will lead to a better understanding of the interaction of this pathogen with its host.

Review Publications
Kuykendall, L.D., Shao, J.Y., Hartung, J.S. 2012. 'Ca. Liberibacter asiaticus' proteins orthologous with pSymA-encoded proteins of Sinorhizobium meliloti: hypothetical roles in plant host interation. PLoS One. 7:e38725.

Li, W., Teixeira, D.C., Hartung, J.S., Huang, Q., Chen, J., Lin, H. 2012. Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis. Journal of Microbiological Methods. 92:79-89.

Last Modified: 05/25/2017
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