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United States Department of Agriculture

Agricultural Research Service

Research Project: EXOTIC PATHOGENS OF CITRUS
2010 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.A new sub-objective will be added to the current Objective 2 in ARS Project no. 1275-22000-251-00D: Sub-objective 2A: Improve sensitivity and specificity of Huanglongbing diagnostics utilizing antibody based assays to supplement existing Q-PCR-based assays for use in host plant and insect diagnostic tests. (NP 303; Component 1; Problem Statement 1A).

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 PCR assay for the citrus greening pathogen previously developed by this project was used in several research activities. We have initiated work on a new objective to characterize changes in gene expression in citrus in response to infection with the citrus greening pathogen. An SCA has been established for this purpose with the University of Florida and the experimental plants have been established and inoculated. Experimental plants and methods of nucleic acid extraction and analysis have been established. We have also isolated RNA from citrus trees infected with Citrus chlorotic dwarf virus. Micro RNA produced by the host in response to the virus has been sequenced using Illumina deep sequencing technology. Bioinformatic analysis of the sequence data is underway to identify any novel virus present in the infected plants. Quantitative PCR assays for the citrus variegated chlorosis and Pierce’s disease pathogens (Xylella fastidiosa) previously developed by this project was used in research activities. Initial experiments monitoring populations of the pathogens in plants with dual infections are complete, but must be repeated. Further research on Xylella fastidiosa in sweet orange seed has begun, funded by a grant from USDA APHIS CPHST. We have also begun work on a project funded by outside funds to develop antibodies against the citrus greening pathogen. This includes recruiting additional staff and setting up several cooperative research agreements. This year we have made a ‘library’ of several million antibodies against infected insect extracts and we are now in the process of screening the library to identify individual antibodies with desired specificity.


4.Accomplishments
1. Citrus greening is a devasting disease that attacks citrus trees and is a major threat to the citrus industry. We have shown that the citrus greening pathogen, ‘Ca. Liberibacter asiaticus’ multiplies in parasitic plants known as dodder, and that the interactions with this host are very much like those with sweet orange. This develops an interesting experimental system for this pathogen. This information will be of interest to scientists looking for a cure to this disease.


Review Publications
Hartung, J.S., Paul, C., Achor, D., Brlansky, R. 2010. Colonization of dodder, Cuscuta indecorans, by Ca. Liberibacter asiaticus and Ca. Liberibacter americanus. Phytopathology. 100(8):756-762.

Last Modified: 12/20/2014
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