2011 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 be 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.
Citrus greening, or huanglongbing, is the most serious disease of citrus, and today it threatens the Florida orange juice industry. This report documents the progress of three related research thrusts related to citrus greening. The pathogen, a bacterium, is injected inside the cells of an orange tree by an insect feeding on the tree. It stays in the infected tree and colonizes other cells eventually inducing tree decline and death. Other pathogens of sweet orange trees also colonize the same cells of the orange tree and produce similar symptoms. We have performed experiments to determine if the genes used by the host tree in response to a pathogen is the same or different among trees infected by the different pathogens. We have found that the expression of an orange tree’s genes is similar, but not identical, when the tree is infected by the different pathogens. We are presently analyzing the data and expect to determine the function of genes that are used in response to the different pathogens. In a separate project, we have developed antibodies against the pathogen that causes citrus greening. These antibodies have been made by a complex laboratory procedure, the result of which is a ‘library’ of millions of antibody molecules that will ‘stick to’ different ‘pieces’ of the pathogen. We are presently isolating antibodies that stick to the different pieces of the pathogen that we think may be important for either disease development or transmission by the insect. These antibodies will be important tools for scientific research. They will also lend themselves to the development of extremely rapid, sensitive and user friendly assays for this pathogen. These will be developed in the coming year through research agreements established with the private sector in the United States and abroad and with USDA-APHIS. The complete sequence of the genome of the pathogen has recently been obtained. This is very useful, but the information is locked in ‘biochemical code’, and is only as useful as the decoding used to interpret the content of the genome. We have carried out an extensive comparison of the code of the citrus greening pathogen with that of other closely related and better known and better characterized bacteria that live in association with plants. In this way we have gained an understanding of the tool kits used by the different bacteria, and in particular the tools that are unique to the citrus greening pathogen. This provides us with a better understanding of the biochemical machinery used by the pathogen to attack citrus and potential opportunities to disrupt the disease process.
Avijit, R., Ananthakrishnan, G., Hartung, J.S., Brlansky, R.H. 2010. Development and application of a hexaplex reverse transcription polymerase chain reaction for screening global Citrus tristeza virus isolates. Phytopathology. 100:1077-1088.
Hartung, J.S., Halbert, S., Pelz-Stelinski, K., Brlansky, R.H., Chen, C., Gmitter, F. 2010. Lack of Evidence of Transmission of 'Candidatus' Liberibacter Asiaticus Through Citrus Seed Taken From Affected Fruit. Plant Disease. 94(10):1200-1205.
Nunney, L., Yuan, X., Bromley, R., Hartung, J.S., Montero-Astua, M., Moreira, L., Ortiz, B. 2010. Population genomic analysis of a bacterial plant pathogen: Novel insight into the origin of Pierce's disease of grapevine in the U.S. PLoS One 5(11):e15488.