2007 Annual Report
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).
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.
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.
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.