2008 Annual Report 1a.Objectives (from AD-416) Determine genetic, epidemiological, biochemical, biological, and serological characteristics of Citrus Tristeza Virus and of the exotic pathogens to improve citrus productivity and to support regulatory actions. Determine nature of host-pathogen interactions, especially as related to host resistance. The following objectives are being proposed for the FY08 funding cycle: This project is to provide a technology and a service that will complement laboratories involved in development of methods to control the citrus canker and HLB bacteria, and development of citrus trees resistant to these diseases. We will provide a tool and a service to those laboratories to allow quick screening of potentially useful genes in citrus trees. 1)Improve the transient expression vector based on citrus tristeza virus (CTV)to be able to express more than one foreign gene or peptide simultaneously. The foreign gene products can then be expressed systemically in small citrus trees and examined for in vivo efficacy. The advantage of this system is that genes can be tested in a range of citrus varieties and species in cycles of weeks instead of years. This technology is required for rapid progress to be made in controlling these diseases. 2) Develop effective agro-inoculation of citrus plants with the vector. Presently, methods to infect citrus trees with recombinant CTV are time consuming and labor intensive. 3) Screen promising antibacterial peptides against citrus canker in citrus trees under simulated field conditions. 1b.Approach (from AD-416) Develop a coordinated, multiphased research effort on Citrus Tristeza Virus and other exotic citrus diseases in Florida with University of Florida (Lake Alfred, Gainesville) and ARS personnel (Ft. Pierce and Beltsville). 3.Progress Report This project is related to Objective 3 of this in-house project: Develop or improve comprehensive integrated disease management strategies. Citrus Tristeza Virus (CTV) and Other Exotic Citrus Diseases. Two experimental approaches were taken to investigate control of citrus canker and huanglungbing (citrus greening). Transient expression vectors based on CTV and tobacco mosaic virus (TMV) were built to produce and test antibacterial peptides in plants. Ten different antibacterial peptides were chosen to be expressed with or without leader peptides to export the peptides outside of cells. We also transformed and expressed the ten peptides with and without leader peptides in tobacco plants and tested efficacy against Psuedomonas syringae pv tabaci 11528. None of the peptides without leader sequences had significant activity against P. syringae, but five of the ten peptides with leader sequences strongly suppressed multiplication and spread of the bacterium. The same peptides with and without leader sequences have been put into the CTV-based expression vector and are being expressed in citrus for activity against the citrus canker and citrus greening bacteria. In 2008, continued to screen antimicrobial polypeptides for efficacy against citrus canker and citrus greening. Antimicrobial polypeptides have been built into transformation cassettes for resistant trees which are being regenerated. Because of the rapid spread of citrus greening in Florida, it has become evident that the juice industry might not be able to survive until transgenic trees can be tested and approved for use in the field. For that reason, we are adapting the citrus tristeza virus-based expression vector as an interim measure to protect trees in the field until resistant trees can be ready. We are applying to do field tests with USDA scientists at Fort Pierce next year as a first step in using this application in commercial citrus. Authorized Departmental Officer's Designated Representative monitoring occurred through repeated phone calls, emails and face-to-face meetings to discuss progress and fulfillment of project objectives.