Characterization and Management of Citrus Pathogens Transmitted By Phloem-feeding Insect Vectors

The three pathogens in this Project Plan cause diseases in citrus ranging in severity from tree death; greatly reduced fruit quality and production; and differential symptoms depending on host cultivar and pathogen strains. The pathogens are CLas; presumed causal agent of Huanglongbing (HLB) (aka citrus greening); CTV, causal agent of tristeza quick decline (QD) and stem pitting (SP); and S. citri, causal agent of citrus stubborn disease (CSD). Key characteristics shared among these pathogens include: i) phloem-association; ii) transmission by phloem-feeding hemipterans; iii) graft-transmissible; iv) incurable by practical means; and v) management practices include removal of infected trees to reduce inoculum and limit pathogen spread by insect vectors. This project plan is designed to evaluate mixed infections of these pathogens on citrus production and pathogen diagnosis, and explore improved management of these diseases. To manage HLB, research will explore using a modified genome of a mild California recombinant CTV (CA-rCTV) isolate to deliver antimicrobial peptides (AMPs) and induce RNA interference (RNAi) against CLas and ACP, respectively, in inoculated plants. Information from this research will assist regulatory agencies, growers, diagnostic laboratories, and integrated pest management practitioners to significantly improve citrus disease management. This research also will improve disease diagnostics and lead to a better understanding of CTV cross-protection. During the next five years, focus will be on the following Objectives and Sub-objectives. Objective 1: Identify the genetic diversity of ?Candidatus Liberibacter asiaticus?, Citrus tristeza virus, and Spiroplasma citri in California and their impact on epidemiology, disease synergism, cross protection, and diagnosis. ? Subobjective 1A: Capture pathogen targets from field trees and/or insect vectors. ? Subobjective 1B: Examine phenotypes of California strains of CTV, S. citri, and CLas and improve pathogen detection. ? Subobjective 1C: Examine cross-protection within and between genotypes of CTV. ? Subobjective 1D: Examine if disease synergism occurs when CLas co-infects citrus with CTV and/or S. citri. Objective 2: Develop and evaluate the potential of mild California Citrus tristeza virus expression vectors for delivery of antimicrobial peptides and RNAi for control of HLB and its respective psyllid vector. ? Subobjective 2A: Develop a CA-rCTV infectious cDNA clone. ? Subobjective 2B: Examine phenotype and stability of the CA-rCTV singly and in mixed infections with California wild-type CTV isolates. ? Subobjective 2C: Incorporate AMPs against CLas and RNAi against ACP in the CA-rCTV vector. ? Subobjective 2D: Develop the CA-rCTV as a virus-induced gene silencing (VIGS) vector to downregulate citrus host genes to ameliorate HLB disease symptoms.