Non Technical Summary
Citrus greening or Huanglongbing (HLB) disease, caused by Candidatus Liberibacter asiaticus (CLas) in the U.S., is the most devastating citrus disease today and has invaded all production areas in the U.S. Developing effective and practical HLB control strategies is critical for the survival of the U.S. citrus industry. In this project, we propose to evaluate two novel systems to deliver antibacterial therapies into the citrus tree vasculature. Additionally,a multi-state teamwill use diverse multi-media outlets to disseminate project information to the stakeholders. We anticipate the project outcomes will have immediate and long-term impacts on the management of HLB. If successful, the project will result in the development of two unique and innovative delivery systems that are not mutually exclusive for delivering antimicrobials or any other products to citrus vasculature.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	0999	1160	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1160 - Pathology;

Goals / Objectives
Citrus greening or Huanglongbing (HLB) disease, caused by Candidatus Liberibacter asiaticus (CLas) in the U.S., is the most devastating citrus disease today and has invaded all production areas in the U.S. Developing effective and practicalHLB control strategies is critical for the survival of the U.S. citrus industry. In this project, our goal isto evaluate two novel systems to deliver antibacterial therapies into the citrus tree vasculature. The objectives are:1.Evaluation of citrus aerial root delivery system for uptake of HLB therapies.2.Evaluation of inarch graft injection system for delivery of HLB therapies. 3.Extension and outreach to inform stakeholders of project results.

Project Methods
Obj. 1. Evaluation of citrus aerial root delivery system for uptake of HLB therapies.Briefly, aerial roots will be induced on suitable branches or portions of the tree trunk through established air layering techniques. This involves peeling portions of the outer bark to expose inner meristematic cambial cells that will regenerate a new aerial root system at the exposed site. The area will be secured with a flexible wrap-around rooting pod/capsule filled with root medium/matrices that support growth and maintain/capture added moisture and liquids. The rooting pod has an access/inlet port that will be used to install the gravity feeder tubing. The growth rate, development, and health of the aerial root system will be monitored throughout the project. After the aerial root system is fully established on the trees,therapies will be delivered to the aerial roots using a controlled gravity-flow feeding system. Systemic uptake and translocation of the antimicrobial will be monitored by sampling distant shoots/phloem sap and below-ground roots. The therapies will be tracked in the different leaves and root tissue samples. Briefly, the tissues will be sampled at several time pointspost-delivery. All tissue samples will be flash-frozen in liquid nitrogen, followed by extraction and spectrophotometric or HPLC/ LCMS-based detection. The impacts of therapies on HLB tree health and CLas titers will be monitored. Tree health is measured based on canopy color and density.The CLas titers within the leaves and root samples will be estimated by qPCR amplification using standard primers specific to CLas.At harvest, fruit yield (lbs./tree) and fruit quality (size and color, % juice, brix, acid, etc.) parameters will be measured. Statistical analyses will be performed using ANOVA/post hoc tests and Student t-tests, as appropriate, to determine significant differences between controls and treatments.Obj. 2.Evaluation of inarch graft injection system for delivery of HLB therapies. After appropriate inarch rootstock/scion combinations are selected, lateralgraft cuts will be made on the inarch rootstock donor and inserted in an area on the acceptor tree trunk above its rootstock graft union. The inarched materials are held together by grafting tape and allowed to form a vascular union. All grafts will be monitored for successful formation (~2-4 months). After fully establishing the inarched grafts, they will be used as delivery conduits for HLB therapies.The therapies will be injected into the inarches using smaller injectors like ChemJetor AvoJect, suitable for the smaller size/diameter of the inarch rootstock trunk diameters.Leaf tissues from different parts of the tree will be sampled to evaluate systemic uptake and translocation of the therapies at various time points after injection. For residual determination, leaf tissues and fruits will be sampled at harvest. All tissue samples will be flash-frozen in liquid nitrogen, followed by extraction and spectrophotometricor HPLC/LC-MS analysis. Simultaneously, the impact of the therapies on HLB symptoms, CLas titers, tree health (canopy color, density), fruit yield (lbs./tree), and fruit/juice quality (size and color, % juice, brix, acid, etc.) parameters will be monitored.All orchards follow management practices, nutritional programs, and psyllid control measures standard for the region. Fruits will be destroyed after harvest per crop destruct protocols for non-registered product testing. Statistical analyses will be performed using ANOVA/post hoc tests and Student t-tests, as appropriate, to determine significant differences between controls and treatments.Obj. 3.Extension and outreach to inform stakeholders of project results. We will leverage severalmeans to engage the stakeholders (growers, industry, and commodity group members)who will be informed of the project findings. Furthermore, our team is affiliated with regional citrus research and extension centers in TX, FL, and CA and regularly interacts with growers to provide HLB disease management recommendations from this study and organize field days and workshops to disseminate the results and benefits of new delivery technologies.We will disseminate project outcomes via oral presentations, posters, peer-reviewed publications, and information booths during grower events in TX, FL, and CA.The presentations/workshops will be tailored to the target audience and will include information on the practical approaches and promising technologies revealed in this project. In addition, we will utilize leading industry newsletters and magazines, such as Citrograph, Citrus Industry, Fruit Gardener, Florida Grower, and respective University Extension Newsletters, to disseminate project highlights periodically.In addition to the target audience-driven presentations, the project findings and updates will be shared through factsheets, brochures, and PowerPoints.Project updates and results will also be shared with various citrus/HLB websites, andprepare blogs for the California Cooperative Extension. The blogs aim to summarize ongoing HLB projects to diverse stakeholders worldwide. We will also provide updates via Twitterand engage social influencers to reach out to end-users who are more active on social media. All the online portals will be updated periodically. Furthermore, we will share the content with our university news and communications teams, local news channels, and newspapers.