Non Technical Summary
Huanglongbing disease (HLB) is one of the costliest diseases worldwide, and is now endemic in commercial citrus in Florida and Texas. Although trunk-injected oxytetracycline (OTC) shows promise in enhancing growth and yield of infected trees, growers are concerned about consistency of efficacy and about observations of phytotoxic accumulation in some parts of the canopy. Growers need information on how, where in the trunk, and when to inject to achieve optimal efficacy and reduce the risk of phytotoxicity. Based on existing knowledge of transpiration, xylem sectoriality, the movement of injected products, and the physiological effects of HLB, we will test three sets of hypotheses in all three citrus producing states to answer these questions using existing technologies and methods that would be immediately available to producers. To achieve better HLB management and reduced phytotoxicity risk, our objectives are to:1. Develop and test high-volume injection to improve distribution of injected materials in trees (how).2. Create and test a region- and species-specific method to improve distribution of injected materials by identifying stem areas connected to low-transpiration portions of the canopy (where).3. Improve production and tree health impact of therapeutics by testing a phenology-based approach to time injection (when).4. Disseminate knowledge to growers and injection companies.Successfully completing these objectives is expected to increase citrus yields in Florida and Texas within in duration of the project, and to prepare the California industry with best practices, should therapeutic injection become necessary. Additionally, delivery of future therapeutic products will also be enhanced.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
205	0999	1160	50%
205	0999	1020	50%

Knowledge Area
205 - Plant Management Systems;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1160 - Pathology; 1020 - Physiology;

Goals / Objectives
1. Develop and test high-volume injection to improve distribution of injected materials in trees (how).2. Create and test a region- and species-specific method to improve distribution of injected materials by identifying stem areas connected to low-transpiration portions of the canopy (where).3. Improve production and tree health impact of therapeutics by testing a phenology-based approach to time injection (when).4. Disseminate knowledge to growers and injection companies.

Project Methods
Objective 1Study 1.1 Test consistency of high-volume injection. Preliminary tests performed in PD-Vincent's lab suggest that > 1300 mL can be consistently injected in 7-year-old 'Valencia' sweet orange using a method that uses only gravitational pressure. We will inject up to 1,500 mL, recording the volume, with 1,000 mL as the high-volume target.Study 1.2 Test the effects of high-volume injection on distribution within the canopy and its subsequent horticultural effects.This study will test the hypotheses that:Higher injection volumes result in more broadly distributed injected materials.Broader distribution reduces the risk of phytotoxicity.Broader distribution improves efficacy against CLas and tree health effects of injection.Experimental design. This study will be replicated in Florida and in Texas. Treatments will be (1) standard (100 mL) injection and (2) high volume (1000 mL) injection in a randomized complete block design with 10 replicates. Each injection will contain the same quantity, the labeled rate (0.55g OTC tree-1) of OTC.Objective 2Study 2.1 Effect of injection location on distribution of injected materials.In California, we will undertake a comprehensive investigation into the efficacy of the injection technique using rhodamine dye. This research will focus on evaluating the effectiveness of this technique across the major citrus varieties in the state. The trials will be conducted at the Lindcove Research and Extension Center (LREC) on mature trees of 'Navel' sweet orange (C. x sinensis), 'Tango' mandarin (C. reticulata), and 'Limoneira 8A' lemon (C. x limon), all grafted onto 'Carrizo' rootstock (C. x sinensis x Poncirus trifoliata). We will test the effect of the trunk injection direction, through injecting the dye on one side in each of the four cardinal directions in each variety and track the dye into the canopy. We will use 16 trees for each treatment, ensuring that our study has sufficient data to draw meaningful conclusions.Study 2.2 Effect of injection location on disease, growth, and yield.Following preliminary results from Study 2.1, we will implement a field study in Florida, to assess the impact of injection location on CLas titer, tree health and yield. Growth of trunk cross-sectional area, canopy, canopy density, yield, and fruit quality will all be measured according to the same methods described for above for Study 1.2.Study 2.3 Branch-specific injection.During early stages of CLas infection, the uneven distribution of bacteria may cause sectional manifestation of HLB symptoms in trees (ie. one branch), while the rest of the tree remains asymptomatic. In this study, we will target symptomatic branches for OTC injection to reduce CLas titers, HLB symptoms, and minimize the distribution of bacteria from the symptomatic branch to the rest of the tree.Objective 3. Study 3.1 Focused study assessing the impacts of injection timing on sink activity.In this study we will use a range of closely spaced injection times to assess the effect of timing between injection and key phenological periods of fruit development. Treatments will be injections of OTC timed at differences of two-week intervals. We will inject the labeled rate (0.55g OTC tree-1) of OTC at 2, 4, 6, 8, or 10 weeks prior to the expected fruit drop, as well as a control with no injection. In addition to injecting OTC we will also inject fruits with a callose inhibitor 2-Deoxy-D-glucose (DDG) at low rate (0.1 mM) and high rate (5 mM)(Radford et al., 1998). Lastly, we will use a combination of OTC and DDG. We will also record the time fruit drop occurs in each treatment, as each year may vary. 10 replicates per treatment will be implemented in bearing trees of 'Duncan' grapefruit which is sensitive to HLB and 'Valencia' sweet orange in a commercial planting.Study 3.2 Multi-year field study to test the effects of injection based on phenological stage.To test the hypotheses that targeting specific phenological stages will result in improved efficacy of therapeutics to improve health of infected plants, we will develop multi-year studies in Texas and Florida (Figure 6). In Florida, we will implement a study using 'Valencia' type sweet orange trees and 'Sugarbelle' a relatively HLB-tolerant mandarin hybrid growing in commercial groves.We will inject the labeled rate (0.55g OTC tree-1) of OTC at different time points to target specific phenological stages:Bloom and flowering flush: Late January, 1 month before bloom is expected.Fruit set and spring root growth: Early March for Sugarbelle, or immediately after harvest for Valencia, 1 month prior to fruit set and the subsequent root flush.Vegetative shoot flush and physiological fruit drop: Mid-April, 1 month prior to expected shoot flush emerging the 2nd or 3rd week of May.Summer root flush and Stage II fruit growth: Early May, 1 month prior to the root flush that succeeds the May shoot flush and the acceleration to fruit expansion.Fall root flush and fruit maturation: Mid-August, 1 month prior to the fall root flush, which broadly coincides with Stage III fruit development.Control: No injection.Based on current observations from other studies, we have designed this study targeting 1 month prior to the start to the expected phenological stage, but this will be adjusted if Study 3.1 indicates a different optimal timing.FloridaExperimental design. In a commercial grove, we will implement the study as a randomized complete block. First observations of tree growth throughout the grove will be made, if gradients in growth or health across the grove are observed, these will be used as the blocking factor. Each experimental unit will consist of 5 treated trees. Because no edge effect is expected with trunk injection treatments, all trees will be used as data trees, however at least one buffer tree will be left between experimental units. The study will have at least 6 blocks. Measurements in this study will include various measurements of growth, CLas titer, tree health, and yield.TexasThe experiment in Texas will use the same experimental design. However, in this study, a smaller subset of measurements will be made: growth measurements, CLas titer, and yield measurements will be collected.Objective 4. The results from this project will not require a commercialization period, and thus will be available for immediate industry use. We have designed this project to have rapid impact on the industry. If the approaches tested are successful the effects on yields in Texas and Florida would be expected before the end of the project.4.1 Inform industry of preliminary resultsExtension efforts will be based on an existing network of channels and venues of communication with the citrus industry in Florida, Texas, and California. We will provide annual updates to growers in Citrus Industry Magazine, Citrograph, and through the All in for Citrus podcast, when available, as well as in presentations in public venues, such as the Citrus Expo, Citrus Show, the Florida Citrus Mutual annual meeting, and Citrus Institute in Florida, the annual Texas Citrus Mutual meeting and the bimonthly Texas Pest and Disease Management Corporation meetings in Texas, and the California Citrus Conference in California.4.2 Injection location and volume recommendationsOnce sufficient results have been developed (beginning as early as the end of Year 1), in addition to communication of recommendations through the channels described above, stand-alone extension documents will be produced in Year 3.4.3 Phenology-based injection timing recommendationsThe recommendations for injection timing will be disseminated in the same manner as those for injection location and volume, though the injection timing results are expected to take longer to develop, not being developed until the end of Year 2.