Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 216 - Integrated Pest Management Systems | 999 - Citrus, general/other | 1160 - Pathology | 100% |
Candidatus Liberibacter asiaticus (CLas) is a phloem-limited, fastidious bacterium that is associated with the most serious citrus disease Huanglongbing (HLB). In most countries affected by HLB, the bacterium is vectored by the Asian citrus psyllid (ACP), Diaphorina citri. Worldwide, the ACP and HLB have spread to most citrus growing regions. In the US, this disease threatens the future of Florida's annual $9 billion industry and the pathogen and vector are continuously spreading in other major production areas in Texas and California.With the devastating impacts of HLB on the citrus industry and no "silver bullet" in sight, growers are seeking alternative solutions to reduce CLas titer levels in trees and prevent disease-induced decline. Controlling HLB presents an unprecedented challenge to both the industry and scientific communities. Efforts to control psyllid and bacteria levels in Florida using chemicals are not successful resulting in most trees in Florida being infected. Current management options for HLB are limited and heavily rely on the application of insecticides for controlling psyllid populations, and more recently, bactericides (i.e., oxytetracycline and streptomycin) in an attempt to suppress or kill CLas in the citrus phloem. However, insecticide resistance among psyllid populations and limited success in delivery of bactericides through the leaves has rendered these strategies ineffective. In addition, there is great threat to the environment and to beneficial organisms.Thus, there is a critical need to develop immediate and sustainable alternative measures for controlling this disease and its vector. The development of such tools is hampered by a lack of knowledge regarding the application method that can effectively deliver potential therapeutics into the citrus phloem. Development of a novel delivery method will have a substantial impact on our ability to design new ways to control CLas in planta and/or in its insect vector, and eventually stop HLB and spread by ACP. To address this challenge, we proposed to develop a practical delivery device that growers can install on existing equipment for applying therapeutics to citrus trees. In the long term, this system can be designed for adaptation to other disease and pest control applications.Our project goal is to develop an automated delivery system (ADS) that will effectively deliver liquid materials (including bactericidal materials, microbial metabolites, RNAi, and other chemicals and biologicals; for simplicity will be referred to as "therapeutics") to citrus phloem through tiny openings in the trunk for improving plant health and performance. The ADS will allow application of therapeutics against plant vascular pathogens, specifically CLas, through trunk and major limbs of a citrus tree. The ADS will be designed, built and tested with an experienced team of Citrus Pathologists, Physiologists, and Horticulturists, Ag. Engineers and Economists and will deliver therapeutics directly to the plant vascular system by facilitating the uptake through the tree's natural hydraulic conducting capacity without causing harm to the tree. This will contribute to an efficient integrated pest management (IPM) for CLas and ACP that will supplement or may even replace foliar sprays.System development will be integrated with the study of tree physiology, specifically vascular transport of liquid materials through the citrus tree to optimize time and mode of delivery. This four-year project addresses the Citrus Disease Sub-committee Priority 1, 3 and 4: Therapies to prevent or suppress CLas bacteria within trees; Improve productivity of infected trees, including root health; and Systems for delivery of therapies into the phloem of citrus trees, including the root system.Our Project Objectives are:Design and development of an automated and economically feasible system to efficiently deliver HLB-therapeutic materials (including, but not limited to, bactericides) to citrus trees.Deciphering the path of citrus vascular transport for uptake, movement, and distribution of therapeutic materials throughout the plant.Optimization of therapeutic application through delineation of daily and seasonal vascular transport dynamics of citrus trees.Evaluation of the ADS in field-grown citrus trees.Evaluation of the economic feasibility of adopting the ADS and comparison of its benefits with currently used disease management strategies in Florida, Texas and California.Development and implementation of an energetic outreach and extension program.In summary, our project will focus on development of a new system of delivery that efficiently and homogenously delivers any type of liquid material with the potential to suppress or cure HLB or other diseases into the vascular transport system of citrus trees. Specifically, we will introduce the materials through numerous small punctures created by multiple small needles surrounding the tree trunk. Numerous openings in multiple locations around the trunk will ensure the even distribution of materials within the vasculature, contrary to single-needle injection. Injury and/or harm to the tree, particularly the destruction of vessel elements at the application site, will be minimized by the smaller size of the needles and the exclusion of high pressure for delivery of material. Rapid delivery will be ensured by using a large number of needles to create numerous openings through which the material will enter into the vasculature. Uptake and transport of the liquid will occur through the tree's natural hydraulic conducting capacity. The latter will be held in a reservoir surrounding the application site and allowing uptake of the material according to the trees natural transport capacity. Automation of the system will facilitate application and greatly reduce any labor-associated costs. We will also conduct detailed investigations on the anatomy and physiology of citrus trees with emphasis on xylem vessel arrangement, daily and seasonal patterns of water conductance, and movement of materials to and within the phloem.
Our project goal is to develop an automated delivery system (ADS) that will effectively deliver liquid materials (including bactericidal materials, microbial metabolites, RNAi, and other chemicals and biologicals; for simplicity will be referred to as "therapeutics") to citrus phloem through tiny openings in the trunk for improving plant health and performance. The ADS will allow application of therapeutics against plant vascular pathogens, specifically CLas, through trunk and major limbs of a citrus tree. The ADS will be designed, built and tested with an experienced team of Citrus Pathologists, Physiologists, and Horticulturists, Ag. Engineers and Economists and will deliver therapeutics directly to the plant vascular system by facilitating the uptake through the tree's natural hydraulic conducting capacity without causing harm to the tree. This will contribute to an efficient integrated pest management (IPM) for CLas and ACP that will supplement or may even replace foliar sprays.System development will be integrated with the study of tree physiology, specifically vascular transport of liquid materials through the citrus tree to optimize time and mode of delivery. This four-year project addresses the Citrus Disease Sub-committee Priority 1, 3 and 4: Therapies to prevent or suppress CLas bacteria within trees; Improve productivity of infected trees, including root health; and Systems for delivery of therapies into the phloem of citrus trees, including the root system.We have established project metrics in the form of milestones that are be expected to be met by the project members at the short, medium and long term:
Our main approach will be to use the trunk of citrus plants as gateway for introducing potential therapeutics into phloem tissues where CLas resides. The automated delivery system will facilitate introduction of therapeutics through numerous small openings created by puncture devices (i.e., small needles or blades) to the tree trunk, scaffold branches, or roots. The therapeutics will be deposited into a reservoir enclosing the openings, allowing uptake through the tree's natural hydraulic conducting capacity without causing harm to the tree. As it is a common misconception that materials injected into a tree will immediately reach the phloem, we will also delineate the exact path of uptake and distribution of liquid materials through the citrus tree's conducting system. This will be done initially through tracking of dyes in stem sections, followed by tracking of therapeutics with fluorescent microscopy, spectrophotometry, and/or liquid chromatography. We will also determine the time of day and season that are most suitable for effective delivery and distribution of therapeutics in the plant, which will be verified by analysis of CLas titers through quantitative PCR (qPCR) and assessment of tree health and productivity. This project will not only provide a practical delivery system that growers will be able to install on existing farm vehicles but also provide new information on the best and most effective time of application. We plan to accomplish the goal of this application by pursuing six specific objectives:1) Design and development of an automated and economically feasible system to efficiently deliver HLB-therapeutic materials (including, but not limited to, bactericides) to citrus trees.2) Deciphering the path of citrus vascular transport for uptake, movement, and distribution of therapeutic materials throughout the plant.3) Optimization of therapeutic application through delineation of daily and seasonal vascular transport dynamics of citrus trees.4) Evaluation of the ADS in field-grown citrus trees.5) Evaluation of the economic feasibility of adopting the ADS and comparison of its benefits with currently used disease management strategies in Florida, Texas and California.6) Development and implementation of an energetic outreach and extension program.In ongoing experiments, we are using simple dyes to track the success of delivery to citrus trees and monitor transport and spread using different delivery methods. Our novel method delivers liquid materials through numerous small punctures opened by tiny needles covering an area of the trunk that is considerable larger than single injection sites from a reservoir (i.e., a funnel or latex balloon mounted on the trunk) covering the application site. This method is a proof of concept and is the principal working mechanism of the proposed delivery system (Automated Delivery System, ADS), henceforth will be referred as Needle Assisted Trunk Infusion (NATI).In initial experiments, 1 ml of rhodamine (1%) was applied onto trunks of grafted and non-grafted young citrus plants grown in the greenhouse using NATI. In one-year-old seed-grown citrus seedlings treated with NATI, a visible red color, indicative of rhodamine uptake and movement, was detected in the upper-most leaves as early as 30-60 min after application, and increase in color intensity was observed 24 hours post-application. Similar results were observed on two-year-old grafted Valencia plants in the greenhouse that received rhodamine by NATI 48 hour-post-application.Additionally, trunks of Valencia plants of similar size and age received rhodamine by NATI either through one or two application sites to facilitate delivery and distribution of more dye in plants. There was noticeable difference in intensity of red coloration between the leaves on plants receiving NATI through one or through two sites on the trunk, the latter being more intense. These differences were more noticeable when plant tissues including leaf petioles and roots from these plants were dissected and observed under a digital microscope. The occurrence of dye in the roots hours after trunk application clearly demonstrates that materials, after being moved upwards through the xylem, are able to move into the phloem and come in contact with CLas. Finally, plants treated with NATI have not shown any stress signs during first two weeks after treatments in which openings on the trunk were completely healed, and the phytotoxicity that was seen on plants having been subjected to trunk injection was not observed.Together, our results indicate that NATI is a viable method and can be used for delivering liquid materials into the citrus vasculature without any detrimental effect to plants. Citrus plants of different size, age, and cultivar, grafted or non-grafted can be treated with NATI as rhodamine dye used in our experiments was shown to cross the graft union and reach the top most leaves as well as roots of the plants. Furthermore, we were able to quantify this dye (and oxytetracycline) by using a fluorescent plate reader with a reliable standard curve (R2=0.99). This will allow us to compare efficiency of NATI technique as well as performance of the automated delivery system (ADS). The ability to track and quantify dyes and other materials will allow us to optimize concentration of liquid materials to be applied by ADS for most effective delivery (see objectives 1-4). In summary, our approach will focus on development of a new system of delivery that efficiently and homogenously delivers any type of liquid material with the potential to suppress or cure HLB or other diseases into the vascular transport system of citrus trees. Specifically, we will introduce the materials through numerous small punctures created by multiple small needles surrounding the tree trunk (NATI). Numerous openings in multiple locations around the trunk will ensure the even distribution of materials within the vasculature, contrary to single-needle injection. Injury and/or harm to the tree, particularly the destruction of vessel elements at the application site, will be minimized by the smaller size of the needles and the exclusion of high pressure for delivery of material. Rapid delivery will be ensured by using a large number of needles to create numerous openings through which the material will enter into the vasculature. Uptake and transport of the liquid will occur through the tree's natural hydraulic conducting capacity. The latter will be held in a reservoir surrounding the application site and allowing uptake of the material according to the trees natural transport capacity. Automation of the system will facilitate application and greatly reduce any labor-associated costs. We will also conduct detailed investigations on the anatomy and physiology of citrus trees with emphasis on xylem vessel arrangement, daily and seasonal patterns of water conductance, and movement of materials to and within the phloem.
Target Audience
Teaching materials, including PowerPoint presentations, video presentations, PDF handouts, news articles, and websites, were prepared for broad audiences, including citrus growers, grove/operation managers, advisory board members, industry representatives, consumers, state/federal regulators, faculty, specialist, staff, undergraduate and graduate students, postdocs, visiting scholars and scientists.
Changes / Problems
Work is still ongoing to eliminate the clogging of the needles of the ADS. We designed two additional prototypes for the end effector. Several field tests were performed with both prototypes, and numerous modifications were made. Although this problem of simultaneous needle removal has yet to be solved, we achieved some marginal success, and current prototype modifications aim to resolve this problem. No other major problems to report.
Training & Professional Development
In 2023, we provided hands-on training to three postdocs, two graduate and one undergraduate student, and five staff. We also trained numerous stakeholders, including growers, faculty, and industry representatives, who were interested in hands-on trunk injection of OTC.
Dissemination Streams
Our project results were disseminated by various activities, including scientific publications, hands-on training, field demos, presentations, seminars, extension media and outlets, and online activities through various websites. We took advantage of the well-established citrus grower and industry meetings in multiple states to engage with and educate citrus growers, industry representatives, and the public at large about the usefulness of trunk injection (and ADS's) to the citrus industry.
Next Reporting Steps
We plan to fine-tune the ADS, fix some of the technical issues, and conduct OTC injection experiments in the grove to assess its effectiveness and compare it with manual trunk injections. We will complete a cost-benefit analysis and finalize all manuscripts about these objectives. Of course, our extension and outreach activities will continue, and new results will be disseminated to stakeholders with our well-established extension program.
Target Audience
Teaching materials, including PowerPoint presentations, video presentations, PDF handouts, news articles, and websites, were prepared for broad audiences, including citrus growers, grove/operation managers, advisory board members, industry representatives, consumers, state/federal regulators, faculty, specialist, staff, undergraduate and graduate students, postdocs, visiting scholars and scientists.
Changes / Problems
Work is still ongoing to eliminate the clogging of the needles of the ADS. We designed two additional prototypes for the end effector. Several field tests were performed with both prototypes, and numerous modifications were made. Although this problem of simultaneous needle removal has yet to be solved, we achieved some marginal success, and current prototype modifications aim to resolve this problem. No other major problems to report.
Training & Professional Development
In 2023, we provided hands-on training to three postdocs, two graduate and one undergraduate student, and five staff. We also trained numerous stakeholders, including growers, faculty, and industry representatives, who were interested in hands-on trunk injection of OTC.
Dissemination Streams
Our project results were disseminated by various activities, including scientific publications, hands-on training, field demos, presentations, seminars, extension media and outlets, and online activities through various websites. We took advantage of the well-established citrus grower and industry meetings in multiple states to engage with and educate citrus growers, industry representatives, and the public at large about the usefulness of trunk injection (and ADS's) to the citrus industry.
Next Reporting Steps
We plan to fine-tune the ADS, fix some of the technical issues, and conduct OTC injection experiments in the grove to assess its effectiveness and compare it with manual trunk injections. We will complete a cost-benefit analysis and finalize all manuscripts about these objectives. Of course, our extension and outreach activities will continue, and new results will be disseminated to stakeholders with our well-established extension program. <br><br>
<br>What was accomplished under these goals? Objective 1 (Partially completed). We developed an Automated Delivery System (ADS) to efficiently and homogenously deliver liquid substances with disease- (not restricted to HLB) therapeutic potential to the vascular system of a citrus tree. A successful water injection was performed, injecting up to 250 ml in about a minute while evaluating the relationship between flow rate and pressure under different conditions. Details of the experiment have been submitted for review in a scientific journal. The ADS design and results from this experiment were also presented at the American Society of Agricultural and Biological Engineering (ASABE) conference. They received the presentation excellence award and the ASABE oral/poster competition award. Work is still ongoing to eliminate the clogging of the needle. We designed two additional prototypes for the end effector. Several field tests were performed with both prototypes, and numerous modifications were made. Although this problem of simultaneous needle removal has yet to be solved, we achieved some marginal success, and current prototype modifications aim to resolve this problem. We automated the extension and retraction of the end effector by the positioning arm, which underwent several changes and was finally completed in October 2023. Currently, sensors, control system architecture, and hardware are being tested and developed for the positioning system. Objective 2 (Completed). Replicated greenhouse experiments were conducted to assess and optimize the uptake and path of liquid materials in the vasculature of healthy and infected citrus trees. Data was collected and used to optimize the delivery technique (trunk infusion and/or injection) and its adaptation by the ADS. Objective 3 (Completed). We studied daily and seasonal patterns of dye uptake and vascular transport in field-grown grafted citrus trees. We used trunk injection and different commercially grown citrus trees of different ages and measured vascular uptake and transport. Based on the knowledge gained from these measurements, we determined the optimal time of the day and seasons for applying therapeutic materials for trunk injections and, ultimately, with the ADS. Objectives 2 and 3 are now accomplished and have already been published (Archer et al. 2022 and other peer-reviewed papers and proceedings in 2022 that are available upon request) with the following major outcomes: •Determined the morning hours and spring and/or fall be optimum times for trunk-injecting citrus trees with minimal injury to the tree. •Developed a new Europium-based detecting and tracing technique for oxytetracycline (OTC) in citrus trees (Hijaz et al. 2021). •Determined that OTC could be injected into citrus as an effective therapeutic, which was uptaken, transferred in the trunk, and distributed in the tree. This was evidenced by reducing the CLas titers in injected trees, reducing the fruit drop, and improving tree health, fruit quality, and yield in Valencia, Hamlin, and Duncan trees. •Screened numerous other potential therapeutics and identified a couple of those showing encouraging results, including gibberellic acid (GA) and SAR-inducers that could be used when OTC could not be injected in the groves. We will continue to work on this to develop more comprehensive integrated pest management (IPM) for effective HLB control in the citrus groves. •The knowledge gathered from greenhouse and field experiments conducted over the last five years allowed us to demonstrate that OTC effectively controls CLas in mature trees if injected immediately after harvest in April-May and/or September-October. Moreover, we also showed stakeholders that when OTC is used in the hot summer months (June through the end of August) or with high doses, it could substantially injure trees. •Finally, our studies in this project (and other studies) showed that trunk injection of OTC could help citrus growers restate tree health in the short term until more effective and sustainable control of HLB with resistant citrus cultivars becomes available. Our findings provided additional evidence on the effectiveness of OTC injection, and we believe that our efforts in educating a wide range of stakeholders on the subject also helped accelerate the issuance of the special local use permits for OTC in citrus groves in Florida. Indeed, our research and extension activities have contributed to the approval of three registrations for using OTC injections in Florida citrus. As a result, millions of trees have been injected this year (30-50% of the active citrus acreage in Florida) with an economic impact of millions of dollars. Objective 4 (Partially completed). We successfully injected water (and various dyes) in multiple citrus trees of varying ages and collected pressure and flow rate data during water injection. We are conducting a field test to determine the flow rate and pressure for injecting OTC into mature orange trees at varying needle penetration depths. We are also resolving the ADS's field capacity and fuel efficiency. Objective 5 (Partially completed). We have developed the partial budgets for OTC trunk injections in orange production. We are currently finishing an extension document (UF-IFAS EDIS document) that summarizes the outcomes from the budgets and discusses initial findings from producers. We are finalizing a choice experiment for the survey respondents to understand the structure of an ideal cost-support program for a citrus producer. For example, the experiment would show whether a respondent preferred a cost-share program to a grant or loan to support their trunk injection operations. The economics team also contributed a section on the economics of antibiotics in fruit production to a paper submitted by Dr. Batuman, which is under the R&R phase at Phytopathology journal. Objective 6 (Ongoing). We developed outreach activities (seminars, hands-on workshops, and field day demonstrations) and materials (handouts, factsheets, websites, videos, magazines, media, and blogs) to engage and update stakeholders on research progress and training. Additionally, we took advantage of the well-established citrus grower and industry meetings in Florida (Citrus Expo, Industry Annual Conference, International Citrus & Beverage Conference, Citrus Show, etc.), Texas (Texas Citrus Mutual Annual Meetings, Texas A&M Kingsville-Citrus Center grower events, etc.) and California (Citrus Research Board grower seminars, California Citrus Mutual, etc.) to engage with and educate citrus growers, industry representatives, and the public at large about the trunk injection (and ADS's) usefulness to the citrus industry. This was achieved with strong interactions between the research teams and the citrus industry. All team members with extension assignments communicated directly with growers, industry, and the public. We created educational events and engaged stakeholders throughout the project to inform citrus communities about the trunk injection (and ADS) and its positive impact on citrus tree health and the environment. Please note that these activities have been ongoing undisturbed since the initiation of this project in 2019. We must continue to train and educate growers on the workings of trunk injection, particularly this and in the next couple of years when the majority of Florida citrus growers (and soon Texas growers) are adopting and using trunk injection to apply OTC to their citrus trees in the grove. <br><br><b>Publications</b><br>
Target Audience
Teaching materials, including PowerPoint presentations, video presentations, PDF handouts, news articles, and websites, were prepared for broad audiences, including citrus growers, grove/operation managers, advisory board members, industry representatives, consumers, state/federal regulators, faculty, specialist, staff, undergraduate and graduate students, postdocs, visiting scholars and scientists.
Changes / Problems
As most COVID-associated difficult situations are being improved in 2022, and with a 12-month no-cost extension granted for our project (thank YOU), we expect to finalize the ADS to use in mature citrus groves in 2023. However, because some components of the ADS (e.g., needles) are having unavoidable technical problems due to breakage or needing further adjustment/improvements, it may impact our progress speed. Nevertheless, as we know the importance of the ADS for the citrus industry in Florida these days, we are concentrating most of our efforts on overcoming these issues as soon as possible. Therefore, we will work with more than a few suppliers for certain supplies (if they become available and the remaining resources permit) to speed up our progress. We expect to accomplish all project objectives and reach our goal, which is to provide an effective and affordable ADS that growers can use in their operation to control HLB by the end of 2023.
Training & Professional Development
We provided hands-on and virtual training to three postdocs, three graduate and one undergraduate student, and five staff. We also trained numerous stakeholders, including growers, faculty, and industry representatives, who were interested in hands-on trunk injection of OTC.
Dissemination Streams
Objective 6. Our progress was continuously communicated to various stakeholders in Florida, California, and Texas. Our in-person activities that were reduced due to COVID-19-associated restrictions in 2021 have increased back to normal in 2022. Nevertheless, we also continued our outreach through alternative online/virtual media as in the past years. Our project progress was presented at our Advisory Board meetings and at additional 15 grower seminars, 1 lecture, 14 international scientific conferences and seminars, 5 national scientific conferences, 7 lab-visit events (domestic and international visitors), and through numerous EDIS/industry magazines and TV/Web media report as well as during numerous individual educational and extension activities. We continued to disseminate our project progress through numerous online and in-person outreach activities such as virtual seminars, in-person and virtual lectures, as well as in-person hands-on workshops and greenhouse demonstrations with accompanying materials (handouts, factsheets, website, Animation videos, magazine, and media news articles). We engaged and updated stakeholders on research progress during grove visits and via phone and virtual meetings, texts, and emails. We presented our project at several virtual and in-person national and international conferences. This information was also disseminated to general audiences through the project webpage and emails (i.e., sending PDF files to meeting attendees).
Next Reporting Steps
We will continue progressing toward our goals, including modifying and developing the automated delivery system (ADS), testing it on mature citrus trees; gathering more information on OTC movement in citrus vasculature; finetuning the best technique, time, and amount to apply OTC (or other therapeutics when become available); and using effective education and extension. Our proposed objectives and development of an ADS are progressing as planned without any major challenges despite long delays and disruptions in supply and logistics-associated activities, as well as personnel resignations (and new hiring) caused by the COVID-19 pandemic. Project progress was demonstrated to a wide range of stakeholders in numerous seminars and through magazine and media reports, as well as other educational and extension activities. Data from 2022 studies were published in 13 peer-reviewed journal articles. Further modifying, testing, and improving the ADS by continuing to work on project objectives are already planned, and some of these activities have been ongoing non-stop. Results that have been gathered from numerous greenhouse and field trials will be validated by repeated and larger-scale field experiments, and numerous new trials will be set up to evaluate both the improved ADS and potential new therapeutics for controlling the HLB in citrus plants. <br><br>
<br>What was accomplished under these goals? Objective 1. We made several key modifications to the previous Automated Delivery System (ADS) prototype, including a new needle, end effector, and support structure. Material for the new needle (hardened 440C stainless steel) was selected to provide maximum strength against bending and machined as a single piece. The new design features a flat tip and holes placed radially along the circumference of the needle. This design is similar to the existing needle design for manual needle-based trunk injection. The needle also featured a slightly longer length (1 inch) and wider diameter (0.155 inches). The number of needles penetrating the stem per injection has been reduced from 4 to 2. To achieve the penetration depth for injection, we designed new actuators that provided the driving force for needle penetration by a hydraulic actuator (maximum push force: 1760 lbs.). The entire mechanism has been redesigned to resist the reactive forces at the injection point. The new support structure design is a custom-made telescoping actuator that is designed to adequately support the weight of the end effector and provide for precise positioning. The ADS is mounted on a utility-type vehicle via the support structure. A stepper motor enclosed in the telescoping frame controls the extension and retraction of the frame to position the end effector such that the opposite-facing needles are positioned radially across the stem. Needle penetration is achieved by the end effector clamp mechanism driven by two opposite-facing hydraulic actuators. The needles are inserted to a depth of 1 inch. The liquid material is then forced into the stem by a metering pump selected to supply injected fluid at a flow rate of 7 ml/s and a pressure of 300 psi. The only remaining part of this objective is to finalize the ADS and use it in several more experimental (and commercial) grove settings to determine its efficiency, which we plan to accomplish during the final (No-cost-extended) year of the project in 2023. Objectives 2 and 3. The goal of these objectives was to gain knowledge on principles including trunk injection, type of material that could be injected, time of injection (i.e., time of the day and best season), screening effective therapeutics, and finally, apply all this knowledge to trunk inject citrus trees to control HLB disease in the grove. These objectives are now accomplished and have already been published (see Archer et al. 2022 and other peer-reviewed papers and proceedings in 2022) with the following major outcomes: Determined the morning hours and spring and/or fall to be optimum times for trunk-injecting citrus trees with minimal injury to the tree. Developed a new Europium-based detecting and tracing technique for oxytetracycline (OTC) in citrus trees. Determined that OTC could be injected into citrus as an effective therapeutic, which was uptaken, transferred in the trunk, and distributed in the tree. This was evidenced by reducing the CLas titers in injected trees, reducing the fruit drop, and improving tree health, fruit quality, and yield in Valencia, Hamlin, and Duncan trees. Screened numerous other potential therapeutics and identified a couple of those showing encouraging results, including gibberellic acid (GA) and SAR-inducers that could be used when OTC could not be injected in the groves. We will continue to work on this to develop more comprehensive integrated pest management (IPM) for effective HLB control in the citrus groves. The knowledge gathered from greenhouse and field experiments conducted over the last four years allowed us to demonstrate that OTC effectively controls CLas in mature trees if injected immediately after harvest in April-May and/or September-October. Moreover, we also demonstrated to stakeholders that when OTC is used in the hot summer months (June through the end of August) or with high doses could lead to substantial injury to trees. Finally, our studies in this project (and other studies) showed that trunk injection of OTC could help citrus growers in the short term to restate tree health until more effective and sustainable control of HLB with resistant citrus cultivars becomes available. Our findings provided additional evidence on the effectiveness of OTC injection, and we believe that our efforts in educating a wide range of stakeholders on the subject also helped accelerate the issuance of the special local use permits for OTC in citrus groves in Florida. Indeed, on October 28, the Florida Department of Agriculture and Consumer Services granted TJ BioTech commercial product ReMedium TI (i.e., injectable OTC) a special local-need registration. Applications for its use are expected to begin post-harvest of the 2022-23 citrus season by our citrus growers in the groves. Objective 4. The goal of this objective was to evaluate the new ADS developed from Obj 1 above in citrus groves. This objective must be accomplished due to delays from unexpected technical challenges associated with engineering, fabrication, and logistics of obtaining new or available materials due to COVID-19-associated supply disruptions. Nevertheless, we tested the ADS on several different age mature trees in our experimental grove and improved its ability to inject liquid materials effectively. In these tests, we were able to redesign and improve needles, end effector, and support structure as outlined above in obj. 1. With more spare parts being ordered, we anticipate testing the ADS on mature tree trunks with different diameters and more rootstock/scion combinations in various grove settings in 2023. Objective 5. Since trunk injection will be used in citrus production in 2023, the economics team will now investigate manual trunk injection of OTC in Florida citrus production. Contractors and UF researchers are now contacted to assess the cost of manual trunk injection, including inputs and labor costs. These costs will be compared to automated trunk injection (i.e., ADS) costs to help assess the feasibility of truck injection for both methods. The next steps include contacting the California Citrus Research Board to get support for the grower's willingness to adopt survey, traveling to California to help develop relationships with citrus producers and specialists, and designing the wiliness to adopt survey. <br><br><b>Publications</b><br>
Target Audience
Teaching materials including PowerPoint presentations, news articles, and a website were prepared for broad audiences including citrus growers, grove/operation mangers, advisory board members, industry representatives, consumers, and state/federal regulators, faculty, specialist, staff, undergraduate and graduate students, postdocs, visiting scholars and scientist.
Changes / Problems
Florida Team: In the beginning of the Covid-19 pandemic in 2020, a few of our post docs, graduate students and staff members resigned from their positions, thus our project, like all other projects, had some setbacks. The team experienced delays in hiring new personnel related to COVID include hiring freezes, travel bands, and delayed visa appointments. However, new personnel were hired and began working on the project in January 2021. In 2021, more substantial problems were occurred with developing and continuously modifying the ADS prototype, which was heavily depending on outsource materials to be purchased and installed. And, again due to COVID, finding, manufacturing or shipping materials was very difficult, which caused unexpected delay in improving the ADS. Texas Team: The freezing temperatures that affected Texas February 14 to 20, 2021, caused significant damage to citrus trees in the area, causing the loss of all the unharvested fruit, defoliation of mature trees, dieback of branches, bark splitting and death of young trees. Due to the poor stage of trees, and the slow recovery, TX team was not able to initiate field studies until the summer months which will continue through 2022. California Team: Covid-19 has not directly limited an extension program in California. The extension program in California is limited by the project's objectives; developing a delivery method for a yet unspecified therapeutics. Therefore, extending information developed in this project is limited to UC academics and extension personnel interested in HLB mitigation. This is being done as updates through the Science for Citrus Health program as well as the continued with verbal project updates at the bimonthly Subtropical Work Group online meetings. A more complete series of seminars is scheduled to start for 2022 with individual presentations by the individual project PIs. We are hoping that all COVID-associated difficult situations will be improved in 2022, and with a hope of getting a 6-12 months no-cost-extension for our project will put us right back on track, and we will be able to accomplish our project goals in 2023.
Training & Professional Development
We provided hands-on and virtual training to four post docs, two graduate and one undergraduate student, and five staff. However, Covid-19 pandemic resulted resignation of several project personnel (i.e., OPS, post doc and graduate student). However, we were able to screen and hire new personnel for this project, and additional personnel will be hired and trained in 2022.
Dissemination Streams
Objective 6. Our progress was continuously communicated to a wide range of stakeholders in Florida, California, and Texas. Although our in-person activities were reduced due Covid-19 associated restrictions, we continued our outreach through alternative online/virtual media in 2021. Our project progress was presented atour Advisory Board meetings and at additional17 grower seminars, 1 lecture, 3 international scientific conferences and seminars, 9 national scientific conferences, 3 lab-visit events (domestic and international visitors) and through 9 EDIS/industry magazine and 3 TV/Web media report as well as during numerous individual educational and extension activities. We continued to disseminate our project progress through numerous online and in-person outreach activities such as virtual seminars, in-person and virtual lectures, as well as limited in-person hands-on workshops and greenhouse demonstrations with accompanying materials (handouts, factsheets, website, Animation videos, magazine and media news articles). We engaged and updated stakeholders on research progress during grove visits as well as via phone and/or virtual (i.e., Zoom, Skype and MS Teams) meetings and emails. We presented our project in several virtual as well as in-person national conferences. This information also disseminated to general audiences through project webpage and emails (i.e., sending PDF files to meeting attendees).
Next Reporting Steps
We continued progressing toward our goals including modifying and developing a prototype of automated delivery system (ADS), testing them on mature citrus trees; gathering basic information on dye and bactericide movement in citrus vasculature; identifying the best technique, time and amount to apply therapeutics; and developing an effective education and extension. Our proposed objectives and development of an ADS are progressing as planned without any major challenges despite long delays and disruptions in supply and logistics associated activities as well as personnel resignations (and new hiring) that caused by the Covid-19 pandemic. Project progress was demonstrated to a wide range of stakeholders in numerous seminars and through magazine and media reports as well as other educational and extension activities. However, most of the outreach and extension activities were presented via online (virtual) settings due Covid-19 pandemic-associated restrictions. Data from 2021 studies were published in eight peer-reviewed journal articles. Further modifying, testing and improving the NATI/ADS by continuing to work on project objectives are already planned, and some of these activities have been non-stop ongoing. Results that have been gathered from numerous greenhouse and field trials will be validated by repeated and larger scale field experiments, and numerous new trials will be set up to evaluate both the improved ADS and potential new therapeutics for controlling the HLB in citrus plants. <br><br>
<br>What was accomplished under these goals? Objective 1.In the first half of the year, the single sided pneumatic punching system prototype attached to the farm vehicle was modified to a two-sided punching system which clamped the tree from both sides, pushed the needles in and subsequently injected liquid at a high pressure. The prototype was successful in creating holes in the tree trunk and delivering high pressure liquid to the needles while avoiding the drawback of the previous prototype. However, there were a few issues with this prototype (e.g., the needles were not long enough to create deeper holes to inject liquid and the actuator used was big and bulky). In the second half of the year, the design was further modified to have a smaller hub with two 13 mm long needles, each aligned vertically. The hubs were fixed to two separate smaller pneumatic actuators that crimped at one end with a through hole drilled for release of pressurized liquid. The support structure for the actuators was also modified to push the clamping and punching mechanism forward. Objective 2. Greenhouse studies showed that NATI provides a more homogeneous delivery of dyes than the traditional trunk injection method in the citrus trunks. The NATI resulted in a higher concentration of the dye at wounded areas and a more uniform distribution throughout the plant. Across all the experiments, the dye movement occurred only vertically up in the plant, with minimal downward movement. In a repeated greenhouse study, NATI application of the antibiotic oxytetracycline (OTC) or a new class of antimicrobial compound, provided significant reduction of HLB symptoms and CLas titer in infected grapefruit trees. We also evaluated the efficacy of a SAR inducer Actigard, a new biocontrol agent, and a novel nanomaterial for their efficacy against citrus canker and HLB, respectively. However, none of them performed better than the water-treated control when applied by NATI. We developed a novel method for the analysis of OTC in the citrus sap, which was measured using the europium (Eu) method directly or after a cleanup step using solid-phase extraction (SPE). The results obtained by the Eu method were similar to the ELISA assay. Recovery of OTC from spiked sap samples purified by SPE was higher than 90%, while recovery from saps without SPE cleanup were nearly 100%. In addition, the Eu method was successfully used to determine OTC levels in the phloem and xylem sap of OTC-injected plants. The citrus tristeza virus (CTV) vector was used to show that NATI can deliver therapies into the phloem. We used NATI to deliver rhodamine into CTV-GFP infected plants, and tetracycline into CTV-RFP infected plants. In both cases, in addition to strong xylem labeling by the NATI delivered material, we also detected a yellow color in phloem cells, indicating that a fraction of the NATI delivered materials ends up in the phloem cells. Finally, NATI was also used to develop an easy agro-infiltration method for transient expression of DNA in citrus. With NATI pretreatment, we were able to easily and effectively express foreign genes in citrus leaves. Field Trial 1 -Wound responses to injection: Preliminary results indicate that 1) injection using medium to high pressure devices allows for rapid and effective delivery of certain compounds into vasculature, but some may also cause a damage to trunk, 2) injection of OTC can significantly reduce bacterial levels and lead to improvements in tree health, and 3) yield differences were noticeable for two seasons after one injection. Field Trial 2 -OTC and Imidacloprid injection in HLB-affected trees: In October 2020 and April 2021 trees were injected with OTC, imidacloprid, or water. Preliminary results indicate that 1) OTC injection can reduce bacterial titer and subsequently fruit drop, 2) imidacloprid injection can temporarily result in psyllid mortality, and 3) OTC residue in leaf, root, and fruit tissue is below threshold levels within 2 months after injection. Field Trial 3 -OTC injection in commercial sweet orange trees: In April 2021 and/or October 2021 two varieties of sweet orange trees in a commercial grove were injected with OTC or water. Here, we would like to analyze efficacy of OTC application either in spring and/or fall injections and determine effective OTC concentration to improve tree health and residues in different citrus tissues. Field Trial 4 -OTC injection in HLB-affected grapefruit: Like above, a trial has been going on in Duncan grapefruit trees. Field Trial 5 - investigating the role of gibberellic acid (GA) on tree health. We are applying GA with foliar or NATI and monitoring flush dynamics and oxidative stress alleviation. Field Trial 6 - assessing therapeutical effect of zinc (Zn) and potassium (K) in the field. Foliar application of Zn and K reduced fruit drop and improved yield by 15% in mature Hamlin trees, which resulted in an increase in harvested boxes per tree. The best dosage of both Zn and K has been determined, which is now being applied by NATI. Objective 3. 1) Greenhouse Conditions: When it comes to distribution and transport of dye, application during the morning (8 am) resulted in greater distribution and transport than applying at noon (12 pm) or later in the afternoon (4 pm). The stomatal conductance in the leaves was also higher during the morning (8 am), than at noon (12 pm) or later in the afternoon (4 pm). This means that the stomata were more open during the morning leading to higher transpiration-pull during the morning and possibly more uptake of material (dye or therapeutics) during the morning. We also evaluated the transport of the dye across different seasons throughout the year and found better uptake during the spring season, and least during winter. 2) Field Conditions: From July 2020 to May 2021 groups of trees were injected with water using the Chemjet tree injectors in both the scion and rootstock. We have also determined how water stress conditions leading to tissue damage depend on the time of the day. Preliminary results indicate that 1) the speed of uptake is associated with tree activity (stomatal conductance), 2) injection in the spring and summer results in more rapid uptake of water, and 3) injection ports in the scion heal more rapidly than injection ports in the rootstock. Objective 4. Multiple tests with the ADS were run on different mature Valencia and Grapefruit tree trunks at a higher punching pressure and a higher injection pressure of liquid and compared to ArborJet injector by checking the dye presence in the leaves. Compared to the ArborJet, the current ADS was not able to penetrate the tree trunk enough for the dye to travel through the xylem. Also, even after trying with multiple types of stainless-steel needles, the needles experienced some type of failure. As a result, the next iteration of the prototype would include needles made of a single piece of metal, fabricated and machined to meet the functional requirement. With the right depth of penetration of the needles and the diameter of the needles, the current system is expected to deliver the fluid at the right pressure. Objective 5. Since trunk injection is not used in citrus production, the economics team investigated manual trunk injection in Florida avocado production. Contractors and UF researchers were contacted to assess the cost of manual trunk injection contracts. While the inputs may not be appropriate for citrus, labor costs should be similar. These costs will be compared to automated trunk injection costs to help assess the feasibility of truck injection for both methods. The team has also contacted the California Citrus Research Board to get support for the grower willingness to adopt survey. Next steps include traveling to California to help develop relationships with citrus producers and specialists and designing the wiliness to adopt survey. <br><br><b>Publications</b><br>
Target Audience
Teaching materials including PowerPoint presentations, news articles, and a website were prepared for broad audiences including citrus growers, grove/operation mangers, advisory board members, industry representatives, consumers, and state/federal regulators, faculty, specialist, staff, undergraduate and graduate students, and postdocs, visiting scholars and scientist.
Changes / Problems
Nothing Reported
Training & Professional Development
We provided hands-on/virtual training to three graduate and one undergraduate student, and three staff. However, Covid-19 pandemic resulted resignation of several project personnel (i.e., OPS, post doc and graduate student). Thus, we are anticipating that new personnel for this project will be hired and trained in 2021.
Dissemination Streams
We disseminated our project progress through numerous online and in-person outreach activities such as virtual seminars, in-person and virtual lectures, as well as limited in-person hands-on workshops and greenhouse demonstrations with accompanying materials (handouts, factsheets, website, Animation videos, magazine and media news articles). We engaged and updated stakeholders on research progress during grove visits as well as via phone and/or virtual (i.e., Zoom, Skype and MS Teams) meetings and emails. We presented our project in two virtual national conferences. This information also disseminated to general audiences through project webpage and emails (i.e., sending PDF files to meeting attendees).
Next Reporting Steps
Further testing and improving the NATI/ADS by continuing to work on project objectives are ongoing. Results that have been gathered from numerous greenhouse and field trials will be validated by repeated and larger scale field experiments, and numerous new trials will be set up to evaluate both ADS and potential therapeutics for controlling the HLB in citrus plants. <br><br>
<br>What was accomplished under these goals? In 2020, we continued progressing toward our goals including modifying and developing a second and third prototypes of automated delivery system (ADS), and testing them on citrus trees; gathering basic information on dye and bactericide movement in citrus vasculature; identifying the best technique, time and amount to apply therapeutics; and developing an effective education and extension. Among the dyes/fluorescent tracers and a few bactericides (i.e., oxytetracycline, streptomycin and penicillin) with or without an adjuvant that we tested showed different patterns of uptake, movement and translocation in treated plants. Our proposed objectives and development of an ADS are progressing as planned without any major challenges despite some delays and disruptions caused by new regulations due the Covid-19 pandemic. Project progress was demonstrated to a wide range of stakeholders in numerous seminars and through magazine and media reports as well as other educational and extension activities. However, most of the outreach and extension activities were presented via online (virtual) settings due Covid-19 pandemic-associated regulations and restrictions on travels and gatherings. Data from these initial studies were also published in three peer-reviewed journal articles. Objective 1.(Ampatzidis, Albrecht and Batuman). · Drill-based second prototype automated delivery system (ADS) was developed and evaluated. The second prototype could penetrate the bark and the trunk up to a depth of 15 mm. The depth and the diameter of the hole could be adjusted and could be used for trees of all ages. Cleaner holes were observed because of the use of drills. The protype was versatile in terms of adjustment of the drill bit size, depth of penetration, and hole location. · Punching-based third prototype ADS was developed and evaluated. The third prototype replaced the drill-based system with a punching system. The target was to penetrate the trunk to a depth between 3-10 mm and work on trees of all ages. This system consists of a pneumatic cylinder as the power unit to provide fast and powerful thrusting motion. The diameter of the holes can be controlled using different hollow needles. This design provides the flexibility of using different sets of needles, different arrangement, and number of needles on the hub. It is versatile in terms of adjustment of the needle size, depth of penetration, and hole location. · Current and future work focusses on modifying the frame of prototype 3 and adding a pump, which would force liquid though the back of the (hollow) needle hub into the tree trunk. An improved version of this prototype is expected to be built by February 2021. Objective 2. (Batuman, Albrecht, Alferez Levy and Killiny). · Analysis of citrus vascular transport using NATI and different delivery methods: In the greenhouse and field, treated citrus plants showed various degrees of responses to applied materials. Among these, size of the wound and healing progress both were extended when pressure of injection was high and/or high concentrations of oxytetracycline (OTC) was used. We made following measurements and data were collected: measured differences in wound closure rates; determined the length of xylem occlusion and discoloration and the physiological impacts due to different injection materials and different methods of application; measured stem water potential and stomatal conductance; analyzed expression of stress-related compounds. Evaluated movement and retention of OTC, dyes and other substances in leaves, roots and other tissues; quantified change in CLas titer, flushing numbers, and psyllid colonization in response to treatments. Investigated susceptibility of citrus plants to different plant growth regulators (PGR), including auxin and homobrassinolides (HBr), when delivered by NATI and studied their effects on modulating fruit yield and quality. The role of the xylem in OTC translocation studied, and a novel method for quantification of OTC residues in different tissues was developed. This method was developed for rapid and more sensitive detection of OTC by complexing it with Europium in alkaline condition. The limit of detection for the developed method was about 0.1 ppm OTC in the final sample solution; ~ 4 µg g-1FW. A GFP-tagged citrus tristeza virus-based vector (CTV-GFP) was used for labelling citrus phloem cells in order to verify whether rhodamine (red) dye reached phloem cells after NATI application. Results indicated that while most of the dye were delivered into xylem, some also were delivered into phloem. Used citrus canker as a model pathogen to study effectiveness of NATI. This approach allowed us to develop a novel and much rapid method to evaluate effectiveness of NATI in delivering substances into citrus vasculature in young citrus trees. Objective 3. (Albrecht and Batuman). · Seasonality in uptake, movement and translocation of substances as well as healing process after injection were studied in greenhouse and field conditions. Here, we conducted various experiments and we: o Studied optimal time of the day and, seasonal differences and environmental conditions that impact uptake, movement and translocation rate o Determined the appropriate location of injection site o Investigated the impacts of wound (i.e., chemical) treatments on healing o Studied wood characteristics in different parts of citrus trees to determine mechanisms associated with variations in uptake. All these studies provided substantial amount of data (and knowledge) that helped us further modify and improve NATI (and ADS). We are now repeating some of these experiments with more substances applied in different time of the day and seasons on different size trees to gather optimal condition for each tree type and season. Objective 4. (Batuman and Ampatzidis). We used both prototypes of ADS described above in number of mature trees in the citrus grove and were effective to perforate the trunk and create multiple holes. Although second prototype (drill-based) was able to drill the bark effectively, it was slow and tedious process to make a single hole on the trunk. Thus, the third prototype with a punching-based ADS using robust and multiple hollow-needles is now being used on mature citrus trees in the grove. The latest prototype was able to effectively penetrate the bark in desired depth and that could be further improved with addition of a pump to inject substances in much faster pace. Objective 5. (Batuman and Wade). The economics team continued to collect information on citrus cultural practices and prices. Growers were contacted to get their expertise on what changes would be necessary for them to consider the adopting a new technology. They note that costs above $10/acre would need to be investigated carefully. The team will continue to meet with growers and gather these insights as this will be used in adoption surveys. Objective 6. (All PIs). The progress of our project objectives was continuously communicated to a wide range of stakeholders. Although our in-person activities reduced due Covid-19 associated restrictions, we continued our outreach through alternative online/virtual media. Our project progress were presented atourAnnual Advisory Board Meeting and at additional9 grower seminars, 4 lectures, 3 international scientific conferences and seminars, 2 national scientific conferences, 5 lab-visit events (domestic and international visitors) and through 3 industry magazine and 3 TV/Web media report as well as during numerous individual educational and extension activities. <br><br><b>Publications</b><br>
Target Audience
Teaching materials including PowerPoint presentations, news articles, and a website were prepared for broad audiences including citrus growers, grove/operation mangers, advisory board members, industry representatives, consumers, and state/federal regulators, faculty, undergraduate and graduate students, postdocs, visiting scholars and scientist, and general public.
Changes / Problems
Nothing Reported
Training & Professional Development
We provided hands-on training on application of NATI in greenhouse and field conditions and trained two postdocs, two PhD students, and three visiting scholars (an engineer and two graduate students), and five private industry representatives.
Dissemination Streams
We disseminated our project progress in numerous outreach activities such as grower and student seminars, lectures, hands-on workshops, field day and greenhouse demonstrations with accompanyingmaterials (handouts, factsheets, website, Animation videos, magazine and media news articles). We engaged and updated stakeholders on research progress and training in grower seminars and grove visits as well as via phone conversations and emails. We presented our project in two international conferences where we had opportunities to engage and educate attendees including scientists, graduate and undergraduate students, postdocs, citrus growers, industry representatives, and the public about the ADS's usefulness to the citrus industry. These information disseminated to general audiences through our project webpage (https://swfrec.ifas.ufl.edu/programs/citrus-path/automated-delivery/).
Next Reporting Steps
To accomplish the short- and mid-term goals, our future work would involve further testing and improving the NATI by continuing to work on project objectives. Results that have been gathered from small-scale greenhouse experiments will be validated by repeated and larger scale field experiments. <br><br>
<br>What was accomplished under these goals? In this period, we made substantial progress toward number of short-term goals that are critical for the project including designing and developing a first prototype of automated delivery system (ADS), and testing on citrus trees, gathering basic information on dye and bactericide movement in citrus vasculature, identifying the best technique, time and amount to apply therapeutics to citrus, and developing an effective education and extension. We used various size microneedle rollers to open numerous tiny holes on the trunk of citrus plants, wrapped around these openings with either small balloons or cloth pads in which various dyes/tracers were applied (needle-assisted trunk infusion method; NATI). Among the dyes/fluorescent tracers and even some bactericides (i.e., oxytetracycline, streptomycin and penicillin) that we tested showed different patterns of uptake, movement and translocation in treated plants. Our proposed ADS is progressing as planned without any major challenges. This automated delivery system will facilitate large-scale and effective application of therapeutics via NATI in citrus groves and help growers to grow citrus environmentally more friendly and sustainably. Project progress was demonstrated to a wide range of stakeholders including Advisory Board members, growers, industry, state and federal regulators, scientists, students and general public in numerous hands-on workshops, seminars and through magazine and media report as well as other educational and extension activities. Data from these initial studies were also published in two peer-reviewed journal articles to even wider (international) audiences. Objective 1. (Ampatzidis, Albrecht and Batuman). Among the tools evaluated, microneedle rollers and wire nails found to be simple and effective for making perforations on the trunk, and both balloons and cloth pads found to be very effective reservoirs for young trees. The first prototype involving a handheld piercing machine utilizing a set of microneedle-based roller mechanism was created. The idea was that the twin rollers would penetrate the bark and create multiple holes on-the-go. All tests showed positive results for small trees, but for big trees (diameter bigger than 20 cm), it wasn't efficient, and microneedles were bent after repetitive applications on trunk of mature trees. A second prototype was created, with a direct hole-punching mechanism using wire nails. This design involves a modular head with multiple wire nails which attaches to a base structure. Objective 2. (Batuman, Albrecht, Levy and Killiny). In the greenhouse and field, movement of dye/tracer and bactericides in citrus trees occurred in both upward and downward direction and was detected throughout vasculature yet more so in the xylem. Our bark thickness and dye movement measurement results indicated that the arrangement of active vessels within the tree xylem is concentrated towards the outermost rings, which is the youngest xylem lying directly beneath the vascular cambium. Uptake of water was significantly (P = 0.02) higher compared with dye/tracer through the IV-type infusion system. This suggests that the uptake of materials will depend on their chemical composition. The HPLC and the ELISA methods were used for the detection of oxytetracycline and streptomycin, however, the ELISA was more sensitive (1.56-50 ng·mL−1) than the HPLC (5-200 µg mL-1method). Oxytetracycline and streptomycin were detected in the leaves, xylem, phloem, and root when delivered via both root and stem (NATI). The level of oxytetracycline in canopy after root drench was higher than that of streptomycin, indicating that streptomycin was bound to the xylem tissues and immobilized. The studies on the uptake and translocation of fluorescence-labeled penicillin (FL-penicillin) showed that FL-penicillin was present in the leaves, and in the stem xylem and phloem tissues above and below the application site. In addition, the presence of FL-penicillin (or rhodamine) in the gut of D. citri that were allowed to feed on the treated plants, confirmed the translocation of this molecule into the vascular tissue. Objective 3. (Batuman). Optimal time of the day for NATI application was in the evening compared to plants treated in the morning or noon. In 30-70% of plants treated in the evening the dye was observed starting 1 to 3h after application whereas in morning or noon treated plants these rates were usually 30% or less. Regardless when plants were NATI treated, the dye was observed up to 9 cm below and 18 cm above the application site on the stem. The results for dye presence in the stem above the NATI application up to 18 cm (top of the plants) was positive for all the plants. Objective 4. (Batuman and Ampatzidis). Although ADS is not yet ready and projected to be completed by the mid or end of second year (2020), a handheld prototype of ADS described in objective 1 above was used in number of mature trees in the citrus grove. This prototype with a set of microneedle-based roller mechanism was used on 6-year-old grapefruit trees to perforate the trunk and create multiple holes on-the-go. Although the prototype was able to perforate the bark, microneedles were bent, and perforations created were not deep enough to allow the uptake of rhodamine dye after NATI application. Thus, a second prototype was created, which replaced the microneedle-based roller mechanism with a direct hole-punching mechanism using wire nails. Now, we are testing this prototype in mature citrus trees in the grove. Objective 5. (Batuman and Wade). The economics team began collecting information on citrus cultural practices before and during widespread HLB infection. This will be complied and used to assess cost of production with the assistance of UF-IFAS citrus budget information. The team also began collecting 10-year yield and price data. Growers were contacted to get their expertise on what changes would be necessary for them to consider the adopting a new technology. They reported that they would consider a new technology, even if it were more expensive, if it were proved to reduce production costs. The team will continue to meet with growers and gather these insights as this will be used in adoption surveys. Immediate next steps are to examine yield and cost trends over time and to determine the cost of the device once developed. Objective 6. (All PIs). Both NATI method and ADS were introduced to a wide range of stakeholders including Advisory Board members, growers, industry, state and federal regulators, scientists, students and general public. We have prepared animations, videos, handouts and PowerPoint presentations about our project progress and these were presented at ourAdvisory Board Annual Meetingand at additional4 hands-on workshops, 13 grower seminars, two lectures, 3 international scientific conferences and seminars, 3 national scientific conferences, 12 lab-visitevents(domestic and international visitors) and through 7 industry magazine and 3 TV/Web media report as well as other numerous and individual educational and extension activities. We created a designated project web page, which has been up and running since September 2019 (https://swfrec.ifas.ufl.edu/programs/citrus-path/automated-delivery/). <br><br><b>Publications</b><br>