Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1100 - Bacteriology | 100% |
Citrus greening, or huanglongbing (HLB), the most serious disease in citrus, is devastating the citrus industry in Florida. HLB has led to a more than 80% decrease in citrus production in Florida since it was first found in 2005. It is currently threatening the industries in California and Texas, which, without relevant tools could experience a similar fate as Florida. Trunk injection of oxytetracycline (OTC) results in high levels of the antibiotic in leaves and reduces the 'Ca. L. asiaticus' (CLas). However, the trunk injection does not specifically target the phloem, and most of the injected antibiotic is introduced into the xylem, where no bacteria is present. The uploading of oxytetracycline into the phloem is a major barrier for efficient phloem targeting. The major phloem transporters, such as SUC1, binds to sugar molecules and upload them into the phloem. Recently, it was discovered that adding a glucose molecule to different fluorescent dyes or insecticides promote them to load into the phloem. This discovery showed that the glucose molecule could be recognized by the phloem transporters and deliver the fluorescent molecules into the phloem. Building on this finding, we suggest adding a glucose molecule to antimicrobials, especially OTC, which could improve their efficacy of against CLas and reduce the effective dose needed for efficient HLB control by increasing loading into the phloem.In this proposal we will test whether adding a glucose molecule to antimicrobials will increase their upload into the phloem and improve their efficiency in killing CLas and reducing HLB symptoms. We will be using oxytetracycline and Bocillin to test the above hypothesis.OBJECTIVE 1: Synthesize glucose-oxytetracycline (Glc-OTC) and glucose-Bocillin (Glc-BOC). We will synthesize oxytetracycline with an added glucose molecule in various positions. We will also link glucose to the oxytetracycline by a linker that can be later cleaved by enzymes inside the phloem to release the free antibiotic. We will test their activity against microbes.OBJECTIVE 2: Test the upload of oxytetracycline-Glc into the phloem and its anti-CLas activity. We will use the hairy root to screen the activity of Glc-OTC and Glc-BOC against CLas. We will use and NATI (Needle Assisted Trunk Infusion) system to test the upload of oxytetracycline-Glc into the phloem of greenhouse sweet orange trees. After infusion, Glc-Bocillinaccumulation will be tested using fluorescence microscopy.Glc-OTC and Glc-Bocillin will be delivered into 10 years old HLB infected field sweet orange trees by trunk injectionand sprays. After treatment, we will also determine the effect of the injections on canopy density, tree healthand yields.OBJECTIVE 3:Stakeholders engagement and dissemination of our findings.We will conduct an outreach plan that will disseminate the knowledge and findings from proposed studies to citrus industry clientele, scientists, and regulators in all major citrus-producing states (FL, CA, TX, and others) by leveraging both conventional as well as social media channels.
Treatments with the antimicrobials, such as trunk injection of oxytetracycline (OTC), reduced CandidatusLiberibacter asiaticus (Clas) titers and improved tree health, fruit quality, and yield. Applying antimicrobials is expected to play a significant role in the current survival of the citrus industry until more long-term solutions come. However, when applied as a foliar spray, OTC did not affectCLas titers, resulting from OTC not reaching the phloem, and even with the trunk injection, OTC does not specifically target the phloem, and most of the injected antibiotic is introduced into the xylem, where no bacteria are present. The loading of antimicrobials specifically into the phloem is a major barrier to efficientCLas targeting. Recently, it was discovered that adding a glucose (Glc) molecule to different non-phloem-mobile insecticides or fluorescent dyes enables the plant to actively load these compounds into the phloem. Building on this finding, we will add a glucose molecule to antimicrobial compounds. This could dramatically improve their efficacy againstCLas and reduce the effective dose needed for efficient HLB control by increasing loading into the phloem. In this application, we will test whether adding glucose to antimicrobials increases their phloem uploading and efficiency.
Objective 1: Link OTC and fluorescent bocillin to glucose and test their anti CLas activity.We plan to have OTC and Bocillinlinked to Glc to generate Glc-OTC conjugates, which can take advantage of the Glc transporter system in the plant for targeted delivery of OTC to the phloem. Thus, it is necessary that the Glc-OTC conjugates are still accepted by the Glc transporters, while the cargo, i.e., OTC, will keep its antibacterial activity. Alternatively, free OTC should be released from the conjugates after they arrive at the phloem. Glycosylation of OTC and Bocillin will be performed with standardsynthetic chemistry methodologies for carbohydrates, glycolipids and glycoconjugates. For this purpose, we design to directly attach Glc to one of the OH groups in OTC through a glycosidic bond. We design to directly attach Glc to one of the OH groups in OTC through a glycosidic bond.In addition, we will also study another design of Glc-OTC conjugates, i.e., having a spacer between Glc and OTC. The advantage of this design is that the linkage form and the distance between Glc and OTC can be manipulated so that we can target specific phloem/ bacterial enzymes for the cleavage of conjugates and adjust the spacer between Glc and OTC to make the conjugate easily recognizable by the Glc transporter and to further improve the conjugate delivery.For the conjugation of Glc to bocillin (BOC; a BODIPY-functionalized penicillin derivative with fluorescence), we plan to attach Glc to the carboxylic group of the penicillin moiety. Synthetic methodologies for complex carbohydrates, glycolipids and other glycoconjugates synthetic intermediates and final products will be fully characterized with technologies including nuclear magnetic resonance (NMR), high-resolution mass spectroscopy (HR-MS), fluorescent spectroscopy, and infrared (IR) spectroscopy. These new glycoconjugates will be tested to check if they maintain their antimicrobial activity. We will test their activity againstE. coliand Liberibacter crescens (Lcr) in culture and compare it to unlinked OTC and BOC. We will use two methods to evaluate the antimicrobial activity of compounds against Lcr andE. coli(1) Agar disk-diffusion method (ADM) and (2) Broth dilution method (BDM). We will select the conjugates that show antimicrobial activity, by measuring the optical density at 600 nm of the culture and the number of the viable bacterial cells per ml.Objective 2: Phloem delivery of Glc-OTC in hairy root system, greenhouse and trees in a commercial field production settingThe active Glc-OTC and Glc-BOC conjugates (top 2 for each) identified in Objective 1 that will retain their activity against E-Coli and Lcr will be tested for their ability to enter the phloem and kill CLas bacteria. We will use the hairy root systemtest to pre-screen/verify that the Glc-linked molecules retained their anti-CLas activity after adding Glc. We will also test their phloem localization by leaf infiltrations.CLas-citrus hairy root cultures will be treated with the respective non-modified and Glc-conjugated chemistries at multiple dosages, using thein vitromulti-well plate format, alongside untreated and DMSO/solvent alone controls. All the treatments are vacuum infiltrated to facilitate penetration into the hairy root matrices and incubated for 72 hours. The hairy root tissues are subsequently sampled for molecular diagnostics. The effectiveness of the treatments will be determined by estimating relative CLas titers among the controls and treatments by qPCR amplification of ribonucleotide reductase β-subunit gene marker of CLas. From these assays, we expect to identify that the Glc-linked molecules retain their anti-CLas activity after adding Glc.For Glc-BocillinWe will also test phloem targeting with sweet orange greenhouse trees. Glc-BOC will be delivered into the tree using the NATI (Needle Assisted Trunk Infusion) system. After infusion, Glc-OTC accumulation in the phloem (bark tissue) and the xylem (wood tissue) will be tested and quantified using fluorescent microscopy.We will compare the amount of Glc-BOC and unbound BOC in the phloem to evaluate loading.Next, Glc-OTC, unbound OTC, Glc-BOC, and unbound BOC will all be delivered into 10-year-old HLB-affected field sweet orange trees.Goals of this objective are to test the field-based efficacy of Glc-OTC conjugation.The delivery will be performed by both trunk injections and foliar sprays.Trunk Injectionwill be performed on 'Hamlin' sweet orange trees in a field managed by UF according to commercial production standards.Each tree will receive dosage according to guidelines for commercial injectable OTC based on trunk cross-sectional area and plant maturity. Treatments will include: (1) commercial product Remedium TI (positive control), (2) Glc-OTC 1, (3) Glc-OTC 2, (4) Glc-BOC 1, (5) Glc-BOC 2, (6) Injection of water (negative control 1), (7) No injection (negative control 2).Canopy volume and density will be measured before treatment and at 6-month intervals. Additionally, measures of leaf health and function, including the yield of photosystem II via chlorophyll fluorescence, will be measured periodically, at least every 6 months. Fruit load will be measured in August by counting all fruits on each tree, and the total pre-harvest fruit drop will be measured at harvest (December) . Yield measurements will assess the total number of fruits, total fruit weight, average fruit size, soluble solids, and titrated juice acidity from a subsample of fruits. For Foilar spray, the treatments will include: (1) Foliar sodium borate+ Glc-OTC 1, (2) Foliar sodium borate + Glc-OTC 2, (3) trunk injection sodium borate+ Glc-OTC 1, (4) trunk injection sodium borate + Glc-OTC 2, (5) No treatment. Individual trees will be the experimental unit, and 10 replicates will be used. Foliar sprays will be applied 2 times (March and August). The canopy growth, yield, and fruit quality data will be collected. Leaves will be collected one month after the treatments to perform nutrient analyses to evaluate the level of Boron and glucose content. Six months after the treatment, leaves will be collected to measureCLas titer and oxidative stress in the leaves.Objective 3: Stakeholders engagement and dissemination of our findingsThe nationwide extension and outreach program for this proposal will have two parts. The first part will develop questionnaires to collect growers' responses about their attitude/acceptance of using glucose-conjugated antimicrobials for a therapeutic cure of HLB. The responses will be collected using Qualtrics online survey system. The questionnaire will be distributed to citrus Industry stakeholders via an email listserv, citrus Industry newsletter, and personal communications. The responses will be compiled, Interpreted, and published. Grower responses and awareness of the proposed technology will inform the second part of the proposal, which is to disseminate information generated. Several approaches will be implemented to disseminate the information generated from this project to citrus industry stakeholders: (1)Webinar/panel discussions through Science for Citrus Health (2)Presentations/extension outreach publications/Newsletter articles and (3) Online and social media tools.
Target Audience
Our target audiance were the the stakeholders of the Florida citrus industry, including the growers and the scientists that are working on citrus in Florida.
Changes / Problems
We had various problems during the work: 1. TheGlc conjugate of Bodipy turned out to be problematic, since it attached to biological membranes and therefore could not be used for assessing its targeting into the phloem. As an alternative, we will inject the GLC conjugate of penicillin, and will measure its phloem targeting by separating the bark (where phloem is) from the wood, and measure the conjugate levels with ELISA 2.We have tried many methods (more than 10) to conjugate Glc to tetracycline without success. The reaction was either too complex to get any specific conjugate in acceptable yield and product purity or resulted in decomposition of tetracycline as it is sensitive to strong base to occur elimination reaction. We therefore changes totobramycine,1,3-diamino-dihydrostreptomycine and penicillin. 3. Addition of Glc conjugate eliminated the activity of the antibiotics, as measured by leaf disk inhibition assays. We are therfore adding a linker between the antibiotic and the Glc, that will separate them.
Training & Professional Development
The project has provided research training and mentoring opportunities to a Hispanic minority undergraduate student and technician. The students, postdocs and staff were routinely mentored by the PIs in laboratory techniques and scientific inquiry. The projects allowed staff to participate in the International Citrus Congress.
Dissemination Streams
Project updates and results were periodically disseminated to the team members, as well as to stakeholders in TX, FL, and CA, and the broader scientific/citrus stakeholder community through publications. Mandadi, K.K. (2024). Novel strategies to screen and evaluate genetic- and chemical-based solutions for crop improvement. Annual Conference of the Society for Experimental Biology (SEB), July 2-5. Prague, CZ. Mandadi, K.K. (2024). Citrus CAP: A NIFA Center of Excellence for Discovery and Development of Citrus Greening Therapies. Invited Webinar for USDA APHIS PPQ-S&T-Domestic & Emergency Scientific Support (DESS), May 10, Washington DC. Mandadi, K.K. (2024). Identification and evaluation of new chemicals and antimicrobial peptides for HLB management. International Research Conference on Huanglongbing (IRCHLB VII). March 26-29. Riverside, CA. Levy, A. (2024). Enhancing the Delivery of Therapeutics into Citrus Phloem by Linking Sugar Molecules (2024). Description of the project in the "Keeping Florida Citrus Growers Informed" booklet and on the University of Florida extention website:https://crec.ifas.ufl.edu/media/crecifasufledu/citrus-research/docs/expo2024/growers-informed/Enhancing_Delivery.pdf
Next Reporting Steps
Objective 1: Design the conjugate using a linker that will separate the antimicrobial and the sugar, so thatthe enzyme betta-glucosidase (which naturally occur in plants), will break the glycosidic bond will hydrolyze the glycosidic bond to release the free antibiotics to become active. Objective 2: After successful synthesis and efficacy testing in surrogate systems, we will pursue efficacy testing of selected glucose-conjugated antimicrobials in CLas-citrus hairy root assays. If successful, we will start field surveys. Objective 3: We will continue outreach activities to disseminate project knowledge and engage stakeholders. We will continue student and staff mentoring.