Source: UNIVERSITY OF FLORIDA submitted to
ZINKICIDE A NANOTHERAPEUTIC FOR HLB
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1005557
Grant No.
2015-70016-23010
Project No.
FLAW-2014-10120
Proposal No.
2014-10120
Multistate No.
(N/A)
Program Code
CDRE
Project Start Date
Mar 1, 2015
Project End Date
Feb 29, 2020
Grant Year
2015
Project Director
Johnson, E. G.
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
AG-CREC-PLANT PATHOLOGY
Non Technical Summary
Huanglongbing (HLB) is an invasive disease of citrus that is devastating the largest citrus industry in the US and threatening the other major citrus producing regions. This disease causes significant crop loss for citrus growers because of increased premature fruit drop and reduced fruit quality (i.e. small, unpallatable flavor). The bacteria that causes the disease (Candidatus Liberibacter asiaticus) is transmitted by insects (Asian citrus psyllid). Control of bacterial plant pathogens is difficult because of the limited bactericides available and because they only act as a protective film on the outside of the plant. Insect transmission bypasses this protective barrier and insect control alone cannot prevent disease spread.This project aims to develop a specially formulated bactericidal particle that is small enough to enter the plant vascular tissue where the bacteria lives. It is made from plant nutrient and plant derived compounds to develop novel bactericidal activity not found in the raw ingredients. These particles are designed to breakdown into these nutrients after it has performed its bactericidal function. In conjunction with testing how effective this new bactericide is in controling HLB in citrus orchards, the safety and fate of the bactericide particles will be investigated to ensure safe use of the product. The effect of this new bactericide on citrus tree health, fruit production and quality will be determined. Crop improvement data from field trials will be used in economic analyses to determine if it will allow citrus growers impacted by HLB to return to profitable production. Once shown to be safe and effective for control of HLB, it will likely be useful for control of many other bacterial pathogens that significantly limit food production and threaten farmers livelihoods.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2120999110045%
6015220301010%
2125220200045%
Goals / Objectives
The goal of this project is to develop an economical bactericide for control of HLB-affected trees in established citrus orchards, allowing growers to maintain production and profitability in the presence of endemic HLB. We have developed a prototype plant nutrient-based nanoparticle (novel vacancy-engineered (VE) Zinc oxide (ZnO) particle formulation) with unique bactericidal activity that can translocate into plant tissue with the goal of providing an economical HLB management option for infected trees that will allow efficient and profitable citrus production in the presence of HLB. We will pursue this goal with the following research and outreach objectives:Research objectivesObjective 1. Development, improvement, and characterization of Zinkicide nanoparticles.Objective 2. Test the efficacy of new Zinkicide formulations and optimize field application for HLB control while minimizing non-target effects on beneficial organisms.Objective 3. Determine residue lifespan of Zinkicide in planta and toxicology on non-target organisms to ensure safety and expedite product registration for grower use to combat HLB.Objective 4. Evaluate the economic feasibility of using Zinkicide to manage HLB compared to existing methods of citriculture in the presence of HLB.Outreach objectivesObjective 1. Develop interactive media tools including a website and related tools to educate citrus growers about the efficacy, viability, and best use practices of Zinkicide as an HLB management optionObjective 2. Provide training for safe handling and field use of nanoparticlesObjective 3. Provide in-service training on Zinkicide for extension agents from major citrus producing regions across the U.S.
Project Methods
Objective 1. Production of the Zinkicide nanoparticles is done in a unique single pot reaction under conditions that don't require a second purification stage. This process will be optimized for large scale synthesis. To be able to identify nanoparticle half-life and location in plants methods will be developed to detect and quantify the Zinkicide particles instead of just their chemical components using multiple microscopy techniques based on the particles unique emission spectra.Objective 2. Greenhouse and field trials on grapefruit and sweet orange will be performed to determine efficacy and the most effective method and timing of application. Currently used field application methods (i.e. foliar spray and soil drench) will be the methods tested to maintain economic sustainability of the treatment. Efficacy will be determined based on fruit production and quality. Samples will also be taken from these trees to determine the systemic movement and residue of the particles using the detection techniques under development in objective 1. Using special microfluidic chamber techniques, developed to study vascular bacterial pathogens, the mode of action of Zinkicide against Liberibacter and/or related bacteria will be determined.Objective 3. Residue analysis of trees treated in objective 2 will be done to determine the duration of effective concentrations in the tree. These residue concentrations will also be incorporated into standard toxicity assays for non-target organisms to provide information on the safety of the treatment.Objective 4. To determine the economic sustainability for the target audience (citrus growers) a benefit-cost analysis will be done to inform the growers on the most cost-effective way of using the treatment to get the best yield productivity return with the minimum input cost.The progress of each method will be evaluated at yearly stakeholder advisory committee meetings where progress on each objective will be presented to the entire research group and stakeholder advisors. Based on these results and the advice of the stakeholders, the plan for each objective will be assessed and modified as needed to ensure the most efficient use of resources towards the final goal of developing an effective management strategy for HLB.Feedback will also be acquired from grower outreach and extension events including field days and workshops to assess the value of the knowledge provided to the grower stakeholders.

Progress 03/01/16 to 02/28/17

Outputs
Target Audience:While the main target audience for this project is the citrus growers. The target audience also includes students, industry partners, regulators and the general public.. In the early stages of experiments, undergraduate and graduate students and post-docs were the main audience this year as they were trained in how to conduct research. Growers, industry partners, and regulators were the target audience of efforts to get Zinkicide commercially manufactured and registered for use on citrus. A website (Zinkicide.org) was created to maintain a platform to disseminate knowledge to growers, industry, and the general public with general information about nanoparticles and specific information about Zinkicide and ongoing research and development. Information about product development research was also presented as part of the UF-IFAS citrus extension booth. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students and post-docs have developed skills in transitioning experimental products into a commercially viable system by transitioning from high quality lab supplies to agricultural grade inputs or in testing antimicrobial compounds in both lab and plant settings. Multiple students, post-docs and staff have presented work at conferences and workshops of their own disciplines and interdisceplenary conferences of collaborators to gain a better understanding of the full spectrum of efforts needed to develop disease control tools. Students, post-docs, and staff from multiple disciplines attended various grower meetings in Florida, including the Citrus Show and Citrus Expo to learn more about conditions in the field and the multiple problems faced by citrus growers. How have the results been disseminated to communities of interest?The results of this project have been disseminated to the scientific community through posters and presentations at multiple conferences, with most presentations occuring at the American Phytopathological Society annual meeting in Tampa, FL and the Materials Inovation for Sustainable Agriculture symposium. To obtain grower and industry interest results of previous field and greenhouse citrus canker trials with Zinkicide were presented to show field efficacy and systemic movement and activity, respectively. What do you plan to do during the next reporting period to accomplish the goals?Field trials on HLB affected grapefruit and Valencia sweet orange trials will transition to use of the fixed ag grade formulation. The decision to consider the stable ag-grade formulation fixed allows for the colleciton of preliminary data for EPA registration without worrying about changes in response due to changes in formulation. Previous efforts to use existing live-dead assays for Liberibacter in planta have proven unreliable. We will develop RNA based viability assays and use them in greenhouse studies to determine the residual efficacy of Zinkicide against Liberibacter and use this informaiton to alter applicaiton patterns. Detection systems will continue to be improved and systemic movement of the nanoparticles will be studied to ensure rapid and even distribution of the nanoparticles with specific focus on phloem tissue. Non-target toxicology studies will become a primary focus now that the fixed formulation has been attained. Modelling of Zinkicide nanoparticles from the fixed formulation will be performed to predict whether Zn ion or reactive oxygen species generation at the surface is the primary mode of action. Preliminary residue analyses will be performed on fruit from the field trials to assist in registration and determine if applicaiton limitations will be likely near harvest. We will produce video and poster materials to disseminate results and information about nanoparticles and our project to growers and the general public.

Impacts
What was accomplished under these goals? Zinkicide nanoparticles have repeatedly shown an improvement in fruit size reversing some of the symptoms of HLB (citrus greening) that leads to lower fruit quality. As we improve the formulation and timing of application, we expect further improvements in reversing HLB damage. The project team has developed an economical version of Zinkicide from agricultural-grade materials that is stable and effective in preliminary tests and found a licensee of the technology to provide larger scale synthesis and begin steps for product registration. Now that a final formulation has been developed, much of the toxicology and field application optimization can be performed. Research objectives Objective 1. Development, improvement, and characterization of Zinkicide nanoparticles. Zinkicide supplied to the field trial was switched from reagent grade to agricultural grade formulation for use in the 2016-17 field season and was provided by an industry partner (Trademark Nitrogen). This formulation was not a final formulation, but was the best version available at the start of the field season. It's main drawback was stability as it sometimes had changed consistency between pickup and use in the field trial Continued improvement incorporating agricultural grade chemicals into the synthesis process has resulted in a stable and effective formulation that will be used for all future experiments and will be considered a fixed formulation for EPA registration purposes (toxicology and efficacy data). Continued improvement in detection methods for nanoparticles in planta is ongoing. Current focus has been on understanding the distribution of nanoparticles to determine if they are systemically distributed throughout the phloem system of the plant and improving detection limits for Zinkicide nanoparticle specific detection. Objective 2. Test the efficacy of new Zinkicide formulations and optimize field applicaiton for HLB control while minimizing non-target effects on beneficial organisms. Field trials in ray ruby grapefruit have completed one year with an early agricultural grade formulation and demonstrated an improvement in fruit size, reversing the small fruit symptom of HLB. The Valencia sweet orange field trial is almost ready for harvest with yield and fruit quality assessment in the next month. Fruit size improvement can now be considered a consistent effect of Zinkicide on HLB as it has been observed in multiple trials over multiple seasons with both reagent and agricultural grade formulations. The preliminary agricultural grade Zinkicide had reduced efficacy compared to reagent grade for both fruit size improvement and citrus canker control. The reduced efficacy was probably due to the poor stability and shelf life of the preliminary ag-grade formulation. Based on initial greenhouse tests with the fixed formulation developed for objective 1, we expect better efficacy in the 2017-18 field trials. Greenhouse trials using root application of Zinkicide and testing of systemic bactericidal activity with leaf inoculations of Xanthomonas citri (citrus canker) suggest that the residual activity of Zinkicide is shorter than originally anticipated. Further testing will be needed to quantify the residual efficacy timeline and inform changes in the field applicaiton rate. This shorter than expected residual efficacy may explain the inconclusive results from qPCR detection of Liberibacter compared to symptom responses. Objective 3. Determine residue lifespan of Zinkicide in planta and toxicology on non-target organisms to ensure safety and expedite product registration for grower use to combat HLB. Honey bee toxicology experiments have begun with agricultural grade formulations and setup has begun for lung cell and aquatic animal toxicology studies with the fixed formulation. Objective 4. Evaluate the economic feasibility of using Zinkicide to manage HLB compared to existing methods of citriculture in the presence of HLB. Economic models to evaluate economic efficacy for growers and consumer acceptance risks are nearly prepared for input of field trial efficacy data. Based on initial efficacy results, modification will likely be necessary to include analysis of fresh market citrus economics based on fruit size. Outreach objectives Objective 1. Develop interactive media tools including a website and related tools to educate citrus growers about the efficacy, viability, and best use practices of Zinkicide as an HLB management option. A website has been deployed and a public outreach video script is being revised to describe the Zinkicide concept to the public. Objective 2. Provide training fro safe handling and field use of nanoparticles. Will be developed as Zinkicide development gets closer to final stages of development and availability to growers. Objective 3. Provide in-service training on Zinkicide for extension agents from major citrus producing regions across the U.S. Will be developed as Zinkicide development gets closer to final stages of development and availability to growers.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: J. H. Graham, E. G. Johnson, M. E. Myers, M. Youngg, P. Rajasekaranp, S. Dasp, and S. Santra. 2016. Potential of Nano-Formulated Zinc Oxide for Control of Citrus Canker on Grapefruit Trees. Plant Disease, 100:12 Pages 2442-2447
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: E. Johnson, M. Myers, K. Gerberich, S. Santra, J. Graham. 2016. Advanced copper and zinc nanomaterials for management of bacterial canker of citrus. Phytopathology 106:S12 p. 165
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: L. Tetard, M. Solimang, M. Youngg, A. Towers, T. Washington, P. Rajasekaranp, S. Dasp, E. Johnson, A. Gesquiere, S. Santra. 2016. Characterization of Zinkicide in plant tissues. Phytopathology 106:S12 p69.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: S. Commerford, K. Gerberich, P. Rajasekaranp, M. Youngg, S. Dasp, J. Graham, S. Santra, E. Johnson. 2016. Citrus canker as a bioassay for systemic bactericidal activity of zinc nanoparticles. Phytopathology 106:S12 p.62.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: H. Mendisp, E. Naranjog, S. Santra, M. Youngg, E. Johnson, P Rajasekaranp, L. De La Fuente. 2016. Evaluation of a novel antimicrobial compound to control growth and biofilm formation in vitro of citrus bacterial pathogens. Phytopathology 106:S12 p.61.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: M. Youngg, M. E. Myers, J. H. Graham, E. G. Johnson, S. Santra. 2016. Mixed-valence coper and copper alternatives: new generation of fungicides/bactericides for citrus disease management. International Citrus Congress. S9-437.


Progress 03/01/15 to 02/29/16

Outputs
Target Audience:While the main target audience for this project is the citrus growers. This early in the project, there was little completed that could be translated into extension and outreach programs. In the early stages of experiments, undergraduate and graduate students and post-docs were the main audience this year as they were trained in how to conduct research. Growers were the target audience of efforts to get Zinkicide commercially manufactured and registered for use on citrus. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Students and post-docs have developed skills in transitioning experimental products into a commercially viable system by transitioning from high quality lab supplies to agricultural grade inputs or in testing antimicrobial compounds in both lab and plant settings. Multiple students, post-docs and staff have presented work at conferences and workshops of their own disciplines and interdisceplenary conferences of collaborators to gain a better understanding of the full spectrum of efforts needed to develop disease control tools. How have the results been disseminated to communities of interest?Initial results of efficacy have been reported at grower meetings through oral presentations. What do you plan to do during the next reporting period to accomplish the goals?We hope to have a fixed formulation using ag grade chemicals that can be used for tests necessary for EPA registration. We will begin comparing lab grade and ag grade formulations in field trials to ensure that efficacy is maintained. We will continue to develop and improve detection systems and further investigate environmental toxicology to ensure safety of applications to non-target organisms. We will have a fully functional website complete and continue to add material to it along with development of extension materials describing the efficacy and development process.

Impacts
What was accomplished under these goals? Research objectives Objective 1. Development, improvement, and characterization of Zinkicide nanoparticles. Increased concentration of Zinkicide nanoparticles in synthesis reaction to make commercially viable. Incorporated agricultural grade chemicals into the synthesis process and began efforts to develop a full efficacy formulation using these ag grade chemicals Identified nanoparticle specific fluroscent and RAMAN signals for detection of nanoparticles in planta. Currently working on improving detection limits Objective 2. Test the efficacy of new Zinkicide formulations and optimize field application for HLB control while minimizing non-target effects on beneficial organisms. Field trials initiated on grapefruit and sweet orange for HLB efficacy. Began greenhouse trials for best application methods. Objective 3. Determine residue lifespan of Zinkicide in planta and toxicology on non-target organisms to ensure safety and expedite product registration for grower use to combat HLB. Initial honeybee toxicology performed and initiated attempts to detect nanoparticles in nectar from flowers of treated trees to determine exposure risk to bees Objective 4. Evaluate the economic feasibility of using Zinkicide to manage HLB compared to existing methods of citriculture in the presence of HLB. Began building economic models to evaluate economic efficacy for growers and consumer acceptance risks Outreach objectives Objective 1. Develop interactive media tools including a website and related tools to educate citrus growers about the efficacy, viability, and best use practices of Zinkicide as an HLB management option. Designed initial template of website. Began developing ideas for videos to demonstrate how nanoparticles work. Began developing posters of efficacy for extension booth for citrus grower meetings. Objective 2. Provide training for safe handling and field use of nanoparticles. Will be developed as Zinkicide development gets closer to final stages of development and availability to growers. Objective 3. Provide in-service training on Zinkicide for extension agents from major citrus producing regions across the U.S. Will be developed as Zinkicide development gets closer to final stages of development and availability to growers.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Commerford, S.L., Gerberich, K.M., Rajasekaran, P., Young, M., Das, S., Graham, J.H., Santra, S., Johnson, E.G., Citrus Canker as a bioassay for systemic bactericidal activity of Zinc nanoparticles. Annual Meeting of the American Phytophathological Society. 2016.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: 1. Rajasekaran P. and Santra S*, "Hydrothermally treated chitosan hydrogel loaded with copper and zinc particles as a potential micro-nutrient based antimicrobial feed additive", Frontiers in Veterinary Science, 2015, 2:62.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: 1. Rajasekaran, P. and Santra, S., Chitosan hydrogel loaded with copper and zinc particles as a micro-nutrient based antimicrobial feed additive for combating antimicrobial resistance, American Society for Microbiology Annual Meeting  FL Branch, Cocoa Beach, FL, October 9-11, 2015.