Source: UNIVERSITY OF FLORIDA submitted to
ZINKICIDE A NANOTHERAPEUTIC FOR HLB
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1005557
Grant No.
2015-70016-23010
Cumulative Award Amt.
$4,613,838.00
Proposal No.
2014-10120
Multistate No.
(N/A)
Project Start Date
Mar 1, 2015
Project End Date
Feb 29, 2020
Grant Year
2015
Program Code
[CDRE]- Citrus Disease Research and Extension Program
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
40%
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%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 601 - Economics of Agricultural Production and Farm Management;

Subject Of Investigation
0999 - Citrus, general/other; 5220 - Pesticides;

Field Of Science
1100 - Bacteriology; 3010 - Economics; 2000 - Chemistry;
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 03/01/15 to 02/29/20

Outputs
Target Audience:The citrus growers were the main target audience and were reached through multiple grower presentations and handouts and posters discussing Zinkicide efficacy and the status of registration and commercialization. Regulators and ag industry members were another key audience this year with the initiation of trials in other crops by IR-4 to help get Zinkicide registered as a pesticide for crop use. They were also the target audience of work to document potential environmental risks or lack thereof. Additionally through training and professional development undergraduate and graduate students and postdocs were a target audience through the performance and presentation of research for this project in interdisciplinary settings such as the MISA symposium and IRCHLB. Animations and videos produced about this project targetted a broad audience including the general public (consumers), growers, regulators, and agrochemical industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Multiple students and postdocs have had the opportunity to present results from this project at professional conferences providing valuable professional development in networking and presenting and receiving feedback on their research. Many of these conferences have been interdisciplinary allowing the students and postdocs a chance to learn about different aspects of research related to food production from engineering, product development, plant pathology, entomology, and horticulture from both academic and industry perspectives. Students, post-docs and staff from multiple nonagricultural disciplines have attended various grower meetings in Florida, including the Citrus Show and Citrus Expo to gain exposure to the challenges that their products will face in real world application of citrus groves and other agricultural fields. At the MISA symposium students and post-docs had the opportunity to interact directly with growers and ag industry representatives to gain a better understanding of the real world problems facing agriculture and the challenges, both economic and regulatory, of bringing scientific discoveries into use. How have the results been disseminated to communities of interest?Information was presented to both growers and researchers in multiple presentations and posters at the International Research Congress on Huanglongbing in March 2019. Posters and handouts were presented and made available at the UF/IFAS extension booth at major Florida citrus grower events. New videos were produced and posted to the project website and spread by the project twitter account to disseminate information developed by the project.The results of this project have also been disseminated to the scientific community through posters and presentations at multiple conferences covering multiple subject areas. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 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. This nanoparticle solution can now be synthesized in agricultural scale based on industrial test batches used for field trials produced by an industry partner (Trademark Nitrogen Inc.). This formulation has shown dramatic improvements in fruit yield and tree health on young trees that pose the greatest risk of economic loss for growers. This improvement results in a net economic gain even when applications are not fully optimized (12 applications per year without tank mixing other products). Early evidence from modified application patterns suggest that the economic benefit could be improved with optimized application schedules. While progress has been made, developing an economically feasible and effective application program for mature trees has not yet been achieved. Non-target toxicology shows that it is less toxic than standard copper bactericide to aquatic animals and that application rates are more than 10 times lower than the level toxic to honey bees. The nanoparticles only stay active in the tree for about 2 weeks before dissolving and becoming a part of the nutrient pool reducing risk of environmental build-up. Zinkicide is currently being tested on other crops by IR-4 to improve the chances of a company funding EPA registration. Zinkicide is currently under a limited license for registration purposes. Research objectives Objective 1. Development, improvement, and characterization of Zinkicide nanoparticles. Zinkicide has been fully developed into an effective, economically viable formulation that can be produced on large scale using existing bulk agricultural industrial processes. The unique features of the nanoparticle have been modelled to understand the unique properties of the nanoparticles to minimize risk of unintended consequences. Objective 2. Test the efficacy of new Zinkicide formulations and optimize field application for HLB control while minimizing non-target effects on beneficial organisms. Zinkicide was shown to directly kill bacteria better than zinc oxide using both Xanthomonas citri and Liberibacter crescens. It did trigger biofilm formation in microfluidic chambers, but killed all but a few of the bacteria present. Many dead bacteria showed complete cellular rupture. Zinkicide has been shown to reduce bacterial titers in trees with a biweekly application schedule and can improve yield by 70% in 5-7 year old trees with only a monthly application through foliar spray or soil drench application, which are the most cost effective application methods currenlty available. A single year of a pulsed biweekly spray at spring or summer flush did not show a yield improvement in declining mature trees, however multiple years of treatment may be needed to observe a turnaround in mature trees already in decline. There is evidence that even early applications to new plantings can slow the progression of HLB in the trees, greatly improving the chances of long term economic viability. 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. Zinkicide did not have significant residue in peel oil or juice at harvest, and Zinkicide is only effective against CLas for less than 2 weeks before it is consumed into the nutrietn pool of the plant making residue a minimal hazard. The only effect observed on soil microbial communities was with the soil drench application, although environment and local soil conditions had a much larger effect. Zinkicide was tested for potential to harm honey bees as a proxy for all pollinators that might encounter it if applied during citrus bloom. Acute toxicity assays were conducted on honey bee worker adults and worker larvae. Topical application of pure Zinkicide concentrate to the thorax using a microapplicator syringe did not result in additional bee mortality over 72 hours with dosages as high as 100 micrograms. However, simulated spray application of Zinkicide onto adult bees using a Potter Spray Tower resulted in significantly elevated mortality at a 1:4 (Zinkicide concentrate:water) dilution rate. Honey bee larvae were the most sensitive life stage, with significantly increased mortality observed when as little as 4.5 micrograms were incorporated into diet fed to in vitro reared larvae. While Zinkicide has the potential to kill bees, risk assessment will need to take into account the concentrations of the product that are actually present in pollen and nectar from treated flowers to determine the risk posed to larvae. Further testing is needed, but bee toxicity is not expected to be a major barrier to use of Zinkicide on blooming crops when bees are present. Objective 4. Evaluate the economic feasibility of using Zinkicide to manage HLB compared to existing methods of citriculture in the presence of HLB. In young producing fresh grapefruit, Zinkicide was found to provide a net profit in some application rates, although the most effective combined foliar spray and soil drench, the application costs were greater than the increased revenue, suggesting that growers may need to balance tree health with costs. These costs will be reduced if future tests show that Zinkicide can be tank mixed with other pest control materials. 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. Multiple videos have been produced showing both grower and researcher perspectives on Zinkicide applications and HLB for outreach and have been posted to youtube and linked on the project website (Zinkicide.org). Additionally, animations explaining the challenges of developing a treatment for HLB have been produced. Objective 2. Provide training for safe handling and field use of nanoparticles Licensing and registration did not develop enough to require this training Objective 3. Provide in-service training on Zinkicide for extension agents from major citrus producing regions across the U.S. Because a product was not available, in-service training was not done, however information was distributed and presented about Zinkicide in both Florida and California with members of the Texas citrus community present.

Publications

  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Murray SK. Effects of used brood comb and propolis on honey bees (Apis mellifera L.) and their associated bacterium, Melissococcus plutonius. The Ohio State University. 2019. Available: https://etd.ohiolink.edu/!etd.send_file?accession=osu1574848132491672&disposition=inline
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Merfa, M.V. *G, E. Perez-Lopez, E. Naranjo*G, M. Jain, D.W. Gabriel, and L. De La Fuente. 2019. Progress and obstacles in culturing Candidatus Liberibacter asiaticus, the bacterium associated with Huanglongbing (HLB). Phytopathology, 109(7):1092-1101. https://doi.org/10.1094/PHYTO-02-19-0051-RVW.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Naranjo, E. *G, M.V. Merfa*G, V. Ferreira*G, M. Jain, M.J. Davis, O. Bahar, D.W. Gabriel and L. De La Fuente. 2019. Liberibacter crescens biofilm formation in vitro: establishment of a model system for pathogenic Candidatus Liberibacter spp.. Scientific Reports 9:5150, https://doi.org/10.1038/s41598-019-41495-5.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Merfa, M.V., and L. De La Fuente. 2019. Enhancing in vitro growth of Candidatus Liberibacter asiaticus by culture medium optimization. Materials Innovations for Sustainable Agriculture Annual Symposium, Orlando, FL, October 24-25, 2019. Oral presentation.
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2020 Citation: Naranjo E., Shantharaj D., Merfa M.V., Santra S., Johnson E.G., De La Fuente L. 2020. Mechanism of action of a zinc-based nanoparticle with activity against vascular plant pathogenic bacteria. Submitted for oral presentation to the 14th International Conference on Plant Pathogenic Bacteria (ICPPB), Azizi, Italy. The meeting was postponed until 2021 due to the COVID-19.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Naranjo, E. *G, M. V. Merfa*G, S. Santra, M. Young, E. Johnson, and L. De La Fuente. 2019. Evaluating the antimicrobial mechanisms of Zinkicide� against Liberibacter crescens as a model for Candidatus Liberibacter spp. MISA (Materials Innovation for Sustainable Agriculture) Symposium, University of Central Florida, Orlando, Florida, October 29-30, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Naranjo, E.*G, S. Santra, M. Young*G, E. Johnson, and L. De La Fuente. 2018. In vitro antimicrobial activity and mode of action of ZinkicideTM against Liberibacter crescens, a surrogate of Candidatus Liberibacter asiaticus. 6th International Research Conference on Huanglongbing, Riverside, CA, March 12-15, 2019.
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Naranjo E, Merfa MV, Santra S, Ozcan A, Johnson E, Cobine P, De La Fuente L. 2020. ZinkicideTM, a ZnO-based nano-formulation, exhibits enhanced antimicrobial activity against Liberibacter crescens in batch cultures and in microfluidic chambers simulating plant vascular systems. Under revision in Applied and Environmental Microbiology. Manuscript number: AEM00788-20.
  • Type: Theses/Dissertations Status: Other Year Published: 2021 Citation: Eber Naranjo. Ph.D. in Plant Pathology, Department of Entomology and Plant Pathology, Auburn University. Dissertation: Antimicrobial mechanisms and environmental impact in soil microbial communities of ZinkicideTM, a ZnO-based nano-formulation to control HLB.. 2016-2021 (anticipated defense Spring 2021). Advisor: Leonardo De La Fuente.
  • Type: Journal Articles Status: Other Year Published: 2021 Citation: Tracking the Movement of Systemic Nanoparticle-Based Antibacterial Treatment to Combat Greening Disease, Mikhael Soliman, Briana Lee, Warren Edmunds, Nicole Labb�, Swadeshmukul Santra, Laurene Tetard
  • Type: Journal Articles Status: Other Year Published: 2020 Citation: Zinc Oxide-based nanotherapeutics to tackle phloem-restricted diseases in plants, Mikhael Soliman, Briana Lee, Mikaeel Young, Ali Ozcan, Hajeewaka Mendis, Parthiban Rajasekaran, Takat Rawal, Andre Gesquiere, Leonardo DeLaFuente, Loukas Petridis, Evan Johnson, Swadeshmukul Santra, Laurene Tetard
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: SDS-PAGE for Monitoring the Dissolution of Zinc Oxide Bactericidal Nanoparticles (Zinkicide) in Aqueous Solutions. ZT Untracht, A Ozcan, S Santra, EH Kang. ACS Omega 2020, 5, 1402?1407.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Interaction of Zinc Oxide Nanoparticles with Water: Implications for Catalytic Activity. TB Rawal, A Ozcan, SH Liu, SV Pingali, O Akbilgic, L Tetard, H ONeill, Swadeshmukul Santra, Loukas Petridis. ACS Applied Nano Materials, 2019, 2(7), 4257-4266.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Applications of nanoscale functional imaging to reveal the role of heterogeneities in complex systems for sustainable applications, L. Tetard, Southeastern Regional Meeting of the American Chemical Society SERMACS, Savannah GA, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Nanoscale infrared spectroscopy to explore materials properties at the nanoscale. L. Tetard, Physics Department (Colloquium), Florida International University, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Manipulating light-matter interaction to explore properties of organic and biological systems at the nanoscale, European Forum on Nanoscale IR Spectroscopy, Amsterdam, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Nanoscale investigation of mode of antibacterial acticivty of zinc oxide nanoparticles, Briana Lee, Hajeewaka Mendis, Ali Ozcan, Swadeshmukul Santra, Laurene Tetard, Nanoflorida International Conference 2019, Tampa, FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Determining the infrared fingerprint corresponding to cell wall degrading and protein inhibiting treatment on E. Coli, K. Wright, B. Lee, S. Santra, L. Tetard, Nanoflorida International Conference 2019, Tampa, FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Comparative Study of Antimicrobial efficacy of Nano-Zinc oxide and Bulk-Zinc oxide on Pseudomonas syringe. Danya Belnour, Hajeewaka Mendis, Swadeshmukul Santra. MISA Symposium 2019, Orlando FL
  • Type: Theses/Dissertations Status: Submitted Year Published: 2020 Citation: K. Wright, Characterization of the Physical and Chemical Effect of Membrane Disruption and Protein Inhibiting Treatments on E. Coli, Honors in the major undergraduate thesis, May 2020.
  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Tyler Maxwell, Coated Quantum Dots: Engineering of Surface Chemistry for Biomedical and Agricultural Applications, PhD Dissertation, 2019
  • Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Ali Ozcan, Advanced multifunctional bactericides for crop protection: A new strategy to reduce Cu pesticide footprint in the environment, PhD Dissertation, 2019
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. M. Murata, S. Santra, E. G. Johnson. 2019. ZinkicideTM: how effective are zinc oxide nanoparticles for HLB control?. Proceedings of the International Research Congress on Huanglongbing.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: M. M. Dewdney, D. B. Bright, K. M. Gerberich, S. Santra, J. H. Graham, E. G. Johnson. 2019. Zinkicide improved yield and fruit size on younger Huanglongbing-affected trees. Proceedings of the International Research Congress on Huanglongbing.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: E. G. Johnson, M. M. Murata, S. Santra, M. M. Dewdney. 2019. Zinc nanoparticles offer systemic efficacy against Candidatus Liberibacter asiaticus improving yield. Plant Health 2019. Phytopathology. 109:S10. 62
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: J. D. Veloso Dos Santos, M. M. Murata, E. G. Johnson. 2019. Movement of Candidatus Liberibacter asiaticus and progression of disease in split-root 'Swingle' trees. Plant Health 2019. Phytopathology. 109:S10. 141
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: J. D. Veloso Dos Santos, M. M. Murata, E. G. Johnson. 2019. Huanglongbing on roots: early movement of Candidatus Liberibacter asiaticus and disease progression. Proceedings of the Materials Innovation for Sustainable Agriculture Symposium 2019. p. 32
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: J. D. Veloso dos Santos Pulici, M. M. Murata, K. M. Gerberich, D. B. Bright, S. A. Lopes, E. G. Johnson. 2019. Translocation of Candidatus Liberibacter asiaticus in split root citrus and progression of Huanglongbing disease in plants. Proceedings of the International Research Congress on Huanglongbing.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: E. G. Johnson, M. M. Murata, S. Santra, M. M. Dewdney. 2019. Zinkicide improves health and yield of HLB-affected trees: limited by dilution and residual efficacy? Proceedings of the Materials Innovation for Sustainable Agriculture Symposium 2019. P. 16
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: J. D. Veloso Dos Santos, M. M. Murata, E. G. Johnson. 2019. Candidatus Liberibacter asiaticus in roots: local and systemic effects of infection. Proceedings of the 16th Biennial meeting of the Florida Phytopathological Society.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: J. D. Veloso dos Santos Pulici, E. G. Johnson. 2019. Huanglongbing in roots: Local and systemic disease effects. Citrus Research and Education Centers Posters and Pastries Research Symposium. Page 45.


Progress 03/01/18 to 02/28/19

Outputs
Target Audience:The main target audience for this project is citrus growers, which was achieved through grower talks and posters and handouts at grower events and trade shows. Other target audiences includ students, industry partneres, regulators and the general public. Undergraduate and graduate students and postdocs continued to be a key audience through training and professional development in the conduct and reporting and presentation of research. Efforts continued with industry partners and regulators as a target audience to work towards getting Zinkicide commercially available for citrus growers, although a change in potential registrant occured during this time. Filming of videos for grower and community outreach was done and are in post production. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Multiple students and postdocs have had the opportunity to present results from this project at professional conferences providing valuable professional development in networking and presenting and receiving feedback on their research. Many of these conferences have been interdisciplinary allowing the students and postdocs a chance to learn about different aspects of research related to food production from engineering, product development, plant pathology, entomology, and horticulture from both academic and industry perspectives. Students, post-docs and staff from multiple nonagricultural disciplines have attended various grower meetings in Florida, including the Citrus Show and Citrus Expo to gain exposure to the challenges that their products will face in real world application of citrus groves and other agricultural fields. How have the results been disseminated to communities of interest?Results have been presented at multiple grower events throughout Florida and to the Citrus Research and Development Foundation Board. Posters, handouts, and an animated video were presented at the UF/IFAS citrus extension booth at major citrus grower events. Information and an introductory video about Zinkicide were available on the Zinkicide.org website. over 400 views of the video were recorded on youtube. The results of this project have also been presented to the scientific community through posters and prsentations at multiple conferences covering multiple subject areas. We have communicated with IR-4 to get testing in other crops to improve the likelihood that a company will fully license Zinkicide as a registrant for EPA registration. What do you plan to do during the next reporting period to accomplish the goals?In the final year of this project we expect to finalize most of the experiments with their final repetitions, finish analyzing the data and have a strong package of data for a registrant to begin moving Zinkicide through the EPA registration process. We will also perform a 1 year field trial with different application frequencies and patterns to improve Zinkicide efficacy on mature citrus trees. Dr. Santra will work with IR-4 to get multiple crop field trials underway and supply Zinkicide through his industrial partner to make product registration economically feasible.

Impacts
What was accomplished under these goals? Zinkicide development and efficacy testing moved forward substantially this year. The efficacy of an agricultural grade formulation was confirmed with a second year of field trials, even after the trees suffered damage from a hurricane the previous year. Tree size is still a limitation and the short residual efficacy became apparent, which is good for product registration, but leads to more challenges with effective application strategies. The UCF team continued to show how Zinkicide particles move systemically in the plant and improving shelf life and reducing off gassing of the concentrated Zinkicide solution for easier storage and distribution. The UF group determined limits of application before phytotoxicity and demonstrated that biweekly application provides the largest reduction in pathogen populations. This has been incorporated into a planned pulse application targeting the spring or summer flush in mature field Valencia orange trees. Pollinator toxicology shows a good margin between honeybee toxicity and application rate. Zinkicide has been confirmed to kill most Liberibacter crescens in microfluidic chambers confirming that efficacy is through direct pathogen kill. Preliminary economic analysis shows a $600/acre increase in revenue from Zinkicide treated grapefruit based on yield and fruit size improvement. Research objectives Objective 1. Development, improvement, and characterization of Zinkicide nanoparticles. Efforts focused on improving shelf-life of the concentrated Zinkicide solution beyond 6 months and reducing off-gassing that requires vented lids for storage and distribution. Objective 2. Test the efficacy of new Zinkicide formulations and optimize field application for HLB control while minimizing non-target effects on beneficial organisms. The fixed agricultural grade formulation was confirmed to be effective on 6 year old trees in the 2nd year of the field trial with this formulation. Mature trees did not see a consistent yield response although tree health was improved. Greenhouse studies were done to demonstrate that biweekly application is more effective than weekly or monthly application and that increasing concentrations to near phytotoxic levels can actually reduce efficacy, which is guiding planning for final year field trials to expand efficacy into mature trees where it is more difficult to deliver and effective dose of Zinkicide nanoparticles to all parts of the phloem in the branches, trunk, and roots. 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. Lifespan of residues in plants and on and in fruit was tested in both seedlings and field trees. The residual efficacy was also tested to look for active nanoparticle activity to infer dissolving of nanoparticles. Non-target toxicology was continued and current toxic levels of Zinkicide on honeybees are well above application rates. Larva are more sensitive than worker bees, so nectar concentrations of Zinkicide will be important to measure. Objective 4. Evaluate the economic feasibility of using Zinkicide to manage HLB compared to existing methods of citriculture in the presence of HLB. Preliminary analysis showed a strong revenue response in grapefruit with an estimated fresh market packout and culled juice grapefruit. As the juice orange trees in the field trial were mature and did not show a yield response, no economic analysis was performed. As the final stages of Zinkicide formulation are developed the costs of materials and application will be incorporated into the model. 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 New videos were filmed and are being editted for release to show the entire Zinkicide development and field testing process including residue and juice quality analysis. Handouts and posters were also developed explaining the differing efficacy of the different Zinkicide formulations and to provide preliminary economic figures on revenue increases due to Zinkicide. Objective 2. Provide training for safe handling and field use of nanoparticles. Will be developed as Zinkicide gets closer to final stages of development and availability for 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 gets closer to final stages of development and availability for growers.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: ZinkicideTM  Preliminary antimicrobial efficacy and toxicity studies. Campos, M.G.N., Parthiban Rajasekaran, Ali Ozcan, Mikaeel Young, Mitsushita Doomra, Tyler Maxwell and Swadeshmukul Santra. Nanoscale Science and Engineering for Agriculture and Food Systems Gordon Research Conference, 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: 7. Evaluation of novel antimicrobial compounds ZinkicideTM and TSOL to control growth and biofilm formation of Candidatus Liberibacter asiaticus surrogates Xanthomonas citri subsp. citri and Liberibacter crescens in vitro and under microfluidic chambers. Hajeewaka Mendis, Swadeshmukul Santra, Mikaeel Young, Ali Ozcan, Evan Johnson, Leonardo De La Fuente. MISA Symposium 2018, Orlando FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: ZinkicideTM  A Systemic Bactericide for Managing Huanglongbing. Maria Campos, Mikaeel Young, Ali Ozcan, Parthiban Rajasekaran, Tyler Maxwell, Monty E. Myers, Evan Johnson, James H. Graham, and Swadeshmukul Santra. MISA Symposium 2018, Orlando FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Tracking and Detection of Bactericidal Quantum Dots. Zachary T. Untracht, Ali Ozcan, Swadeshmukul Santra, Hyeran Kang. MISA Symposium 2018, Orlando FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: ZinkicideTM: A systemic bactericide for managing Huanglongbing. Maria Campos, NanoFlorida 2018, Melbourne/FL-USA, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Van der Waals Density Functional Study of Structural and Electronic Properties of ZnO Nanoparticles and their Interaction with Small Molecules. Takat B. Rawal and Loukas Petridis. MISA Symposium 2018, Orlando FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: In vitro antimicrobial activity and mode of action of ZinkicideTM against Liberibacter crescens, a surrogate of Candidatus Liberibacter asiaticus. Eber Naranjo, Swadeshmukul Santra, Mikaeel Young, Parthiban Rajasekaran, Evan Johnson, Leonardo De La Fuente. MISA Symposium 2018, Orlando FL
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: N. Ciaffone, Interplay of Molecular and Nanoscale Behaviors in Biological Soft Matter, MS Thesis, May 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Challenges of quantifying death of phloem-limited pathogens: Zinkicide and thermotherapy as case studies. Evan G. Johnson, Mayara M. Murata, Naweena Thapa, Megan M. Dewdney. MISA Symposium 2018, Orlando FL
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: B. Lee, A Multisystem Approach for the Characterization of Bacteria for Sustainable Agriculture, MS Thesis, May 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Gleason, J. B., Annual Meeting of Enhancement of Microbial Food Safety by Risk Analysis 1077 USDA multi-state Team in Traverse City Oct 9-11, 2018. Invited Presentation: Using Visuals, Animations and Interactive Media to Increase the Effectiveness of Food Safety Outreach Programs.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Chamberlin, B. A., Martinez, P. N., NC 1023 Annual Food Engineering Meeting, University of Maine, Orono, Maine, October 15-16, 2018. Invited Presentation: NMSU Station Report.,
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Antimicrobial nano-zinc oxide-2S albumin protein formulation significantly inhibits growth of "Candidatus Liberibacter asiaticus" in planta. Ghosh, D. K., S. Kokane, P. Kumar, A. Ozcan, A. Warghane, M. Motghare, S. Santra and A. K. Sharma. Plos One, 2018, 13(10).
  • Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: M. Soliman, Advanced Nanoscale Characterization of Plants and Plant-Derived Materials for Sustainable Agriculture and Renewable Energy, PhD Dissertation, May 2018
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Gleason, J. B., USDA-NIFA Food Safety Outreach Project Director National Meeting Blacksburg, VA, Aug 20-22, 2018. Invited Presentation: Using Innovative Media to Reach Key Audiences with Food Safety Messages
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: E. G. Johnson, M. M. Dewdney, M. Danyluk, J. H. Graham, S. Santra. 2018. Zinc nanoparticles mitigate some fruit symptoms of Huanglongbing on citrus. Phytopathology. 2018. 108(10): S.157
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: M. M. Murata, S. Santra, E. G. Johnson. 2018. Zinc nanoparticles for potential control of Huanlgongbing on citrus. Phytopathology. 108(10): S.149
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: J. D. Veloso Dos Santos, M. M. Murata, K. Gerberich, D. B. Bright. E. G. Johnson. 2018. Limited movement of Candidatus Liberibacter asiaticus in split-root citrus provides a model system for local and systemic effects of Huanlgongbing. Phytopathology. 108(10): S.127
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: H. Mendis, S. Santra, M. Young, P. Rajasekaran, E. G. Johnson, L. De La Fuente. 2018. Evaluation of novel Zinc-based antimicrobial formulations to control growth and biofilm formation of Xanthomonas citri and Liberibacter crescens. Phytopathology. 108(10): S151


Progress 03/01/17 to 02/28/18

Outputs
Target Audience:The main target audience for this project is citrus growers. Other target audiences include students, industry partners, regulators and the general public. Undergraduate and graduate studentand post-doc training was a key audience with a focus onhow to conduct research and the exposure to interdisciplinary fields ofnanotechnology, chemistry, engineering, and agriculture. This included general understanding of the challenges of each and the value of bringing different disciplines together to solve a larger problem. Growers, industry partners, and regulators were the target audience of efforts to get Zinkicide commercially manufactured and registered for use on citrus. Animations, videos and the webpage target citrus growers and citrus industry stakeholers, consumers, school children, local news media in Florida and as introductory support to graduates, scientific peers and scholarly audiences. They were shown at grower trade shows and school outreach and were available for general audiences on the project website. Information continued to be 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?Multiple students and postdocs have had the opportunity to present results from this project at professional conferences providing valuable professional development in networking and presenting and receiving feedback on their research. Many of these conferences have been interdisciplinary allowing the students and postdocs a chance to learn about different aspects of research related to food production from engineering, product development, plant pathology, entomology, and horticulture from both academic and industry perspectives. Students, post-docs and staff from multiple nonagricultural disciplines have attended various grower meetings in Florida, including the Citrus Show and Citrus Expo to gain exposure to the challenges that their products will face in real world application of citrus groves and other agricultural fields. How have the results been disseminated to communities of interest?Results have been presented at multiple grower events throughout Florida and presented at the UF/IFAS citrus extension booth at some of these events, which has also provided interaction with potential industry partners for EPA registration. The website has provided a platform to provide information including a new animated video describing Zinkicide to the general public. The results of this project have also been disseminated to the scientific community through posters and presentations at multiple conferences covering multiple subject areas. What do you plan to do during the next reporting period to accomplish the goals?We will determine the optimum application frequency for Zinkicide to provide the most efficacy against CLas in greenhouse plants in preparation for field trials to improve Zinkicide efficacy on mature citrus trees. We will continue monitoring field trials treated with the fixed agricultural grade fromulation. Understanding Zinkicide movement in planta will continue with surrogates as necessary.R.Johnson: To establish potential effects on honey bees we will perform USEPA Tier 1 testing on individual honey bee adults through topical application, 10-day feeding in sugar syrup and larvae administration to in vitro reared larvae. This work will be performed with the final formulation for Zinkicide.Additional videos and animations are in development. The NMSU team is working with the Zinkicide team to finalize the next round of media tools by July, 2018. Economic analysis will become a larger focus as yield results come in.

Impacts
What was accomplished under these goals? Zinkicide development has moved from a research material to a commercially viable formulation that is currently made in small batches by an industry partner from fertilizer feedstocks. The latest formulation of agricultural grade Zinkicide actually outperforms reagent grade Zinkicide in improving fruit quality and quantity on young citrus trees with an up to 70% increase in fruit yield per tree. Tree size is emerging as a major limitation for efficacy, which is likely due to dilution effects as the nanoparticles move from the site of application to the phloem in the branches, trunk, and structural roots. This is supported by the fact that foliar spray and soil drench are more effective combined than separate. Initial nontarget toxicology experiments are promising for minimized environmental impact, normally lower than standard copper bactericide. Objective 1 Development, improvement, and characterization of Zinkicide nanoparticles. (Santra, Tetard, Gesquire) Optimization and characterization of Zinkicide (Research grade and Agri-grade) We pursued the characterization of the materials synthesized by Dr. Santra's team to determine the stability and other properties of the material. Chemical states of Zn in Zinkicide TMN 110 and TMN 113 We have developed a X-Ray-Excited Auger Electron Spectroscopy (XAES)/Photo Electron Spectroscopy (XPS) study to analyze Zn in TMN 110 and TMN 113 materials. XAES/XPS is a surface characterization technique that can provide valuable information on the chemical nature of Zn in the sample. Computational analysis was performed to model the chemical activity of Zinkicide particles to better understand mode of action Objective 2 Test the efficacy of new Zinkicide formulations and optimize field application for HLB control while minimizing non-target effects on beneficial organisms. (E. Johnson, Dewdney, Graham, Danyluk, De La Fuente) Field trials This was the first year using a final fixed agricultural grade formulation provided by Dr. Santra's industrial partner, that has limited license for Zinkicide, Trademark Nitrogen Inc. We have not completed the mature Valencia harvest, but the young (5 year old) grapefruit field trial has been harvested demonstrating up to 70% yield improvement over the untreated control that is receiving standard grower practices. Fruit size also showed a significant increase, although juice quality was not measurably different. We also collected juice and peel oil for Zn residue analysis by the Santra and Tetard lab groups. De La Fuente (this paper will be submitted in May) The in vitro antimicrobial activity of a Zinkicide™, a ZnO based nanoformulation, was evaluated in batch cultures and under flow conditions, using Liberibacter crescens (Lcr) as a biological model for Liberibacter spp. 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. (Santra, R. Johnson, Petridis, Tetard, Gesquire, E. Johnson, De La Fuente) Time course inoculations with citrus canker were performed after soil drench application to determine the length of efficacy. Formulation differences were observed to give highly variable results, suggesting that activity of systemic Zinkicide in the apopoplast is highly dependent on formulation. These results did not always match up with field HLB data, suggesting that leaf apoplast is not a perfect bioassay for systemic activity of a material in phloem tissue. Experiments to test the direct residual activity lifespan against CLas have been initiated in the greenhouse using RNA as a reporter and different frequencies of application. De La Fuente With the goal of studying the effect of Zinkicide™ on natural soil microflora, we have been collecting soil samples in the experimental plot (Thayer block, Florida), where Zinkicide™ is being assessed. Samples were collected in July and August 2016, August 2017, and the last sampling is planned for August 2018. At each time point, we sample two trees per each treatment (n=4, N and S per tree, two trees): untreated control, Zinkicide™ Drench application, and Zinkicide™ spray inoculation. All samples have been frozen, and DNA was extracted. After the last sampling this year we will conduct a metagenomics analysis to understand the impact of Zinkicide™ in non-target microorganisms. R. Johnson: Conducted preliminary bioassays testing spray application on honey bee adults, consumption by honey bee adults through contaminated pollen and in vitro rearing of larval honey bees. Effect of dialysis on the TMN 110 particle size. We studied the effect of dialysis on the TMN 110 particle size. Zinkicide is designed to be degraded with time and eventually metabolized in the plant system. It is extremely challenging to study the rate of degradation of Zinkicide in planta due to their ultra-small size and lack of sophisticated characterization tools. Zinkicide releases Zn ions in solution. It is therefore expected that Zinkicide will release Zn ions once delivered to plant system. In general, rate of dissolution of nanoparticles is higher than their micron size particles[6]. 24hr after root drench the plant was sectioned for analysis. For Raman studies, we used plant extract collected from leaves of the upper and lower parts of the seedlings (Figure 8). For the SEM and EDS studies, we collected leaf sections from bottom part of the leaf and stem section from the middle part of the shoot. The remaining leaves of the seedlings were grounded to powder form (using the spice and nut grinder) for XRF measurements. ?Objective 4 Evaluate the economic feasibility of using Zinkicide to manage HLB compared to existing methods of citriculture in the presence of HLB. (Singerman) Based on data from trials on fruit weight by tree for 10 different treatments provided by Dr. Johnson, I estimated grapefruit on-tree revenue per tree and the differential revenue with respect to the control. The results were then extrapolated to obtain estimates on a per acre basis. Such results were presented as part of a poster displayed at the UF booth at the Florida Citrus show in January 2018. So far extension efforts at the beginning of the project will be focused on surveying growers to establish the costs of citrus production under the current standard practices. ?Outreach 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. An animated video was produced describing the major threat of HLB and how Zinkicide nanoparticles can overcome many of the barriers to developing an effective bactericide treatment. It also explains the efforts by the research team to produce ensure that Zinkicide will be environmentally safe. Outreach objective 2. Provide training for safe handling and field use of nanoparticles Will be developed as Zinkicide gets closer to final stages of development and availability for growers Outreach objective 3. Provide in-service training on Zinkicide for extension agents from major citrus producing regions Will be developed as Zinkicide gets closer to final stages of development and availability for growers

Publications

  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2018 Citation: Mendis, H. S Santra, M. Young, P. Rajasekaran, E. Johnson, and L. De La Fuente. Evaluation of novel Zinc-based antimicrobial formulations to control growth and biofilm formation of Xanthomonas citri and Liberibacter crescens. International Congress of Plant Pathology, Boston, USA, July 29-August 3, 2018.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Takat Rawal, Computational Study of Zinc Oxide Nanoparticle: Insights from Quantum Mechanical Calculation, MISA Symposium; Orlando, FL 11/06/17
  • Type: Other Status: Published Year Published: 2017 Citation: E. Johnson. 2017. Citrus canker management for 2017. Citrus Industry. 98:3 p. 16-18.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: J. Orrock, H. Davis, E. G. Johnson. 2017. Rapid lateral movement between sieve tubes by Candidatus Liberibacter asiaticus in split root trees. International Research Congress on Huanglongbing. Orlando, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: E. G. Johnson, M. M. Dewdney, S. Santra, J. H. Graham, M. Danyluk. 2017. Zinkicide: A nanotherapeutic for HLB. MISA (Materials Innovation for Sustainable Agriculture) Symposium, University of Central Florida, Orlando, Florida, November 6-7, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Johnson, E. G. 2017 Citrus Huanglongbing in the roots: the hidden half of a systemic disease. Georgia Association of Plant Pathology. March 8, 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Gleason, J. B. & Martinez, P. N., Annual Meeting of the USDA S-1056 Multi-State Project on Enhancing Microbial Food Safety by Risk Analysis, Portland, Maine. Using Innovative Media to Reach Key Audiences (October 2017).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Gleason, J. B. & Martinez, P. N., Annual Meeting of the USDA NC-1023 Multi-State Project on Enhancing Microbial Food Safety by Risk Analysis, Washington State University, Pullman, WA. (December 2017). How educational media enhance public understanding of new scientific development.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Gleason, J. B., Muise, A. S., & Dewdney, M.M. Zinkicide SCRI Annual Meeting, January 8, 2018, Citrus Research & Education Center, Lake Afred, FL, "Outreach and online website for the Zinkicide Project."
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Naranjo, E., S Santra, M. Young, P. Rajasekaran, E. Johnson, and L. De La Fuente. In vitro antimicrobial activity of ZinkicideTM against Liberibacter crescens, a surrogate of Candidatus Liberibacter asiaticus. Journal of Citrus Pathology, iocv_journalcitruspathology_34714, p. 31. Proceedings of the 5th International Research Conference on Huanglongbing, Florida, March 15-17 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Mendis, H. S Santra, M. Young, P. Rajasekaran, E. Johnson, and L. De La Fuente. Evaluation in vitro of novel antimicrobial compounds to control growth and biofilm formation of citrus bacterial pathogens. Journal of Citrus Pathology, iocv_journalcitruspathology_34714, p. 29. Proceedings of the 5th International Research Conference on Huanglongbing, Florida, March 15-17 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Naranjo, E., H. Mendis, S. Santra, M. Young, P. Rajasekaran, E. Johnson, and L. De La Fuente. In vitro antimicrobial activity of ZinkicideTM against Liberibacter crescens, a surrogate of Candidatus Liberibacter asiaticus. MISA (Materials Innovation for Sustainable Agriculture) Symposium, University of Central Florida, Orlando, Florida, November 6-7, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: MRS Fall 2017: Soliman, M., Edmunds, W., Rajasekaran, P.,Young, M. Santra, S., Labbe, N., Tetard, L., Understanding the Uptake and Translocation of Zinc-Based Treatments in Citrus Plants to Combat Citrus Greening Disease, Boston, MA, USA, November 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: NanoFlorida 2017: Soliman, M., Tetard, L., Studying Different Modes of Uptake of Zinc-based Treatments Designed to Combat Citrus Greening Disease at Florida International University, Miami, Florida, USA, September 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: NanoScience Technology Center Graduate Student Seminar: Soliman, M., Tetard, L., Studying the Uptake and Translocation of Zinc-based Treatments Aimed at Tackling Phloem Restricted Diseases in Citrus Plants at NanoScience Technology Center, Orlando, Florida, USA, April 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Florida AVS 2017: Soliman, M., Tetard, L., Zinc Based Treatment to Combat Greening Disease in Plants - A Nanoscale Study at University of Central Florida, Orlando, Florida, USA, March 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Materials Innovation for Sustainable Agriculture Symposium 2017: Soliman, M., Khater, A., Edmunds, W., Rajasekaran, P.,Young, M. Santra, S., Labbe, N., Tetard, L., Analyzing the Uptake and Translocation of Systemic Zinc-based Treatment Developed to Combat Citrus Greening Disease, at University of Central Florida, Orlando, Florida, USA, November 2017  Awarded 2nd place
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: NanoFlorida 2017: Soliman, M., Edmunds, W., Rajasekaran, P.,Young, M. Santra, S., Labbe, N., Tetard, L., Tracking the Uptake and Translocation of Zinc-based Treatments Designed to Combat Citrus Greening Disease at Florida International University, Miami, Florida, USA, September 2017  Awarded 3rd place
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 2017 Joint Nanoscience and Neutron Scattering User Meeting: Soliman, M., Edmunds, W., Rajasekaran, P.,Young, M. Santra, S., Labbe, N., Tetard, L., Tracking the Uptake and Translocation of Zinc-based Treatments Designed to Combat Citrus Greening Disease at Oak Ridge National Laboratory, Knoxville, Tennessee, USA, August 2017  Awarded 1st place
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 2nd Annual UCF Society of Emerging Nanotechnology Conference: Soliman, M., Edmunds, W., Rajasekaran, P.,Young, M. Santra, S., Labbe, N., Tetard, L., Zinc Based Treatment to Combat Greening Disease in Plants - A Nanoscale Study at University of Central Florida, Orlando, Florida, USA, April 2017  Awarded 1st place
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: MRS Fall 2017: Tetard, L., A nanoscale view of plant tissues and their response to external stresses, Boston, MA, USA, November 2017
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Graduate Research Forum 2017: Soliman, M., Edmunds, W., Rajasekaran, P.,Young, M. Santra, S., Labbe, N., Tetard, L., Zinc Based Treatment to Combat Greening Disease in Plants - A Nanoscale Study at University of Central Florida, Orlando, Florida, USA, April 2017  Awarded 2nd place
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Florida AVS 2017: Soliman, M., Edmunds, W., Rajasekaran, P.,Young, M. Santra, S., Labbe, N., Tetard, L., Zinc Based Treatment to Combat Greening Disease in Plants - A Nanoscale Study at University of Central Florida, Orlando, Florida, USA, March 2017
  • Type: Theses/Dissertations Status: Submitted Year Published: 2018 Citation: Mikhael Soliman, Advanced Nanoscale Characterization of Plants and Plant-derived Materials for Sustainable Agriculture and Renewable Energy, Spring 2018
  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2018 Citation: Naranjo, E., E. P�rez-L�pez, Merfa, M.V., M. Jain, M.J. Davis, D. Gabriel, and L. De La Fuente. Liberibacter crescens, a presumed bacterial plant pathogen, forms biofilm in vitro. International Congress of Plant Pathology, Boston, USA, July 29-August 3, 2018.
  • Type: Theses/Dissertations Status: Submitted Year Published: 2018 Citation: Eber Naranjo (Advisor L. De La Fuente) M.S. Thesis. Novel ZnO nanoparticle activity against Liberibacter crescens growth and biofilm formation. Department of Entomology and Plant Pathology, Auburn University. Defense scheduled for 4/27/2018


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.