Grant Information

NANOTECHNOLOGY FOR IMPROVING LOCAL SYSTEMIC ACTIVITY OF COPPER (CU)PESTICIDES AND MEASURING SUB-SURFACE CU BIOAVAILABILITY IN PLANT TISSUE

Sponsoring Institution National Institute of Food and Agriculture

Program A1511 - Agriculture Systems and Technology: Nanotechnology for Agricultural and Food Systems

Status COMPLETE

Funding Source AFRI COMPETITIVE GRANT

Division NIFA Non Formula

Reporting Frequency Annual

Project Director Santra, Swadeshmukul

Accession Number 1009091

Grant Number 2016-67021-24992

Project Number FLAW-2015-07823

Proposal Number 2015-07823

Dates 2016-03-01 - 2020-02-29

Grant Year 2016

Cumulative Award Amount $477,000.00

Animal Health Component 10%

Performing Department NanoScience Technology Center

Recipient Organization UNIVERSITY OF CENTRAL FLORIDA
12722 RESEARCH PARKWAY
ORLANDO,FL 32826

Keywords copper
copper-tolerant
locally-systemic
pesticide
quat
tomato
x. perforans

Research Effort Applied (10%)
Basic (80%)
Developmental (10%)

Classification Parameters

Knowledge Area	Subject of Investigation	Field of Science	Percent
212 - Pathogens and Nematodes Affecting Plants	5220 - Pesticides (includes herbicides, insecticides, fungicides, etc.)	2000 - Chemistry	70%
212 - Pathogens and Nematodes Affecting Plants	1460 - Tomato	1100 - Bacteriology	20%
212 - Pathogens and Nematodes Affecting Plants	1460 - Tomato	2020 - Engineering	10%

Non-technical Summary

Bacterial spot is a common disease of tomatoes. In many tropical and sub-tropical regions worldwide, the disease represents a major production constraint due to high temperatures, and high moisture. Infections usually result in leaf and fruit lesions, leaf defoliation, and yield loss of marketable fruits. In regions where the weather conditions are optimal for infection and spread of the disease, bacterial spot disease has been estimated to cause yield losses of up to 50%. Monetary losses due to bacterial spot in southwest Florida alone were estimated at $3,090 per acre based on 2007-08 production costs and market value.Currently, management options for this disease are quite limited. Chemical treatments using the antibiotic streptomycin was successfully used in 1950s. As strains developed resistance to streptomycin in due course, the antibiotic becameineffective andcopper (Cu)-based bactericides became the chemical of choice for disease management. However, as a result of repeated applications and continuous use, Cu resistance in bacterial spot strainsis nowadays the norm.To overcome this, we propose touse advances in nanotechnology tointroduce a new disease management strategy for bacterial spot of tomato. We will focus on materials innovation to develop a locally-systemic pesticide that will exhibit higher efficiency against Cu resistant bacteria, even at concentrations that are markedlylower than the current best practices, thereforereducing impact of Cu accumulation in the environment.The interdisciplinary team will (1)develop industrially-viable bi-modallocally-systemic pesticideformulations efficient to target Cu resistant bacterial strains, (2)evaluate efficacy of thelocally-systemic pesticideand grower standard against Cu-resistant bacteriain vitroand tomato bacterial spot disease in greenhouse and field conditions,and(3)perform high resolution structural, subsurface imaging and spectroscopy studies to measure distribution and composition oflocally-systemic pesticideand grower standard in plant tissues.The fundamental understanding of the nanoparticle-bacteria interactions and their effect on plants at the molecular as well as at the system level will have a strong impact on plant science and sustainable agriculture, providing a strong experimental framework for the nascent field of material innovation for sustainable agriculture.

Goals / Objectives

The overall goal of this project is to use advances in nanotechnology to design and develop novel Locally-Systemic Pesticide (LSP) particle for improving efficacy of Cu bactericides against Cu resistant plant pathogens and reducing impact of Cu accumulation in the environment. To achieve this we will push the boundaries of nanometrology to enable measuring LSP particle distribution and composition in plant tissue at the molecular/nanoscale level.This proposal introduces a novel concept of developing LSP particles and will meet at least two program objectives. (i) Innovative ideas/fundamental science and (ii) Enhanced crop protection/food security. The optimized LSP formulation composition (including active and inerts) will contain chemicals, which are EPA approved for "Food Use" only. If successful, the LSP technology will revolutionize the agriculture industry by providing powerful tools to farmers for combating "difficult to manage" bacterial diseases such as tomato bacterial spot.Specific objectives are:

Develop industrially-viable bi-modal LSP formulations containing mixed-valence Cu nanoparticles and Quaternary Ammonium compound (Quat) (Santra)We will (1) synthesize silica "seed" particles of three different sizes (50 - 100 nm, 100 - 300 nm and 400 - 600 nm) using StÃ¶ber sol-gel method with some modifications; (2) grow Cu loaded silica shell using our previously published synthesis protocol with further modifications to create mixed-valence Cu; (3) apply Quat coating over the mixed-valence core-shell Cu loaded silica nanoparticles to obtain the proposed LSP particles.All LSP formulations will be characterized for size distribution, morphology and composition using standard analytical techniques (FE-SEM, AFM, DLS, XPS, FT-IR and Raman spectroscopy, TGA and DTA, and AAS).To evaluate plant tissue damage (phytotoxicity) potential of the LSP materials, in growth chamber, plants will be sprayed with LSP test samples. Plants will be monitored for potential tissue damage for one week.Rainfastness of LSP formulations will be evaluated by spraying the LSP formulation on tomato plants until it runs off. Once dried, plant will beexposed to a series of rain conditions. Next, leaves and fruits will be collected to quantify the concentration of metallic Cu using AAS and Quat selective chemical assay.
Evaluate in vitro efficacy of LSP and grower standard against Cu-resistant X. perforans (Santra, Paret, Jones)In this task, preliminary antibacterial efficacy studies will be done for screening a series of LSP formulationsusing Turbidity assay and Microplate Alamar Blue Assay (MABA)and determine the MIC values. We will then select 2-3 most promising candidates for evaluating efficacy in greenhouse and field conditions.CFU assays will be used to study two X. perforans strains, GEV485 (Cu-resistant) and 91-118 (Cu-sensitive), isolated from tomato in Florida. The effect of treatments including LSP at 100, 200, 500, and 1000 ppm and copper prepared from KocideÂ® 3000 at 1000 ppm will be compared.
Evaluate efficacy of LSP and controls against tomato bacterial spot disease in greenhouse and field conditions (Paret, Jones)The following treatments will be used: 100, 200, 500 and 1000 ppm of LSP; KocideÂ® 3000 (2.1 g/liter) by itself, a combination of KocideÂ® 3000 (2.1 g/liter) and PenncozebÂ® 75DF (United Phosphorus, Inc., King of Prussia, PA, USA; 1.2 g/liter); and sterile tap water. We will use 16 plants per treatment in each experiment and the experiment will be repeated twice. The LSP materials and control suspensions will be applied as foliar sprays onto both the underside and upper sides of three to four week old Bonnie Best tomato leaves until run off and will be allowed to dry thoroughly before bacterial inoculation. Cu-resistant X. perforans strain GEV485 will be used in the greenhouse experiments. The bacterial suspension will be prepared and sprayed onto both sides of the tomato leaves. The inoculated seedlings will be then placed in plastic bags to maintain high humidity for 48 h. The bags will be carefully removed and the inoculated plants will be rated using the Horsfall-Barratt disease severity scale every other day for 14 days. The area under disease progress curve (AUDPC) will be then calculated by directly taking the midpoint of the percent ranges for each disease severity value.The effective LSP from greenhouse experiments will be tested on field grown tomatoes based on methods established by the co-PIs. Five field trials will be conducted in Florida in Spring of 2016, 2017, 2018 and Fall of 2015 and 2016 at the University of Florida North Florida Research and Education Center in Quincy, FL. Experimental plots will consist of fumigated raised beds covered with white polyethylene mulch. Plots will be maintained throughout the season using standard fertility, irrigation, and crop protection practices recommended for commercial tomato production in Florida. Inorganic fertilizers will be applied to experimental plots based on soil test results and cooperative extension recommendations. Seedlings of 'BHN 602' (BHN Seeds, Immokalee, FL), a bacterial spot susceptible variety will be used for the study. Each entry will consist of 5 replications with 18 plants in each replication. The plants will be sprayed in the early morning at weekly intervals (8 times) starting from 2nd week after transplanting. Disease severity will be rated at weekly intervals using the Horsfall-Barratt scale, to assess the percentage of canopy affected by bacterial spot. Twelve plants from the center of each plot will be marked, and fruit will be harvested from these plants 2 to 3 times during the trial at a mature green/early breaker stage typical of tomato production in Florida and graded based on USDA Grades. Leaves and fruits will be examined for phytotoxicity during the experiment and rated (see Task 4).
Perform high resolution structural, subsurface imaging and spectroscopy studies to measure distribution and composition of LSP and controls in plant tissue (Tetard, Santra)Tomato plants will be grown in Dr. Santra's lab to use for our optimization of the platform. Later in the project, tomato tissues will be provided from field efficacy trials coordinated by Drs. Santra and Paret. Optimization of sectioning of plant tissue cross sections for high resolution analysis will be performed. This step is critical to access the nanoscale feature inside the plant cell wall. We will use a custom-made microtome to section fresh plant samples without the need to freeze, fix or embed the tissues.We will use confocal Raman microscopy as a preliminary screening of the samples to screen large areas of plant tissues to identify changes in plant structure, composition or indicators of nanoparticle presence.We will use high resolution imaging to study possible changes in morphology of the NPs and plant tissues, as well as possible changes in composition of the NPs and plant tissues. We will especially focus on the advancing the fundamental understanding of the mechanisms involved in nanoparticle uptake for locally systemic activity, phytotoxicity and the effect of rainfastness treatments on the plant leaves and fruits.

Methods (unparsed)

The project will generate samples and experimental data from research activities related to:LSP formulations and their optimizationSamples and experimental data resulting from the comprehensive characterization of all LSP formulationsSamples and experimental data resulting from the characterization of formulation properties and residues in tomato plants and fruitsExperimental data from greenhouse and field efficacy studiesThe following data will be collected during the characterization of the samples:Optical, FESEM, AFM, DLS, XPS, FTIR and Raman spectroscopy, TGA and DTA, and AAS data (images or spectra).Assays: Bacterial growth, Minimum Inhibition Concentration (MIC), CFU assaysAt least, three sets of data will be acquired for each set of experiments.The data will be analyzed and presented in graphic and numerical form. Graphics will be generated using Origin, PowerPoint and Adobe Creative Suite software. Images that will be generated from SEM, AFM and optical microscopy will be processed using the software available for respective instruments (Zeiss for SEM, and Witec Project Four, Nanoscope and Gwyddion for AFM).All numerical data will be saved in txt files. Figures will be produced in .eps, .jpg or .tif formats. Documents and presentation will be created using Tex, Word, Portable Document Format (PDF), and PowerPoint.Throughout the project, students will be taught the ethical responsibilities towards data management and sharing. The team of PDs will regularly meet student to discuss experimental design and optimization, data acquisition, data evaluation, numerical calculations to evaluate the progress of the project based on collected data.Data management responsibility:Each co-PD will be responsible for storing the data acquired from the project, under a folder that is accessible to PD. Data developed under the proposed project will be deposited onto a data server at the University of Central Florida's NanoScience Technology Center. Access to these data will be controlled by a username/password scheme for faculty and student working on the proposed project. Physical access to the server is controlled by key entry and is limited to IT staff only. In addition to this a copy will be stored on an additional computer and a third copy will be stored on back-up storage equipment. The team will share data regularly and the data and research plan will be discussed at joint group meetings.Archiving of data and samples:The data server will have a space reserved to preserve the data produced over the duration of the proposed project. The team will maintain their access to the data during and after the completion of the proposed project. In addition, data will also be archived through backup on external hard drives to preserve data in case of loss of server issues. When students collect data at the labs of collaborators, they will be responsible for storing and archiving that data according to policies outlined above. Samples will be stored at the labs were analyses occurred according to safety regulations. All the samples will be labeled and stored.

Methods

The project will generate samples and experimental data from research activities related to:

LSP formulations and their optimization
Samples and experimental data resulting from the comprehensive characterization of all LSP formulations
Samples and experimental data resulting from the characterization of formulation properties and residues in tomato plants and fruits
Experimental data from greenhouse and field efficacy studies

The following data will be collected during the characterization of the samples:

Optical, FESEM, AFM, DLS, XPS, FTIR and Raman spectroscopy, TGA and DTA, and AAS data (images or spectra).
Assays: Bacterial growth, Minimum Inhibition Concentration (MIC), CFU assays
At least, three sets of data will be acquired for each set of experiments.

The data will be analyzed and presented in graphic and numerical form. Graphics will be generated using Origin, PowerPoint and Adobe Creative Suite software. Images that will be generated from SEM, AFM and optical microscopy will be processed using the software available for respective instruments (Zeiss for SEM, and Witec Project Four, Nanoscope and Gwyddion for AFM). All numerical data will be saved in txt files. Figures will be produced in .eps, .jpg or .tif formats. Documents and presentation will be created using Tex, Word, Portable Document Format (PDF), and PowerPoint.Throughout the project, students will be taught the ethical responsibilities towards data management and sharing. The team of PDs will regularly meet student to discuss experimental design and optimization, data acquisition, data evaluation, numerical calculations to evaluate the progress of the project based on collected data.

Data management responsibility: Each co-PD will be responsible for storing the data acquired from the project, under a folder that is accessible to PD. Data developed under the proposed project will be deposited onto a data server at the University of Central Florida's NanoScience Technology Center. Access to these data will be controlled by a username/password scheme for faculty and student working on the proposed project. Physical access to the server is controlled by key entry and is limited to IT staff only. In addition to this a copy will be stored on an additional computer and a third copy will be stored on back-up storage equipment. The team will share data regularly and the data and research plan will be discussed at joint group meetings.

Archiving of data and samples:The data server will have a space reserved to preserve the data produced over the duration of the proposed project. The team will maintain their access to the data during and after the completion of the proposed project. In addition, data will also be archived through backup on external hard drives to preserve data in case of loss of server issues. When students collect data at the labs of collaborators, they will be responsible for storing and archiving that data according to policies outlined above. Samples will be stored at the labs were analyses occurred according to safety regulations. All the samples will be labeled and stored.

Project Timeline Tracking

03/01/16 to 02/29/20
03/01/18 to 02/28/19
03/01/17 to 02/28/18
03/01/16 to 02/28/17

Outputs

Changes / Problems
Nothing Reported

Training & Professional Development
MISA symposium (yearly 2016-2019) PIs Santra and Tetard organized yearly symposia on the topic of Materials Innovation for Sustainable Agriculture. Over the years, all students participating in the projects had the opportunity to present their work (oral and/or poster presentation) and were involved in professional development activities including conference organization, session chairing, etc. PI Paret also organized a session in the 2019 Symposium. The symposium was designed to provide opportunities for students to interact with stakeholders. A special time was set aside during the poster session. In the last 2 editions of the Symposium, a "Young Leader Session" was organized to showcase the work by graduate students and postdoctoral fellows working on projects related to Materials Innovation for Sustainable Agriculture. Students were involved with the organization of conferences, including MISA. A. Ozcan was a session chair to discuss nanotechnology and societal impacts in the "Young Scientists Series". Florida Agriculture Expo held on Nov 2, 2016 in the Gulf Coast Research and Education Center, Balm, Florida. The team interacted directly with several vegetable farmers and other farming community members and shared information about the project. Team members also interacted with several pesticide companies and obtained the latest product information. Education: Tetard and Santra incorporated findings for the research to several lectures offered in undergraduate (ISC3424 Chemical and Life Science Nanotechnology, BSC3424 Nanobiotechnology) and graduate courses (IDS6250 Introduction to Nanoscience and Nanotechnology, IDS6261 Nanotechnology for Sustainable Agriculture). Outreach: UCF team offered a number of lab tours to promote research for undergraduate students. In additional, the team participated in STEM Days on a yearly basis, to engage K-12 students from the Orlando community in science. Several students volunteered in the PIs' labs as a result of these activities. Some of the students won science fair prizes at the local, county and national levels. Awards: Liao Y-Y. 2nd Place, Graduate Student Oral Presentation Competition, Southern Division - American Phytopathological Society Annual Meeting, 2020 - Ying-Yu Liaog; Liao Y-Y. Graduate Student Council Travel Award, University of Florida for attending at presenting at the Southern Division - American Phytopathological Society Meeting, 2020 - Ying-Yu Liaog; A, Khater. Poster Presentation Finalist Awardee, Showcase of Undergraduate Research Excellence (2019). "A multimodal evaluation of copper uptake to combat bacterial spot disease in tomato plants." Ozcan, A. Exemplary Oral Presentation Award; "Development of Locally Systemic Pesticide (LSP) particles for bacterial spot disease of tomato", May 2019, UCF Graduate Forum 2019. Ozcan, A. 2nd prize, MISA 2018 annual symposium, Orlando, October 31st 2018 - November 1st 2018, "Development of Locally Systemic Pesticide (LSP) particles against bacterial spot disease of tomato." Khater, A. 1st Place Overall Undergraduate Oral Presentation. NanoFlorida 2018. "A multimodal evaluation of copper uptake to combat bacterial spot disease in tomato plants." Ozcan, A. 2nd prize, Summer workshop on industry application of Nanotechnology, July 29 - August 4, 2018, "Boosting antimicrobial activity of copper-based pesticides with nanotechnology. Lee, B. Finalist Awardee, Poster presentation at Materials & Research Society (MRS) Fall Conference (2017). "Materials innovation for sustainable agriculture and its applications for bacterial diseases." Lee, B. 1st Place Poster Presentation in Energy and Environment, NanoFlorida 2017, Florida International University, Miami, FL. "Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans." Lee, B. Finalist Awardee, Poster Presentation for Annual Joint Nanoscience and Neutron Scattering Meeting at Oak Ridge National Laboratory (2017), Oak Ridge, TN. "Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans."

Dissemination Streams
2019 B. Lee, A. Ozcan, A. Khater, M. Doomra, M. Young, S. Santra, L. Tetard. (2019). AFRI: Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring subsurface Cu bioavailability in plant tissues. Vanderbilt University, Nashville, TN for the 2019 USDA AFRI Grantees Conference. Ozcan, "Development of Locally Systemic Pesticide (LSP) particles for bacterial spot disease of tomato", May 2019, UCF Graduate Forum 2019. A. Khater, B. Lee, A. Ozcan, M. Soliman, S. Santra, L. Tetard. "A multimodal evaluation of copper uptake to combat bacterial spot disease in tomato plants." Showcase of Undergraduate Research Excellence, University of Central Florida, April 2019. 2018 A. Ozcan, M. Doomra, M. Young, S. Santra," Development of Locally Systemic Pesticide (LSP) particles against bacterial spot disease of tomato", MISA symposium, Orlando, FL, October 31st 2018- November 1st 2018. A. Khater, B. Lee, A. Ozcan, M. Soliman, S. Santra, L. Tetard. "A multimodal evaluation of copper uptake to combat bacterial spot disease in tomato plants." MISA symposium, University of Central Florida, October 31st 2018- November 1st 2018. A. Ozcan, "Boosting antimicrobial activity of copper-based pesticides with nanotechnology", Summer workshop on industry application of Nanotechnology, July 29th 2018 - August 4th, 2018. A. Ozcan, M. Doomra, M. Young, S. Santra, "Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue", Gordon Research Seminars, June 2- June 3, 2018. A. Ozcan, M. Doomra, M. Young, S. Santra," "Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue", Gordon Research Conference, June 3- June 8, 2018. B. Lee, A. Ozcan, M. Doomra, N. Castaneda, P. Rajasekaran, H. Kang, S. Santra, & L. Tetard. Physicochemical characterization of Soft Matter: Applications for studying Bacterial Pathogenicity. Poster session presented at the University of Central Florida Graduate Research Forum, April 2018. A. Khater, B. Lee, A. Ozcan, M. Soliman, S. Santra, L. Tetard. "A multimodal evaluation of copper uptake to combat bacterial spot disease in tomato plants." Showcase of Undergraduate Research Excellence, University of Central Florida, April 2018. A. Khater, B. Lee, A. Ozcan, M. Soliman, S. Santra, L. Tetard. "A multimodal evaluation of copper uptake to combat bacterial spot disease in tomato plants." American Vacuum Society, University of Central Florida, March 2018. 2017 Lee, B., Ozcan, A., Rajasekaran, P., Santra, S., Tetard, L. "Materials innovation for sustainable agriculture and its applications for bacterial diseases". Materials Research Society Fall Meeting & Exhibit, Boston, MA, November 26 - December 1, 2017. Ozcan, A., Lee, B., Young, M., Fan, Q., Strayer, A., Liao, Y., Smith, S., Doomra, M., Rajasekaran, P., Jones, J., Paret, M., Tetard, L., Santra, S. "Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring subsurface Cu bioavailability in plant tissue". Materials Research Society Fall Meeting & Exhibit, Boston, MA, November 26 - December 1, 2017. Khater, A., Solimon, M., Lee, B., Ozcan, A., Santra, S., Tetard, L. "Evaluating Copper uptake to combat Bacterial Spot Disease in tomato plants using elemental analysis". 2nd annual Materials Innovation for Sustainable Agriculture (MISA) symposium, University of Central Florida, Orlando, FL, November 6 - 7, 2017. Lee, B., Ozcan, A., Rajasekaran, P., Santra, S., Tetard, L. "Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans". 2nd annual Materials Innovation for Sustainable Agriculture (MISA) symposium, University of Central Florida, Orlando, FL, November 6 - 7, 2017. Ozcan, A., Lee, B., Young, M., Fan, Q., Strayer, A., Liao, Y., Smith, S., Doomra, M., Rajasekaran, P., Jones, J., Paret, M., Tetard, L., Santra, S. "Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring subsurface Cu bioavailability in plant tissue". Annual NanoFlorida Conference, Florida International University, Miami, FL, September 23 - 24, 2017. Lee, B., Ozcan, A., Rajasekaran, P., Santra, S., Tetard, L. "Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans". Annual NanoFlorida Conference, Florida International University, Miami, FL, September 22 - 24, 2017. Khater, A., Solimon, M., Lee, B., Ozcan, A., Santra, S., Tetard, L. "Evaluating Copper uptake to combat Bacterial Spot Disease in tomato plants using elemental analysis". Annual NanoFlorida Conference, Florida International University, Miami, FL, September 23 - 24, 2017. Lee, B., Ozcan, a., Rajasekaran, P., Santra, S., Tetard, L. "Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans". Oak Ridge National Laboratory Annual Joint Nanoscience and Neutron Scattering User Meeting, Oak Ridge, TN, July 27 - August 4, 2017. Briana Lee, Laurene Tetard. Nanoscale investigation of bacterial responses to stress. Florida American Vacuum Society (FLAVS), University of Central Florida, Orlando, Florida, March 6, 2017 Ali Ozcan, Young, M., Bittman, J., and Santra, S. Nanotechnology for Improving Local Systemic Activity of Copper (Cu) Pesticides. Florida American Vacuum Society (FLAVS), University of Central Florida, Orlando, Florida, March 6, 2017 Briana Lee, Laurene Tetard. Nanoscale investigation of biophysicochemical responses to induced stress in bacteria. 2017 Scientista Symposium, New York, April 8-9, 2017 Briana Lee, Laurene Tetard. Nanoscale investigation of biophysicochemical responses to induced stress in bacteria. 2nd Annual Emerging Research Showcase, UCF Society of Emerging Nanoscience and Nanotechnology, University of Central Florida, Orlando, Florida. April 17, 2017 (Awarded second place). Ali Ozcan, Briana Lee, Mikaeel Young, Q Fan, A Strayer, Y-Yu Liao, S Smith, JB Jones, ML Paret, L Tetard, S Santra, Nanotechnology for Improving Local Systemic Activity of Copper (Cu) Pesticides and Measuring Sub-Surface Cu Bioavailability in Plant Tissue, NIFA-AFRI Nanotechnology Grantees' Conference 2017, NIFA Building, Washington, DC, May 18-19, 2017 2016 Ali Ozcan, Mikaeel I. Young, John Bittman, Briana Lee, Amanda Strayer, Ying Yu Liao, Laurene Tetard, Mathews L. Paret, Jeffrey B. Jones, Swadeshmukul Santra. Nanotechnology for improving for local systemic activity of copper (Cu) pesticide and measuring sub-surface Cu bioavailability in plant tissues, Materials Innovation for Sustainable Agriculture symposium, University of Central Florida. Orlando, Florida. Oct 3-4, 2016. Mikaeel Young, M., Ozcan, A., Lee, B., Strayer, A., Liao, Y., Tetard, L., Paret, M. L., Jones, J. B. and Santra, S., Nanotechnology for Improving Local Systemic Activity of Copper (Cu) Pesticides and Measuring Sub-Surface Cu Bioavailability in Plant Tissue, USDA Nanotechnology Grantees Annual Meeting, The Millennium Science Building, Agricultural Sciences and Industries Building, Penn State University, University Park, PA, June 6 - 7, 2016. Briana Lee, Laurene Tetard. Combined spectrographic and microscopic characterization of bacteria on the nanoscale level, NanoFlorida 2016, University of Central Florida, Orlando, Florida, September 25-26, 2016, Awarded first place for Graduate poster in "Health and Environmental Applications"

Next Reporting Steps
Nothing Reported

Outputs

Target Audience
The target audience of this project includes tomato growers, production managers, tomato and other food crop researchers, industry partners, consumers, regulators and general public. The audience was reached trought the following activities: 2018 NanoFlorida Conference: The 11th Annual NanoFlorida Conference was held during October 5 - 7 at the Florida Institute of Technology in Melbourne, FL. The conference is designed for young scientists in the field of nanotechnology, providing a platform for networking and exchanging scientific knowledge. A full session on Nanotechnology and Agriculture was organized by the PI. 2018 MISA Annual Symposium: 2018 Materials Innovation for Sustainable Agriculture (MISA) Symposium was conducted in the University of Central Florida Campus on October 31-November 2, 2018. The symposium had representatives from all the target audience population. About 70 attendees participated to the event. Scientists from the US (states represented: Florida, Alabama, Tennessee, Texas, California, Ohio, New York, Washington D.C.) as well as international visitors from Brazil, Costa Rica, and Indiawere in attendance. The symposium also hosted the local growers and packers who appreciated the opportunity to interact with scientists. 2018 Gordon research conference "Nanoscale Science and Engineering for Agriculture and Food Systems - Nano-Enabled Technologies to Improve Efficiency, Quality, and Health in Food and Agriculture": the team participated to this event and presented several oral presentations, and a poster to highlight the work carried out in the course of the project.

Changes / Problems
Nothing Reported

Training & Professional Development
MISA 3rd Annual Symposium (Orlando, Florida) - October 31st - November 1st, 2018 - The 2018 MISA Symposium had 69 attendees. Represented by 6 Countries, 8 Universities, 4 Governmental Agencies, 5 Industry Partners and 24 Invited Speakers. MISA Session at NanoFlorida Conference (Melbourne, FL) - October 5-6, 2018. 6 Faculties, 2 postdocs and 16 Student gave presentation at the Nanotechnology for agriculture session. Awards: Exemplary Oral Presentation Award;"Development of Locally Systemic Pesticide (LSP) particles for bacterial spot disease of tomato", May 2019,UCF Graduate Forum 2019, 2nd prize, MISA 2018 annual symposium, Orlando, October 31st 2018- November 1st 2018, "Development of Locally Systemic Pesticide (LSP) particles against bacterial spot disease of tomato" 2nd prize, Summer workshop on industry application of Nanotechnology, July 29 - August 4, 2018, "Boosting antimicrobial activity of copper-based pesticides with nanotechnology

Dissemination Streams
Contributed oral presentations "Development of Locally Systemic Pesticide (LSP) particles for bacterial spot disease of tomato", May 2019,UCF Graduate Forum 2019 "Nanotechnology for improving local systemic activity of copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissues", June 2018,Gordon Research Seminars, Massachusetts. "Nanoscale Imaging and Spectroscopy of Plant Systemsand Pathogens", June 2018,Gordon Research Conference, Massachusetts. "Locally Systemic Pesticide (LSP) Approach against Bright Spot Disease of Tomato", May 2018, Florida Chapter of theAVS science and Technology Society, Young Leaders Session "Development of Locally Systemic Pesticide (LSP) particles for bacterial spot disease of tomato", May 2018,UCF NSTC Graduate Research Seminar Series "Nanoscale investigation of mode of antibacterial activity of multivalent nanoparticles on Xanthomonas perforans." Invited talk, Florida Institute of Technology for NanoFlorida 2018. "Multiscale Exploration of Plants' Responses to External Physicochemical Factors." Invited talk, Florida Institute of Technology for NanoFlorida 2018. "A multisystem approach for the characterization of bacteria for sustainable agriculture." Physics Women Society at the University of Central Florida, 2018. Contributed posterpresentations B. Lee, A. Ozcan, A. Khater, M. Doomra, M. Young, S. Santra, L. Tetard. (2019). AFRI: Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring subsurface Cu bioavailability in plant tissues. Vanderbilt University, Nashville, TN for the 2019 USDA AFRI Grantees Conference. A. Ozcan,M. Doomra, M. Young,S. Santra," Development of Locally Systemic Pesticide (LSP) particlesagainst bacterial spot disease of tomato",MISA symposium, Orlando, FL,October 31st2018- November 1st2018 B. Lee, A. Ozcan, M. Doomra, N. Castaneda, P. Rajasekaran, H. Kang, S. Santra, & L. Tetard," Materials innovation for sustainable agriculture and its applications for bacterial diseases.",MISA symposium, Orlando, FL,October 31st2018- November 1st2018 A.Ozcan, "Boosting antimicrobial activity of copper-based pesticides with nanotechnology",Summer workshop on industry application of Nanotechnology, July 29th2018 - August 4th, 2018 A. Ozcan,M. Doomra, M. Young,S. Santra, "Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue", Gordon Research Seminars, June 2- June 3, 2018 A. Ozcan,M. Doomra, M. Young,S. Santra," "Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue", Gordon Research Conference, June 3- June 8, 2018 B. Lee, A. Ozcan, M. Doomra, N. Castaneda, P. Rajasekaran, H. Kang, S. Santra, & L. Tetard. Physicochemical characterization of Soft Matter: Applications for studying Bacterial Pathogenicity. Poster session presented at the University of Central Florida Graduate Research Forum, 2018

Next Reporting Steps
LSPII and LSPIII will be evaluated under field conditions in fall 2019, spring 2020 and fall 2020 for reduction in bacterial spot disease severity, phytoxicity and yield impacts.

Impacts (unparsed)

What was accomplished under these goals? Task 1: Develop industrially-viable bi-modal LSP formulations containing mixed-valence Cu nanoparticles and Quaternary Ammonium compound (Quat) (Santra): Morphological characterization: LSP sizes were characterized using Dynamic Light Scattering, Scanning Electron Microscopy, High-Resolution Transmission Electron Microscopy. Meanwhile, HRTEM imaging revealed the presence of ultra-small size (~5 nm) Cu hydroxide nanoparticles embedded in the silica shell. The composition was confirmed by infrared spectroscopy. Kinects of active ingredients release: Release rates of Cu and Quat actives from LSP were quantified using AAS and LC-MS, respectively. 95% of Quat molecules were released from LSP after 4 washes with DI water. Meanwhile, Cu release remained fairly constant after 8 washes. Therefore, when compared with bulk CuO and Cu2O materials, LSP continuously demonstrated higher release of soluble Cu overall. Rainfastness: leaf adherence properties of LSP particles were assessed using a leaf-washing experiment following foliar spray. Cu active concentrations were quantified using AAS in the washed-off solution. Results indicated leaves treated with LSP particles still retained 90% of Cu material on the leaf surface after 3 washes, while only 36% of Cu remained for the commercial standard. Phytotoxicity: toxicity of LSP against ornamental Vinca plants was evaluated 72 h after foliar spray. LSP was determined to be non-phytotoxic with concentrations as high as 1,000 Î¼g/mL Cu and 250 Î¼g/mL Quat. This is compared to bulk Cu sulfate, which was phytotoxic at 1,000 Î¼g/mL Cu. Phytotoxicity was also assessed in 2 greenhouse studies on tomatoes conducted at UF-NFREC in Quincy, FL, and no phytotoxicity was observed for LSP particles (I, II and III). Task 2: Evaluate in vitro efficacy of LSP and grower standard against Cu-resistant X. perforans (Santra, Paret, Jones) In-vitro antimicrobial efficacy was tested on Cu-tolerant X. perforans. LSP was found effective in inhibiting growth at as low as 10 Î¼g/mL Cu, and 2.5 Î¼g/mL Quat after 24 h of treatment. Comparatively, commercial Cu standard did not demonstrate inhibition even at the highest tested concentration (100 Î¼g/mL Cu). In-vitro, metallic copper from LSP at 100 Î¼g/mL inhibited the copper-tolerant X. perforans strain within 1 h of exposure. The study is complemented by nanoscale studies of the effect of the treatment on the mechanical and chemical properties of the bacteria. The work unveiled significant differences in mechanical properties of copper-tolerant and copper-sensitive bacteria, as well as changes in chemistry and mechanical properties in the bacteria undergoing treatments. The effect of having only Quat,, only Cu or both in the LSP formulation was also studied. The results suggest a synergistic effect of the two active ingredients in the LSP formulation. The in vitro study was repeated again, which validated the antibacterial effects of LSP particles demonstrated in the first experiment. Task 3: Evaluate efficacy of LSP and controls against tomato bacterial spot disease in greenhouse and field conditions (Paret, Jones) Two greenhouse experiments were conducted to evaluate the effect of LSP on bacterial spot disease severity. The first greenhouse experiment study indicated that LSP II particle exhibited the best disease management ability compared to water control. This was followed by LSPIII particle. The second greenhouse experiment was inconclusive with high disease severity across all treatments including untreated, Kocide 3000 and all LSP particles. Task 4: Perform high-resolution structural, subsurface imaging and spectroscopy studies to measure distribution and composition of LSP and controls in plant tissue (Tetard, Santra) The distribution of the treatment at the surface of the leaves was evaluated. Washing protocols were optimized to selectively remove the treatment residues at the surface of the leaves without affecting the inside composition of the leaves. Studies to determine an efficient washing procedure was carried out. Electron microscopy and x-ray fluorescence spectroscopy were used to determine the distribution of treatment on the leaves surface before and after washing. Once the washing protocol was established, high-resolution structural, subsurface imaging and spectroscopy were carried out to evaluate Cu uptake in the plants. The effect of having one or two actives in the formulation was evaluated and revealed that the presence of Quat improves the uptake. Cu was found to move in the plant. The content is sink vs source leaves was also evaluated. Publications

Outputs

Target Audience
The target audience of this project includes tomato growers, production managers, tomato and other food crop researchers, industry partners, consumers, regulators and general public. The audience was reached trought the following activities: 2017 NanoFlorida Conference: The 10th Annual NanoFlorida Conference was held during September 23-24, 2017 at Miami, FL. The conference is designed for young scientists in the field of nanotechnology, providing a platform for networking andexchanging scientific knowledge. 2017 MISA Annual Symposium: 2017 Materials Inovation for Sustainable Agriculture (MISA) Symposiumwas conducted in the University of Central Florida Campus on November 6 and 7 of 2017. The symposium had representatives from all the target audience population. Visiting scientists from states such as Alabama, Tennessee, Arizona, California, Washington D.C., and all across Florida, as well as international visitors from France and Brazil were in attendance. The symposium also hosted the local growers and packers who appreciated the opportunity to interact with scientists. More than 30 faculty members, 15 industry affiliates and 35 students (21 graduates and 14 undergraduates) joined the symposium. Materials Reseach Society (MRS) meeting: 7 graduate students and 3 faculty members of MISA team joined the MRS Fall Conference in Boston, MA, on November 2017. All members participate on the special symposium entitled "Materials Innovation of Sustainable Agriculture and Energy". This special symposium was selected as the broader impact session by the MRS Organizing Committee (http://www.mrs.org/fall-2017-symposium-sessions).

Changes / Problems
Nothing Reported

Training & Professional Development
1. NanoFlorida 2017 Conference (Miami, Florida) - September 23-24. NanoFlorida brings together people from academiaandindustry in the field of nanoscience and nanotechnology accross Florida, providing a platform for networking andexchanging scientific knowledge. The member of this team, UCF students and Faculty participated in this event. 2. 2017 MISAAnnual Symposium (Orlando, Florida) - November 6-7, 2017. Participants:32 Faculty members, 15 Industry Affiliates,35 Students (21 Graduates and14 Undergraduates). 3. MRS Fall Conference (Boston, MA) - November 2017. Atendees from MISA team:3 Faculty members and7 Graduate students.

Dissemination Streams
Contributed oral presentations Khater, A., Solimon, M., Lee, B., Ozcan, A., Santra, S., Tetard, L. "Evaluating Copper uptake to combat Bacterial Spot Disease in tomato plants using elemental analysis". Annual NanoFlorida Conference, Florida International University, Miami, FL, September 23 - 24, 2017. Ozcan, A., Lee, B., Young, M., Fan, Q., Strayer, A., Liao, Y., Smith, S., Doomra, M., Rajasekaran, P., Jones, J., Paret, M., Tetard, L., Santra, S."Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue". Annual NanoFlorida Conference, Florida International University, Miami, FL, September23 - 24, 2017. Contributed posterpresentations Lee, B., Ozcan, A., Rajasekaran, P., Santra, S., Tetard, L. "Materials innovation for sustainable agriculture and its applications for bacterial diseases". Materials Research Society Fall Meeting & Exhibit, Boston, MA, November 26 - December 1, 2017. Ozcan, A., Lee, B., Young, M., Fan, Q., Strayer, A., Liao, Y., Smith, S., Doomra, M., Rajasekaran, P., Jones, J., Paret, M., Tetard, L., Santra, S."Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue". 2017 Materials Research Society Fall Meeting & Exhibit, Boston, MA, November 26 - December 1, 2017. Khater, A., Solimon, M., Lee, B., Ozcan, A., Santra, S., Tetard, L. "Evaluating Copper uptake to combat Bacterial Spot Disease in tomato plants using elemental analysis". 2nd annual Materials Innovation for Sustainable Agriculture (MISA) symposium, University of Central Florida, Orlando, FL, November 6 - 7, 2017. Lee, B., Ozcan, A., Rajasekaran, P., Santra, S., Tetard, L. "Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans". 2nd annual Materials Innovation for Sustainable Agriculture (MISA) symposium, University of Central Florida, Orlando, FL, November 6 - 7, 2017. Lee, B., Ozcan, A., Rajasekaran, P., Santra, S., Tetard, L. "Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans". Annual NanoFlorida Conference, Florida International University, Miami, FL, September 22 - 24, 2017. Lee, B., Ozcan, a., Rajasekaran, P., Santra, S., Tetard, L. "Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans". Oak Ridge National Laboratory Annual Joint Nanoscience and Neutron Scattering User Meeting, Oak Ridge, TN, July 27 - August 4, 2017.

Next Reporting Steps
For the next report period, we plan to dothe following activities in order to accomplish the project goals: The translocation of LSP particles as well as Cu and Quat actives in tomato plant system will be characterized to understand the efficacy of LSP materials. The greenhouse studies will be continued to understand the role of actives and size dependency efficacy for field trials. The most promising 1-2 LSP materials including proper controls will be sent for the evaluation.

Impacts (unparsed)

What was accomplished under these goals? Task 1: Develop industrially-viable bi-modal LSP formulations containing mixed-valence Cu nanoparticles and Quaternary Ammonium compound (Quat) (Santra): In the previous reporting period, 3 different sizes of LSP particles with average sizes 50nm, 200 nm, 600 nm weresuccessfully synthesized from 3 different sizes of silica seed particles. In this reporting period LSP particles were furtheroptimized to achieve (i) high active loading, (ii) high dispersion properties in DI water upon mixing and (iii) improved surface hydrophobic surface wetting properties mimicking leaf surface. LSP particles with active concentration up to 13.5% metallic Cu and 3.4%Quat (w/w) were successfully developed. HR-TEM characterization proved that ultra-small CuNPswere embedded in the silica gel around LSP particles ranging size between 3 to 8 nm. Fast Fourier Transform (FFT) analysisof CuNPs were further carried out and lattice fringes 1.93A correspond to CuO and 1.79A correspond to Cuprite were found. LSP particles' hydrophobic surface wettingproperties were tested on a parafilm covered microscope slide and LSP particles demonstrated better surface wettingcompare to Kocide 3000 (film-forming commercial copper hydroxide based bactericide). Retention of LSP particles on tomatoplant leaf surface were tested upon foliar-spraying LSP particles to 4.5-inch tomato plants and quantifying Cu content on theleaf surfacevia AAS after drying. It was found out that tomato leaves sprayed with LSPparticles demonstrated average0.70 mg Cuper gram of leaves, while untreatedleaves had 0.275 mg of Cu per grams of leaves. Leaves treated withKocide 3000 demonstrated 0.675 mg Cu per grams of leaves, which is comparable to to LSP particles.The rainfastness properties of LSPparticles in different rain conditions for tomato plants will be tested this year. Task 2: Evaluate in vitro efficacy of LSP and grower standard against Cu-resistant X. perforans (Santra, Paret, Jones) LSP particles in-vitro antimicrobial efficacy was tested against both Cu tolerant (GEV485) and sensitive strains of X. perforans(91-118) via broth microdilution method. LSP particles demonstrated Minimum Inhibitory Concentration (MIC) 2 ppm Cu &0.5ppm Quat while MIC values for Kocide 3000 are 250 ppm for Cu sensitive and 500 ppm for Cu tolerant strains. In order to understand the role of actives and release of actives from LSP particles,in-vitro antimicrobial efficacy was further tested after washingwith DI water. It was found out that LSP particles had MIC value of 32 ppm even after 5 washes and 10washes.Thisindicates that Quat molecules are released from LSP particles after each washes and the efficacy was preserved further dueto loaded CuNPs. The colony-forming unit (CFU) assay was carried out against Cu tolerant X. perforans bacteria. The testing was carried outagainst 3 of LSP materials and Kocide 3000 at concentrations 100, 200, 500 and 1000 ppm copper. Commercialcontrol Kocide 3000 was tested at concentrations similar to LSP formulations, while DI water was used as the negativecontrol. The materials were incubated with bacteria suspension for 24 hours and number of colonies formed were countedafter plating. The results demonstrated that all 3 LSP formulations fully eradicated the bacteria at all tested concentrations. Incomparison, Kocide 3000 were not aseffective at even 1000 ppm concentration of treatment, where 16% of bacteria was stillviable compare to untreated control. Task 3: Evaluate efficacy of LSP and controls against tomato bacterial spot disease in greenhouse and field conditions (Paret, Jones) Greenhouse efficacy of 3 LSP materials (LSP 1; 50 nm, LSP 2; 200 nm. LSP 3; 600 nm)were carried out against Cu tolerant X. perforans (GEV 485)bacteria strain using four week old Bonnie Best tomato plants. The controls includesteriletap water and commercial Cu control Kocide 3000. 16 plants were foliar sprayedwith 3LSP materials andcontrolat spray rates 100, 200 and 500 ppm Cu concentration. The materials were allowed to dry before the bacteria inoculation.Plants were sprayed with bacteria suspension atconcentration 5x10^8 CFU/mL. The disease severity scale wasassessed every 2 daysfor 14 days. The area under disease progress curve (AUDPC) was calculated and the results were shown that LSP 1 and LSP 3 at 100 ppm providedsimilar protection as Kocide 3000 at 200 ppm. Among all the materialsand control, LSP 2 provided the best protection at 500 ppm spray rate, followed by LSP 1 and LSP 3 at the same spray rate. Kocide 3000 did not providedisease controlat 500 ppm compare to water treatment. Task 4: Perform high-resolution structural, subsurface imaging and spectroscopy studies to measure distribution and composition of LSP and controls in plant tissue (Tetard, Santra) During this reporting period, we have continued to successfully measure molecular interactions between (i) LSP particles and bacteria and (ii) LSP particles and plant tissue. Characterization of LSP particle with cell membrane components of bacteria was divided into (a) chemical analyses and (b) physical modifications. (i) LSP particles and bacteria Chemical analyses were conducted at various scales. Population studies were performed using Fourier Transform Infrared spectroscopy (FTIR) and Raman spectroscopy to identify successful cell wall disruption by LSP particles. Single bacteria studies were pursued, first using Raman confocal microscopy (~300 nm in lateral resolution) and later using infrared nanospectroscopy (Nano-IR) was used to determine chemical variations on the nanoscale. By these chemical analyses, we successfully identified significant variations caused to cell membrane polysaccharides, structural proteins, and lipids which corroborated expected results based on the properties of the particle. Physical analysis was conducted using optical microscopy and Atomic Force Microscopy (AFM). Through nanoscale morphology imaging with AFM we compared surface roughness with various treatments. Results suggested evidence of significant destruction occurring with the tandem LSP Copper and Quaternary Ammonium particle to the bacterial membrane surface. Lastly, we have begun probing the changes in observations made between bacterial membrane/particle interactions in an ambient environment and within liquid/in-vivo environments. With this comparison, we can investigate the associated changes to the mechanical properties, such as flexural rigidity, which will determine other behavioral responses to treatment, such as mobility. For this we developed a novel protocol combining persistence length measurements (previously done on bio-fibrils with fluorescence microscopy) to AFM. We evaluated and validated the approach and used it to show that the mechanical properties of the bacteria vary upon treatment. (ii) LSP particles and plant tissue Characterization of the interaction of the LSP particle with plant tissues was performed by applying the treatments by foliar spray. Leaves, stems and roots were characterized separately. X-Ray fluorescence spectroscopy (XRF) was performed to determine the content in copper in the tissues. Raman and IR spectroscopy was carried out on the midrib to study potential changes in the tissue composition as a result of treatments. The results will be analyzed with PCA during the next reporting period. Publications

Outputs

Target Audience
The target audience of this project includes, citrus growers, production managers, citrus and other food crop researchers, industry partners, consumers, regulators and general public. This project is listed as one of the funded ressearch projects under UCF - Materials Innvation for Sustainable Agriculture (MISA; http://nanoscience.ucf.edu/misa/). The UCF MISA is a Center of Excellence recongnized by the USDA-NIFA. Disseminating information to growers and farmers: A team of 19 students and staff members attended the Florida Agriculture Expo held on Nov 2, 2016 in the Gulf coast research and education center, Balm, Florida. 4 students and 2 faculty members of this project team attended this expo. This visit provided the members an opportunity to inform the farming community including but not limited to citrus growers, vegetable farmers and other farming community members alike in the state about the MISA center and its activities. The industry entities were also present in the expo and they were also informed about the center. MISA inaugural symposium: A two-day symposium was conducted in the University of Central Florida Campus on October 3 and 4 of 2016. The symposium had representatives from all the target audience population. The two-day symposium provided the platform for researchers to present their findings pertaining to HLB disease prevention and treatment. Visiting scientists from states such as Alabama, Tennessee, Arizona, California, Washington D.C., and all across Florida were in attendance. The symposium also hosted the local growers and packers who appreciated the opportunity to interact with scientists and more importantly they were excited to learn about the current work that can result in field-usable products to cure citrus canker and citrus greening. 6 graduate students and 3 faculty members of this reesrach team attended the MISA symposium. World wide web: To help with disseminating the information about the activities of the MISA center, a website has been created that can be accessed from anywhere in the world (http://nanoscience.ucf.edu/misa/). This website provides information about all our constituent researchers that are spread across the state of Florida, and rest of the country here in United States and international collaborators from Canada, France and India. The website can also act as a source of information for all the target audience about the work that are currently underway and what products can come out of the projects in the future.

Changes / Problems
Nothing Reported

Training & Professional Development
(i) Eight graduate students (including two Profession Science Masters in Nanoscience and Nanotechnology)were involved in this project and mentored by the PDs. (ii) A number ofteam members attendedthe Florida Agriculture Expo held on Nov 2, 2016 in the Gulf coast research and education center, Balm, Florida. The teaminteracted direcly with several vegetable farmers and other farming community membersand shared information about the project. Team members also interacted with several pesticide companies and obtained latest product information. (iii) The team attended the UCF-MISA inaugural symposium (http://nanoscience.ucf.edu/misa/) in 2016. Scientists, engineeres, growers, agrochemical industry partners and government agencies attended the symposium.Apart from presenting poster, students had the opportunity to interact with thisinterdisciplinary community.

Dissemination Streams
Disseminating information to growers and farmers: A team of 19 students and staff members attended the Florida Agriculture Expo held on Nov 2, 2016 in the Gulf coast research and education center, Balm, Florida. 4 students and 2 faculty members of this project team attended this expo. This visit provided the members an opportunity to inform the farming community including but not limited to citrus growers, vegetable farmers and other farming community members alike in the state about the MISA center and its activities. The industry entities were also present in the expo and they were also informed about the center. MISA inaugural symposium: A two-day symposium was conducted in the University of Central Florida Campus on October 3 and 4 of 2016. The symposium had representatives from all the target audience population. The two-day symposium provided the platform for researchers to present their findings pertaining to HLB disease prevention and treatment. Visiting scientists from states such as Alabama, Tennessee, Arizona, California, Washington D.C., and all across Florida were in attendance. The symposium also hosted the local growers and packers who appreciated the opportunity to interact with scientists and more importantly they were excited to learn about the current work that can result in field-usable products to cure citrus canker and citrus greening. 6 graduate students and 3 faculty members of this reesrach team attended the MISA symposium. World wide web: To help with disseminating the information about the activities of the MISA center, a website has been created that can be accessed from anywhere in the world (http://nanoscience.ucf.edu/misa/). This website provides information about all our constituent researchers that are spread across the state of Florida, and rest of the country here in United States and international collaborators from Canada, France and India. The website can also act as a source of information for all the target audience about the work that are currently underway and what products can come out of the projects in the future.

Next Reporting Steps
(i) Scale up and optimize the synthesis protocol. Transition from reagent grade chemicals to agri-grade chemicals without compromizing product quality. (ii)Deliver 1-2 most promising reagent-grade LSP product formulations to University of Florida collaborators to support preliminary greenhouse studies and field efficacy testing. (iii)Sub-surface imaging and uptake in the plants studies will be completed using reagent-grade LSP product formulation to understand the localization and distribution of particles in the plant tissues. LSP-bacteria interactions will be characterized using the protocols developed in order to establish a deeper understanding of the mechanisms of action of the formulation for bacterial killing. (iv) Based on the feedback from greenhouse and field trial results, we will further improve the LSP product formulation for rainfastness and efficacy.

Impacts (unparsed)

What was accomplished under these goals? Task 1: Develop industrially-viable bi-modal LSP formulations containing mixed-valence Cu nanoparticles and Quaternary Ammonium compound (Quat) (Santra): As proposed, 3 different sizes silica seed particles (to control the overall size of LSP) were synthesized using the StÃ¶ber sol-gel method. Dynamic Light Scattering (DLS) measurements indicated the average (z-average) hydrodynamic size of the seeds particles to be (A) 45 nm, (B) 150 nm and (C) 530 nm. The size of the seed particles was also confirmed using Scanning Electron Microscopy (SEM) and High Resolution Transmission Electron Microscopy (HR-TEM), which indicated particle sizes of 35-50 nm (A), 130-160 nm (B), and 500-600 nm (C). Next, the copper (Cu) loaded silica shell was grown on the seed particles using acid-catalyzed hydrolysis of silica source (TEOS) by adjusting the pH of the solution. The Cu loading and the overall size of particles were characterized using FE-SEM and HR-TEM. Overall core-shell particle sizes were found to be 30 nm-50 nm (A), 130 nm-170 nm (B) and 500-600 nm (C). The shell thickness was found to be in the 10-30 nm range with HR-TEM. HR-TEM also revealed Cu nanoparticles (NP) embedded and distributed in the silica shell with an average diameter of 5 nm. Lattice spacing measurements on Cu NPs revealed an interlayer spacing of ~1.87 Ã…, which corresponds to copper oxide. Finally, the three different formulation of LSP particles, using the three different seed particles with the Cu loaded shells, were achieved upon application of Quat coating (Quaternary ammonium compound) on the synthesized core-shell Cu loaded particles. The interactions between Quat molecules and silica shell were characterized using Fourier Transform Infrared (FT-IR) and Raman spectroscopy. In the IR spectrum, the stretching at 1039 cm-1 was identified as Si-O vibration from Si-O-Si. Pure Quat solution exhibited strong bands corresponding to the N-C bend at 1468 cm-1, which shifted to 1431 cm-1 in LSP, thus confirming the electrostatic interactions between Quat and the core-shell Cu loaded particles. The initial phytotoxicity evaluation of LSP material was conducted on 4.5-inch (height) tomato plants obtained from Home Depot store. The plants were foliar sprayed with different concentrations of LSP particles and were placed in a plant growth chamber (Panasonic MLR-352H; temperature 90 Â°F and humidity 60-70%) for 72 hours after treatment. The phytotoxicity grading was carried out using the following criteria: (-) non-phytotoxic (no brown spots), (+) minimal (a few scattered brown spots), (++) moderate (multiple brown spots on multiple leaves), (+++) severe (large brown spots on most leaves). LSP particles are non-phytotoxic until a concentration of 300 Âµg Cu/mL (75 Âµg Quat /mL). Plants exhibited the first signs of phytotoxicity at 500 Âµg Cu/mL (125 Âµg Quat/mL) for LSP material, while the control plants sprayed with Quat alone showed signs of phytotoxicity at 75 Âµg Quat/mL. The results suggest that silica is an attractive host material to reduce the toxicity of Quat on tomato plants. In addition, we showed that LSP is non-phytotoxic to tomato plants at the commercial spray rate commonly used for Cu-based pesticides (300 Âµg Cu/mL). Task 2: Evaluate in vitro efficacy of LSP and grower standard against Cu-resistant X. perforans (Santra, Paret, Jones) In-vitro antimicrobial efficacy of LSP was tested against Copper (Cu) tolerant Xanthomonas perforans (GEV485) and Cu sensitive Xanthomonas perforans (91-118) bacteria strains. The Minimum Inhibitory Concentration (MIC) determination of LSP particles was completed using broth microdilution method. Serially diluted concentrations of candidate antimicrobial agents were added to Mueller-Hinton broth that was inoculated with 5 x 105 CFU/mL (0.5 McFarland Standards) of bacteria. The lowest dilution of the test agent that exhibited complete inhibition of bacterial growth was assigned as MIC value. MIC results revealed LSP particles inhibiting bacteria growth completely at 2 Âµg Cu /mL (0.5 Âµg Quat /mL) for Cu sensitive Xanthomonas perforans and 2 - 4 Âµg Cu /mL (1-0.5 Âµg Quat/mL) for Cu tolerant Xanthomonas perforans, while Quat alone inhibited bacteria growth completely at 2 Âµg/mL for both bacteria strains. This could be due to the fact that combining Quat and Cu as antimicrobial agents introduces multiple modes of action for killing with a possible "synergistic effect". This aspect will be investigated further in the next reporting period. Overall, LSP particles demonstrated superior antimicrobial efficacy compared to KocideÂ® 3000 (growers standard, insoluble Cu(OH)2 product) which revealed MIC values of 250 Âµg Cu /mL for Cu sensitive and 500 Âµg Cu /mL for Cu resistant bacteria strains. Based on MIC results it can be concluded that LSP particles constitute a very attractive material to overcome Cu tolerance and increase antimicrobial efficacy of standard copper-based materials, in addition to offering multiple modes of action. Task 3: Evaluate efficacy of LSP and controls against tomato bacterial spot disease in greenhouse and field conditions (Paret, Jones) Greenhouse and field efficacy testing will be initiated in Fall 2017. We will evaluate efficacy of a couple of most promising LSP candidates based on in vitro efficacy and phytotoxicity results. We will update our findings in the coming reporting periods. Task 4: Perform high-resolution structural, subsurface imaging and spectroscopy studies to measure distribution and composition of LSP and controls in plant tissue (Tetard, Santra) SEM and Energy-dispersive X-ray Spectroscopy (EDS) measurements were carried out to determine the LSP particle distribution on tomato leaves upon treatment. SEM measurements on leaf surface demonstrated that LSP particles form large aggregates around the stomata supporting our initial hypothesis of a possible movement of LSP particles inside the stomata for locally systemic activity. EDS elemental mapping for Cu and Silica (Si) confirmed the co-localization of the two elements in the material distributed on the tomato leaves, with some LSP particle aggregates distributed and concentrated around the stomata. The distribution of LSP particles in the tomato leaves upon treatment was characterized using X-ray Fluorescence Spectroscopy (XRF) measurements. Tomato plants were foliar sprayed at multiple concentrations of LSP particles and kept in plant growth chamber for 72 hours. At that time, all leaves were collected, washed, air-dried for 96 hours and ground using a spice and nut grinder. XRF measurements demonstrated relative Cu concentration in the Cu treated leaves after treatment when compared to DI water treated leaves. Based on XRF measurements, leaves treated with LSP particle at 300 Âµg Cu /mL (75 Âµg Quat /mL) contained 3 times the Cu content compared to untreated control leaves. The results constitute the initial calibration of the methods for LSP uptake characterization. Quantitative measurements of uptake will be carried out during the next reporting period. Finally, the protocol development for the characterization of LSP-bacteria interactions was completed. A combination of mechanical and infrared spectroscopy measurements can be used to study the bacterial response to the treatment. The localized measurements will allow us to infer the variations present in the LSP-bacteria interactions to be correlated to the MIC and CFU measurements. The measurements will be completed during the next reporting period. Publications

Publications Inventory

Conference Papers and Presentations

A Ozcan, Development of nanotechnology enabled locally systemic pesticide (LSP) particles April 2019, ACS Spring 2019 National Meeting, Orlando FL, 2019 [Other 2019]
A. Ozcan "Development of Locally Systemic Pesticide (LSP) particles for bacterial spot disease of tomato", May 2018, UCF NSTC Graduate Research Seminar Series [Other 2018]
A. Ozcan, "Development of Locally Systemic Pesticide (LSP) particles for bacterial spot disease of tomato", May 2019, UCF Graduate Forum 2019 [Other 2019]
A. Ozcan, "Development of Locally Systemic Pesticide (LSP) particles for bacterial spot disease of tomato, May 2019, UCF Graduate Forum 2019 [Other 2019]
A. Ozcan, Boosting antimicrobial activity of copper-based pesticides with nanotechnology, Summer workshop on industry application of Nanotechnology, July 29th 2018 August 4th, 2018 [Other 2018]
A. Ozcan, Development of Locally Systemic Pesticide (LSP) particles for bacterial spot disease of tomato, May 2018, UCF NSTC Graduate Research Seminar Series [Other 2018]
A. Ozcan, Locally Systemic Pesticide (LSP) Approach against Bright Spot Disease of Tomato, May 2018, Florida Chapter of the AVS science and Technology Society, Young Leaders Session [Other 2018]
A. Ozcan, M. Doomra, M. Young, S. Santra, Development of Locally Systemic Pesticide (LSP) particles against bacterial spot disease of tomato, MISA symposium, Orlando, FL, October 31st 2018- November 1st 2018 [Other 2018]
A. Ozcan, M. Doomra, M. Young, S. Santra, Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue, Gordon Research Conference, June 3- June 8, 2018 [Other 2018]
A. Ozcan, M. Doomra, M. Young, S. Santra, Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue, Gordon Research Seminars, June 2- June 3, 2018 [Other 2018]
A. Ozcan, Nanotechnology for improving local systemic activity of copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissues, June 2018, Gordon Research Seminars, Massachusetts. [Other 2018]
Ali Ozcan, Briana Lee, Mikaeel Young, Q Fan, A Strayer, Y-Yu Liao, S Smith, JB Jones, ML Paret, L Tetard, S Santra, Nanotechnology for Improving Local Systemic Activity of Copper (Cu) Pesticides and Measuring Sub-Surface Cu Bioavailability in Plant Tissue, NIFA-AFRI Nanotechnology Grantees Conference 2017, NIFA Building, Washington, DC, May 18-19, 2017 [Accepted 2017]
Ali Ozcan, Mikaeel I. Young, John Bittman, Briana Lee, Amanda Strayer, Ying Yu Liao, Laurene Tetard, Mathews L. Paret, Jeffrey B. Jones, Swadeshmukul Santra. Nanotechnology for improving for local systemic activity of copper (Cu) pesticide and measuring sub-surface Cu bioavailability in plant tissues, Materials Innovation for Sustainable Agriculture symposium, University of Central Florida. Orlando, Florida. Oct 3-4, 2016. [Accepted 2016]
Ali Ozcan, Young, M., Bittman, J., and Santra, S. Nanotechnology for Improving Local Systemic Activity of Copper (Cu) Pesticides. Florida American Vacuum Society (FLAVS), University of Central Florida, Orlando, Florida, March 6, 2017 [Accepted 2017]
B. Lee "A multisystem approach for the characterization of bacteria for sustainable agriculture." Invited talk, Physics Women Society at the University of Central Florida, 2018. [Other 2018]
B. Lee "Nanoscale investigation of mode of antibacterial activity of multivalent nanoparticles on Xanthomonas perforans." Invited talk, Florida Institute of Technology for NanoFlorida 2018. [Other 2018]
B. Lee, "Multiscale Exploration of Plants' Responses to External Physicochemical Factors." Invited talk, Florida Institute of Technology for NanoFlorida 2018. [Other 2018]
B. Lee, A multisystem approach for the characterization of bacteria for sustainable agriculture. Physics Women Society at the University of Central Florida, 2018. [Other 2018]
B. Lee, A. Ozcan, A. Khater, M. Doomra, M. Young, S. Santra, L. Tetard. (2019). AFRI: Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring subsurface Cu bioavailability in plant tissues. Vanderbilt University, Nashville, TN for the 2019 USDA AFRI Grantees Conference. [Other 2019]
B. Lee, A. Ozcan, M. Doomra, N. Castaneda, P. Rajasekaran, H. Kang, S. Santra, & L. Tetard, Materials innovation for sustainable agriculture and its applications for bacterial diseases., MISA symposium, Orlando, FL, October 31st 2018- November 1st 2018 [Other 2018]
B. Lee, A. Ozcan, M. Doomra, N. Castaneda, P. Rajasekaran, H. Kang, S. Santra, & L. Tetard," Materials innovation for sustainable agriculture and its applications for bacterial diseases.", MISA symposium, Orlando, FL, 2018. [Other 2018]
B. Lee, A. Ozcan, M. Doomra, N. Castaneda, P. Rajasekaran, H. Kang, S. Santra, & L. Tetard. Physicochemical characterization of Soft Matter: Applications for studying Bacterial Pathogenicity. Poster session presented at the University of Central Florida Graduate Research Forum, 2018 [Other 2018]
B. Lee, Multiscale Exploration of Plants' Responses to External Physicochemical Factors. Invited talk, Florida Institute of Technology for NanoFlorida 2018. [Other 2018]
B. Lee, Nanoscale investigation of mode of antibacterial activity of multivalent nanoparticles on Xanthomonas perforans. Invited talk, Florida Institute of Technology for NanoFlorida 2018. [Other 2018]
B. Lee. Nanoscale Characterization for sustainable agriculture: A closer look at Xanthomonas perforans. November 2017, UCF NSTC Graduate Research Seminar Series. [Other 2017]
Briana Lee, Laurene Tetard. Combined spectrographic and microscopic characterization of bacteria on the nanoscale level, NanoFlorida 2016, University of Central Florida, Orlando, Florida, September 25-26, 2016, Awarded first place for Graduate poster in Health and Environmental Applications [Accepted 2016]
Briana Lee, Laurene Tetard. Nanoscale investigation of bacterial responses to stress. Florida American Vacuum Society (FLAVS), University of Central Florida, Orlando, Florida, March 6, 2017 [Accepted 2017]
Briana Lee, Laurene Tetard. Nanoscale investigation of biophysicochemical responses to induced stress in bacteria. 2017 Scientista Symposium, New York, April 8-9, 2017 [Accepted 2017]
Briana Lee, Laurene Tetard. Nanoscale investigation of biophysicochemical responses to induced stress in bacteria. 2nd Annual Emerging Research Showcase, UCF Society of Emerging Nanoscience and Nanotechnology, University of Central Florida, Orlando, Florida. April 17, 2017 (Awarded second place). [Accepted 2017]
Khater, A., Lee, B., Ozcan, A., Soliman, M., Santra, S., Tetard, L. A multimodal evaluation of copper uptake to combat bacterial spot disease in tomato plants. . NanoFlorida 2018, Florida Institute of Technology, Melbourne, FL. [Other 2018]
Khater, A., Solimon, M., Lee, B., Ozcan, A., Santra, S., Tetard, L. Evaluating Copper uptake to combat Bacterial Spot Disease in tomato plants using elemental analysis. 2nd annual Materials Innovation for Sustainable Agriculture (MISA) symposium, University of Central Florida, Orlando, FL, November 6 7, 2017. [Other 2017]
Khater, A., Solimon, M., Lee, B., Ozcan, A., Santra, S., Tetard, L. Evaluating Copper uptake to combat Bacterial Spot Disease in tomato plants using elemental analysis. Annual NanoFlorida Conference, Florida International University, Miami, FL, September 23 24, 2017. [Other 2017]
L. Tetard "Nanoscale Imaging and Spectroscopy of Plant Systems and Pathogens", June 2018, Gordon Research Conference, Massachusetts. [Other 2018]
L. Tetard, Nanoscale Imaging and Spectroscopy of Plant Systems and Pathogens, June 2018, Gordon Research Conference, Massachusetts. [Other 2018]
Lee, B., Ozcan, A., Rajasekaran, P., Santra, S., Tetard, L. Materials innovation for sustainable agriculture and its applications for bacterial diseases. Materials Research Society Fall Meeting & Exhibit, Boston, MA, November 26 December 1, 2017. [Other 2017]
Lee, B., Ozcan, A., Rajasekaran, P., Santra, S., Tetard, L. Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans. 2nd annual Materials Innovation for Sustainable Agriculture (MISA) symposium, University of Central Florida, Orlando, FL, November 6 7, 2017. [Other 2017]
Lee, B., Ozcan, A., Rajasekaran, P., Santra, S., Tetard, L. Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans. Annual NanoFlorida Conference, Florida International University, Miami, FL, September 22 24, 2017. [Other 2017]
Lee, B., Ozcan, a., Rajasekaran, P., Santra, S., Tetard, L. Nanoscale investigation of biophysicochemical responses to multivalent nanoparticle treatment on Xanthomonas perforans. Oak Ridge National Laboratory Annual Joint Nanoscience and Neutron Scattering User Meeting, Oak Ridge, TN, July 27 August 4, 2017. [Other 2017]
Mikaeel Young, M., Ozcan, A., Lee, B., Strayer, A., Liao, Y., Tetard, L., Paret, M. L., Jones, J. B. and Santra, S., Nanotechnology for Improving Local Systemic Activity of Copper (Cu) Pesticides and Measuring Sub-Surface Cu Bioavailability in Plant Tissue, USDA Nanotechnology Grantees Annual Meeting, The Millennium Science Building, Agricultural Sciences and Industries Building, Penn State University, University Park, PA, June 6 - 7, 2016. [Accepted 2016]
Ozcan, A., Lee, B., Young, M., Fan, Q., Strayer, A., Liao, Y., Smith, S., Doomra, M., Rajasekaran, P., Jones, J., Paret, M., Tetard, L., Santra, S. "Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue". Annual NanoFlorida Conference, Florida International University, Miami, FL, September 23 24, 2017. [Other 2017]
Ozcan, A., Lee, B., Young, M., Fan, Q., Strayer, A., Liao, Y., Smith, S., Doomra, M., Rajasekaran, P., Jones, J., Paret, M., Tetard, L., Santra, S. "Nanotechnology for improving local systemic activity of Copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissue". Materials Research Society Fall Meeting & Exhibit, Boston, MA, November 26 December 1, 2017. [Other 2017]
S. Santra, "Nanotechnology for improving local systemic activity of copper (Cu) pesticides and measuring sub-surface Cu bioavailability in plant tissues", June 2018, Gordon Research Seminars, Massachusetts. [Other 2018]

Journal Articles

A. Ozcan "Locally Systemic Pesticide (LSP) Approach against Bright Spot Disease of Tomato", May 2018, Florida Chapter of the AVS science and Technology Society, Young Leaders Session [Other 2018]
Fan Q., Liao Y., Kunwar S., Young M., Santra S., Minsavage J., Sujan T., Freeman J., Jones J.B. Paret M.L. Antibacterial effect of copper composites against Xanthomonas euvesicatoria (in preparation) [Other 2020]
Kah, M.; Divia, N., Kookana, R. S., Kirbi, J. K., Santra, S., Ozcan, A., Shervin, K. "Impact of (nano)formulations on the distribution and wash-off of copper pesticides and fertilisers applied on citrus leaves". Environmental Chemistry, 2019 [Published 2019]
Kah, M.; Divia, N., Kookana, R. S., Kirbi, J. K., Santra, S., Ozcan, A., Shervin, K.; "Impact of (nano)formulations on the distribution and wash-off of copper pesticides and fertilisers applied on citrus leaves". Environmental Chemistry, 2019, https://doi.org/10.1071/EN18279 [Published 2019]
Khater, A., Lee, B., Ozcan, A., Soliman, M., Santra, S., Tetard, L. Evaluating copper uptake through nanotechnology-assisted delivery to combat bacterial spot disease in tomato plants (in preparation) [Other 2020]
Lee, B., Castaneda N., Doomra, M., Modha, N., Santra, S., Thornton, J., Zhang, T., Kang, E. H., Tetard, L. Nanoscale quantification of longitudinal and transverse mechanics of bacterial bodies, Applied Physics Letters, 2020, 116, 053701 [Published 2020]
Lee, B., Castaneda, N., Doomra, M., Santra, S., Thornton, J., Zhang, T., Kang, H., Tetard, L. Nanoscale quantification of longitudinal and transverse mechanics of bacterial bodies , Submitted to Applied Physics Letters, 2019 [Submitted 2019]
Lee, B., Ozcan, A., Doomra, M., Castaneda, N., Modha, N., Kang, E. H., Santra, S., Tetard, L. Resolving multiscale physicochemical changes to Xanthomonas perforans by multimodal core-shell (in preparation) [Other 2020]
Ozcan, A., Young, M, Lee, B., Liao, Y.-Y., Sa Silva, S., Godden, D., Colee, J., Mendis, H.C., Huang, Z., Campos, M., Jones, J.B., Freeman, J.H., Paret, M. L., Tetard, L., Santra, S. Multifunctional locally systemic pesticide particles for the management of bacterial spot disease of tomato [Submitted 2020]
Young, M., Ozcan, A., Rajasekaran, P., Kumrah, P., Myers, M., Johnson, E., Graham, J., Santra, S. Fixed-Quat: An attractive non-metal alternative to copper biocides against plant pathogens, J. Agric. Food Chem., 2018, 66 (50), pp 1305613064 [Published 2018]
Young, M., Ozcan, A., Rajasekaran, P., Kumrah, P., Myers, M., Johnson, E., Graham, J., Santra, S. Fixed-Quat: An attractive non-metal alternative to copper biocides against plant pathogens, J. Agric. Food Chem., 2018, 66 (50), pp 1305613064. doi: 10.1021/acs.jafc.8b04189 [Published 2018]

Theses/Dissertations

Ali Ozcan, Advanced multifunctional bactericides for crop protection: A new strategy to reduce Cu pesticide footprint in the environment. PhD Dissertation 2019 [Published 2019]