Sum ummary o of C CRDF f funded unded R Resea esearch h - PowerPoint PPT Presentation

Sum ummary o of C CRDF f funded unded R Resea esearch h Projec jects: s: 2009 09 - pr presen esent Swadeshmukul Santra University of Central Florida Presented to Citrus Research and Development Foundation February 22, 2017 5/12/2017

Ackn knowled edgem emen ents UCFRF Confidential Document 5/12/2017

Cont ntent nts 1) Description of the materials 2) Evidence of efficacy 3) Commercial readiness (using ag grade chemicals and the like, regulatory) 4) Patent family reference 5) Commercial Sponsored Research and Licensing status 6) Reference to CRDF support (grant titles, period and amount) 5/12/2017 UCFRF Confidential Document

New B Bactericide/Fungi gicide p product c concepts Mixed-Valence Copper-Silica Nanogel Core-Shell Copper-Silica Nanoparticle Core-shell copper-silica nanoparticle (MV- Mixed-valence copper-silica nanogel CuSiNP) is a composite biocide material in which (MV-CuSiNG) is a composite biocide Cu active is embedded in the shell region of a material in which Cu active is core-shell silica particle where the core is pure embedded within the silica gel matrix. silica. The objective is to further reduce Cu per MV-CuSiNG is sprayable non- application. MV-CuSiNP is sprayable non- phytotoxic liquid formulation that uses phytotoxic liquid formulation that uses EPA EPA approves chemicals (active and approves chemicals (active and inerts) and meets inerts) and meets all the industry all the industry requirements for commercial requirements for commercial viability. viability. MV-CuSiNG are shown to be effective MV-CuSiNG are shown to be effective against citrus canker, melanoses and scab even at against citrus canker, melanoses and lower metallic Cu rate in comparison to industry scab even at lower metallic Cu rate in standards. comparison to industry standards. 5/12/2017 UCFRF Confidential Document

New B Bactericide/Fungi gicide p product c concepts Fixed-Quat: A Cu Alternative Copper-Fixed Quat Composite Fixed-Quat is a composite biocide material Copper-Fixed-Quat (Cu-Fixed-Quat) is a in which Quaternary Ammonium composite biocide material in which Cu and Compound (active) is integrated with silica Quat actives are blended with silica gel at the gel matrix. Fixed-Quat is sprayable non- molecular level. The objective is to further phytotoxic liquid formulation that uses EPA reduce metallic Cu per application. Cu-Fixed- approves chemicals (active and inerts) and Quat is sprayable non-phytotoxic liquid meets all the industry requirements for formulation that uses EPA approves chemicals commercial viability. Fixed-Quat are shown (active and inerts) and meets all the industry to be effective against citrus canker, requirements for commercial viability. Cu- melanoses and scab in comparison to Fixed-Quat are shown to be effective against industry standards. citrus canker, melanoses and scab even at lower metallic Cu rate in comparison to industry standards. 5/12/2017 UCFRF Confidential Document

New B Bactericide/Fungi gicide p product c concepts Zinkicide TM T-SOL ZinkicideTM is a ZnO nanoparticles based T-SOL is a Zn-chelate. It is sprayable non-phytotoxic liquid formulation that biocide material. It is sprayable non-phytotoxic liquid formulation that uses EPA approves uses EPA approves chemicals and meets chemicals (active and inerts) and meets all the all the industry requirements for industry requirements for commercial viability. commercial viability. T-SOL is shown to Zinkicide is shown to be effective against citrus be effective against citrus canker, melanoses and scab in comparison to canker, melanoses and scab even at lower metallic Cu rate in comparison to industry industry standards. standards. 5/12/2017 UCFRF Confidential Document

Zinkicide TM : Transition from reagent grade to agri-grade Zinkicide TM Zinkicide TM TMN TMN SG-6 110 SG-4 113 5/12/2017 UCFRF Confidential Document

HRTEM of SG6 (2015 version) Aggregates of <10 nm size crystalline Gel-like morphology showing aggregates particles of particles 5/12/2017 8 Zinkicide TM - UCFRF Confidential Document

HRTEM of TMN 110 (2016-2017 version) Fairly dispersed particles ~2 nm size crystalline particles 5/12/2017 UCFRF Confidential Document

Minimal Inhibitory Concentration (MIC) Conducted using a broth microdilution protocol E. coli X. alfalfae P. syringae Materials (ATCC 10536) (ATCC 49120) (ATCC 19310) (µg/mL) (µg/mL) (µg/mL) 16-31 31-62 31-62 Batch 110 – As synthesized 250 31-62 31-62 Batch 113- As synthesized 125 31-62 31-62 Batch 110 - Dialyzed and lyophilized Batch 113 - Dialyzed and lyophilized 125-250 31-62 31-62 16-31 31-62 31-62 Batch 110 - Rotary evaporated Batch 113 - Rotary evaporated 250 31-62 31-62 250-500 250-500 250 Kocide 3000 125-250 (Cu), 125 (Cu), 125 (Cu), Nordox 30/30 125-250 (Zn) 125 (Zn) 125 (Zn) 5/12/2017 UCFRF Confidential Document

Microfluidic chambers: ‘artificial’ vascular system: Zinkicide efficacy study Flow Outlet Inlets s Inlet for Bacteria Credit: Leonardo De La Fuente, Auburn University 5/12/2017 UCFRF Confidential Document

Evaluation of the preventive antimicrobial activity of Zinkicide TM against L. crescens in microfluidic chambers Materials and Methods 24 hrs post innoculation, the waste of the collecting syringe was aseptically removed to eliminate residual inoculated cells. 2 3 1 Time lapse videos were captured in order to detect Lcr cells Colony Zinkicide TM 30ppm cell division. counting Flow direction After seven days of treatment, the effluent of the microfluidic chamber was collected, OD 600 nm measures were performed, and 100µl of each effluent was streaked in mBM7 agar plates by mBM7 Colony triplicate. Lcr cells Control counting Credit: Leonardo De La Fuente, Auburn University 5/12/2017 UCFRF Confidential Document

Evaluation of the preventive antimicrobial activity of Zinkicide TM against L. crescens in microfluidic chambers 7 days post innoculation Zinkicide TM mBM7 30ppm Control 50 µm Zinkicide T mBM7 Zinkicide TM M 30ppm Control 30ppm Flow direction direction Flow Control 80 µm Flow direction Credit: Leonardo De La Fuente, Auburn University 5/12/2017 UCFRF Confidential Document

Microfluidic chamber: X. citri pv. citri + TMN Zinkicide TM TMN Zinkicide SB Medium 20 µm Credit: Leonardo De La Fuente, Auburn University 5/12/2017 UCFRF Confidential Document

TSOL-SP ( X. citri ) 1.2 Total growth 1.0 Planktonic growth Biofilm 0.8 0.6 OD600 0.4 0.2 0.0 -0.2 0 30 60 90 TSOL-SP concentration (ppm) N=6, Error bars show SE, this experiment was repeated twice Credit: Leonardo De La Fuente, Auburn University 5/12/2017 UCFRF Confidential Document

X. citri Control media before chemical treatment Credit: Leonardo De La Fuente, Auburn University 5/12/2017 UCFRF Confidential Document

X. citri UP: Chemical treatment TSOL-SP DOWN: Control media 5/12/2017 UCFRF Confidential Document Credit: Leonardo De La Fuente, Auburn University

Phytotoxicity conducted on Sour Orange Plants were treated and results recorded after 72 hrs Treatment (ppm) Result - 300 - TMN 110 500 - 800 - 300 - TMN 111 500 - 800 - 300 - TMN 112 500 - 800 - 300 - TMN 113 500 - 800 Result scaling : (-) No observed phytotoxicity, (+) low phytotoxicity, (++) moderate phytotoxicity 5/12/2017 UCFRF Confidential Document

MS3T T formulation on c concep ept • MS3T is a composite material which contains two active ingredients (A.I.), a Zn-chelate (T-SOL) and Quaternary ammonium compound (Fixed-Quat) • A.I. are stabilized and delivered using natural clay (EPK clay; inactive ingredient) • Clay also serves as ACP repellent • T-SOL is micronutrient based, designed for systemic activity. • Fixed-Quat is non-phytotoxic and designed to control surface/sub- surface restricted bacterial and fungal diseases 5/12/2017 UCFRF Confidential Document

SEM of of MS3T an and EP EPK-clay EPK-clay MS3T  MS3T is film-forming  Fixed-Quat and T-SOL contribute to colloidal stability of MS3T formulation 5/12/2017 UCFRF Confidential Document

MS MS3T Mi Minimu mum Inhibitor ory C Concen centr tration ( (MIC) Month 1 Month 2 Month 3 X. alfalfae E. coli P. X. alfalfae E. coli P. syringae X. alfalfae E. coli P. syringae (ppm) (ppm) syringae (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) Material MS3T < 20 (zinc) < 20 < 20 < 20 (zinc) < 20 (zinc) < 20 (zinc) < 20 (zinc) < 20 (zinc) < 20 (zinc) (zinc) (zinc) < 5 < 5 < 5 < 5 < 5 < 5 < 5 (DDAC) < 5 < 5 (DDAC) (DDAC) (DDAC) (DDAC) (DDAC) (DDAC) (DDAC) (DDAC) Clay+T-SOL 80 (zinc) 170 (zinc) - 170 (zinc) 340 (zinc) 80 (zinc) 80 (zinc) 170 (zinc) 80 (zinc) Clay+DDAC 5 (DDAC) 5 (DDAC) - 5 (DDAC) 10 10 5 (DDAC) 10 10 (DDAC) (DDAC) (DDAC) (DDAC) TSOL-U 80 (zinc) 80 (zinc) 170 (zinc) 40 (zinc) 170 (zinc) 80 (zinc) 80 (zinc) 340 (zinc) 80 (zinc) TSOL-UP 40 (zinc) 80 (zinc) 170 40 (zinc) 170 (zinc) 80 (zinc) 40 (zinc) 340 (zinc) 80 (zinc) (zinc)  Antimicrobial efficacy of A.I. in MS3T is not compromised in presence of clay.  Improved efficacy of TSOL is observed in MS3T  MS3T antimicrobial efficacy is maintained for at least 3 months.  Formulation contains all agri-grade chemicals except DDAC. 5/12/2017 UCFRF Confidential Document