Zinkicide for HLB: Where Efficacy and Registration Stand. Evan Johnson, Megan Dewdney, Jim Graham, Swadesh Santra. Citrus Expo August 16, 2018

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1 Zinkicide for HLB: Where Efficacy and Registration Stand Evan Johnson, Megan Dewdney, Jim Graham, Swadesh Santra Citrus Expo August 16, 218

2 Zinkicide what is it? Zinc oxide based nanomaterial Developed by Dr. Swadesh Santra at UCF Plant metabolizable building blocks Designed for cell to cell movement Size of GFP protein <6 nm for systemic movement Stronger bacterial inhibition than copper Immobile surface particle Systemic particle

3 Zinkicide Systemic activity Root uptake (soil drench) Phloem availability confirmed in UCF experiments 4x reduction in total metal for local systemic activity This data led to too low application rate in early field trials Canker lesions (% of control) A a B Firewall a BC b Zinkicide SG6 Spray C Zinkicide SG6 Drench b 1/4 Zinkicide SG6 Spray b * * 1/4 Zinkicide SG6 Drench a Trial 1 Trial 2 C Actigard c

4 Zinkicide Reagent to Ag grade materials Reagent grade Agricultural grade 11 th attempt <1 nm particles Stability - Slowly settles ~4 nm particles Stability - No settlement In proper storage container

5 Zinkicide Canker efficacy Not all ZnO nanoparticles are the same Reagent grade Early Ag grade formulation Canker incidence (%) a b b 1. Copper A.5 Copper B.5 Zinkicide SG4.5 Zinkicide SG c c a Treatment (lbs metallic/acre) b cd c de 1. Copper A.5 Copper B.25 Copper B.25 Zinkicide SG4.25 Zinkicide SG6.14 Zinkicide SG4.14 Zinkicide SG6 e e c Canker incidence (%) Copper A old canker new canker C C C C.5 Copper B 1. Copper B 1. Copper C Zinkicide SG6 Ag grade.5 Zinkicide SG6 Ag grade Treatment (lbs metallic/acre) B B D 1. Zinkicide SG6 A

6 Zinkicide HLB field Trials Monthly application year round Expect management, not cure Grapefruit 6 year old trees in Indian River Randomized design with 25 trees in 5 plots Spray only, drench only, spray+drench at multiple rates Valencia 2+ year old trees on Ridge Randomized complete block design 2 rates for spray only, 1 rate for drench and spray+drench Yield, fruit size, and juice quality assessed at harvest

7 Zinkicide Fruit size increase 1..8 Reagent grade Lab-Grade Zinkicide application (lbs metallic Zn/acre) Early Ag grade formulation Preliminary Ag-grade Zinkicide application (lbs metallic Zn/acre) Final Ag grade formulation Early Ag grade formulation Grapefruit 215 Grapefruit 216 Grapefruit 217 small fruit Valencia 216 medium fruit large fruit Proportion of Fruit foliar.5 foliar Lab-Grade Zinkicide application (lbs metallic Zn/acre) Dose dependent fruit size increase Max effect not reached No yield increase..125 foliar + drench.25 foliar + drench.5 foliar + drench Only highest rate spray+drench increased fruit size No yield increase..125 foliar + soil.25 foliar + soil.5 foliar + soil Final Ag-grade Zinkicide application (lbs metallic Zn/acre) Strong fruit size effect. 2. foliar 1. foliar 2. foliar + soil 2. soil Preliminary Ag-grade Zinkicide application (lbs metallic Zn/acre) Only highest rate spray+drench increased fruit size No yield increase

8 Zinkicide HLB yield response 6 year old Grapefruit Harvested fruit weight (lbs fruit/tree) Preliminary Ag-grade Zinkicide.125 foliar.25 foliar.5 foliar.125 soil.25 soil.5 soil.125 foliar + soil.25 foliar + soil.5 foliar + soil Zinkicide applicaiton (lbs metallic Zn/acre) Harvested fruit weight (lbs fruit/tree) D C Fixed Ag-grade Zinkicide BC AB C BC BC BC AB.125 foliar.25 foliar.5 foliar.125 soil.25 soil.5 soil.125 foliar + soil.25 foliar + soil.5 foliar + soil Zinkicide applicaiton (lbs metallic Zn/acre) A

9 Zinkicide HLB Yield Response year old Valencia 218 Harvest Why a less consistent response? Dilution effect Size of tree lbs Fruit per Tree lb/A Spray 1.lb/A Spray Full Spray/Drench Full Drench Zinkicide improves crop Yield Juice quality Response is application dependent Brix-Acid Ratio A B BC BC C 2. lb/a spray 1. lb/a spray 2. lb/a spray+drench 2. lb/a drench Immobile surface particle Phytotoxicity limit to rate (peel damage observed) Greater variability in HLB decline Application timing May have tree size limit Systemic particle

10 Zinkicide reduced hurricane damage in young trees New planting trial Ft. Pierce Trees treated monthly since planting Originally with SG4, 217 with fixed ag-grade formulation Spray + Drench Zinkicide treatment kept majority of leaves and fruit Most other trees stripped of leaves, some of fruit

11 Zinkicide reduced hurricane damage in young trees New planting trial Ft. Pierce Trees treated monthly since planting Originally with SG4, 217 with fixed ag-grade formulation Spray + Drench Zinkicide treatment kept majority of leaves and fruit Most other trees stripped of leaves, some of fruit Foliage Rating E B Zinkicide Spray C Zinkicide Drench Zinkicide Spray/Drench A TSol Spray Treatment C D TSol Drench B TSol Spray/Drench % foliage maintained 1 = -2% 2 = 21-4% 3 = 41-6% 4 = 61-8% 5 = 81-1% Tsol is a soluble (nonnano) zinc formulation that did not show a yield response

12 Efficacy is short-lived Loss of efficacy between sprays Preliminary evidence from greenhouse trials suggests efficacy lost after ~7 days Timing depends on formulation Better for registration Short active residual Need to optimize application schedule Cluster few sprays Would 4 or 8 sprays in 2 month interval be more effective? Las populations may rebound in remaining 3 weeks

13 Zinkicide Optimizing application Bacterial activity RNA based Clas cells per mg (Log) Clas titer DNA based ns ns * ns Control Monthly Biweekly Weekly a ab Before application 5 weeks 2 weeks Old leaves 1-fold reduction in bacterial titer after 2 weeks of biweekly application RNA activity reduced in young leaves Plugging in old leaves may reduce access RNA activity in new growth predicts bacterial titer reductions Biweekly application is most effective Clas cells per mg (Log) b b Control Monthly Biweekly Weekly Young leaves

14 Efficacy Conclusions Zinkicide has systemic activity Functional Agricultural-grade formulation developed Not all ZnO nanoparticles act the same Not just a Zinc response Fixed for EPA registration purposes Zinkicide can improve yield on HLB-affected trees Tree size dependent? Application rate and timing needs to be optimized Cluster fewer sprays more frequently?

15 Registration Status Zinc and Zinc oxide are not registered as actives for any pesticide use Full registration requires a Registrant (Ag-chem company) Original registrant backed out when couldn t get a me too registration (existing active) Final negotiations underway for new registrant Full registration process (GLP certified lab testing) Toxicology Residue Environmental Fate

16 Registration Process Example: Honey Bees Difficult to predict timeline Depends on tier triggered in each case Existing literature on Zn and ZnO from nonagricultural uses may help Tier 1: Laboratory studies on adults and larvae Tier 2: Semi-field studies with whole colonies in tents Tier 3: Full field studies with treated crop

17 Registration Status Prelim Data Not GLP certified Toxicology Adult Honeybees not affected by Zinkicide Honeybee larva sensitive to feeding on rates 1 times in field rate Less toxic to aquatic animals (Fathead minnow larvae) than commercial copper products Residue No significant increase in Zinc content of juice or peel oil

18 Conclusions Zinkicide has systemic activity Zinkicide can improve yield on HLB effected trees Tree size dependent Application schedule needs optimization Functional Agricultural-grade formulation developed Not all ZnO nanoparticles act the same Not just a Zinc response Fixed for EPA registration purposes Registration timeline difficult to predict Preliminary data looks good

19 Team Effort Monty Myers Tony McIntosh Mayara Murata Diane Bright Kayla Gerberich Jian Wu Johnathan Alexander UCF Santra Lab Design and characterization Tetard Lab Detection and characterization Gesquire Lab Detection and characterization UF Dewdney Lab Field Trials Tracey Hobbs and Telva Aguilar Jim Graham Danyluk Lab/Packing house Yield analysis Singerman Lab - Economics Auburn University OSU NMSU De La Fuente Lab Mode of Action/Microfluidics Johnson Lab Pollinator Toxicology Gleason Program Extension and outreach materials ( Oak Ridge National Laboratory Funding Petridis Lab Nanoparticle modelling CRDF USDA-NIFA-SCRI-CDRE