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Multimodal GRAS ZnO/nano-Copper composite: A novel antimicrobial material for the management of citrus phytopathogens Mikaeel Imran Young, Ali Ozcan, Monty E. Myers, Evan G. Johnson, James H. Graham, and Swadeshmukul Santra J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02526 • Publication Date (Web): 23 Aug 2017 Downloaded from http://pubs.acs.org on August 23, 2017
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Journal of Agricultural and Food Chemistry
Multimodal GRAS ZnO/nano-Copper composite: A novel antimicrobial material for the management of citrus phytopathogens Mikaeel Young1, 2, Ali Ozcan1, 3, Monty E. Myers5, Evan G. Johnson6 James H. Graham6, * and Swadeshmukul Santra1, 2, 3, 4,*
1
NanoScience Technology Center, 2Burnett School of Biomedical Sciences, 3Department of Chemistry and 4Department of Materials Science and Engineering
University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, FL 32826, USA E-mail:
[email protected] (Santra) 5
Indian River Research and Education Center, University of Florida, 2199 South Rock Road, Fort Pierce, Florida 34945, USA 6
Citrus Research and Education Center, University of Florida, 700 Experiment Road, Lake Alfred, FL 33850, USA. E-mail:
[email protected] (Graham)
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Abstract
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Copper (Cu) bactericides/fungicides are used extensively for crop protection in agriculture.
3
Concerns for Cu accumulation in soil, Cu leaching into the surrounding ecosystem and
4
development of Cu resistance in phytopathogenic bacteria are evident. While there is no suitable
5
alternative to Cu available to date for agricultural uses, it is possible to reduce Cu per application
6
by supplementing with Zn and improving Cu bioavailability using nanotechnology. We have
7
prepared a non-phytotoxic composite material consisting of GRAS (Generally Recognized As
8
Safe) ZnO 800 particles and nano-Copper loaded silica gel (ZnO-nCuSi). The morphology of the
9
ZnO-nCuSi material was characterized using SEM, showing ZnO particles dispersed in the silica
10
gel matrix. The ZnO-nCuSi demonstrated strong in-vitro antimicrobial properties against several
11
model plant bacterial species. Two consecutive year field efficacy results showed that agri-grade
12
ZnO-nCuSi was effective in controlling citrus canker disease at less than half the metallic rate of
13
the commercial cuprous oxide/zinc oxide pesticide.
14
Keywords: copper, silica gel, zinc oxide, nanotechnology, antimicrobial, citrus canker
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Journal of Agricultural and Food Chemistry
Introduction
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The citrus industry maintains a strong agricultural presence in the United States despite
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setbacks from a variety of diseases including citrus canker and Huanglongbing (HLB, also known
19
as citrus greening). The United States produced more than 4.8 million tons of fresh oranges,
20
600,000 tons of grapefruit and 800,000 tons of tangerine/mandarin in 2016-2017
21
(http://www.fas.usda.gov/ psdonline/circulars/citrus.pdf). Even though new challenges are rising,
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technological improvements to protect against infection are at a slower pace. Copper (Cu) based
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pesticides continue to be the most commonly used formulation for crop protection. Cu exhibits
24
antimicrobial properties only at higher concentration (>50 µM) 1, and it is an essential
25
micronutrient for many organisms including plants and animals 2. Copper bactericides/fungicides
26
are the most frequently used metal-based crop protectants for managing bacterial and fungal
27
diseases of a wide variety of crops including citrus, vegetables, stone fruit, legumes, cereals, pome
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fruit and berries
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plague citrus industries worldwide. Application rates and frequency interval of foliar spray vary
30
per crop as well as climate conditions. In Florida citrus groves, citrus crops receive approximately
31
5-15 applications per season, starting from March through October 6-7. Long-term usage of Cu has
32
created numerous limitations: (i) soil Cu accumulation over time that reduces uptake of other
33
essential elements such as Zn, (ii) development of Cu resistance to target microbial species 8-9 and
34
(iii) higher risk of Cu toxicity to the target crop and non-target soil microbiome.
2-4
. Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc)5 continues to
35
Most marketable Cu products such as Cu oxides, Cu hydroxides, Cu oxychlorides have limited
36
solubility in water and therefore form a water-stable film after foliar application. Low solubility
37
greatly minimizes the risk of phytotoxicity but also diminishes Cu bioavailability. In contrast,
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soluble forms of Cu such as Cu chelates, Cu salts have higher bioavailability but may cause severe 3 ACS Paragon Plus Environment
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phytotoxicity if applied at the rate of film-forming Cu. To address these limitations of Cu
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phytotoxicity and bioavailability, we have developed a composite of GRAS (Generally
41
Recognized as Safe) Zinc oxide-nano-Copper silica gel (ZnO-nCuSi) following a modified version
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of our previously published copper silica gel
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materials including TiO2, silica particles, graphene oxide and carbon nanoparticles demonstrate
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the strong potential for nanomaterials in agriculture for crop protection and pathogen detection 11-
45
12
46
protection. However, recent innovations are promoting the development of ZnO as an
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antimicrobial due to its ability to release antimicrobial Zn ions 11, 13. ZnO nanoparticles are being
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investigated for the potential role it may play in agriculture to improve crop nutrient and production
49
14-15
50
possess antimicrobial properties. The copper silica gel coating will additionally serve as a reservoir
51
for both soluble and insoluble Cu compounds.
10
. In the last decade, a series of next generation
. ZnO has traditionally been used as a fertilizer but not labeled as bactericides/fungicides for crop
. The ZnO-nCuSi is therefore a multi-modal antimicrobial system as both copper and zinc
52
We hypothesize that the ZnO-nCuSi will display different modes of action due to the presence
53
of both Cu and Zn. As a result, it is possible to reduce the amount of Cu per application without
54
compromising overall efficacy. Less Cu per application will reduce Cu loading into the
55
environment.
56 57
Materials and Methods
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Materials
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All chemicals were reagent grade and used without any further purification. Copper sulfate
60
pentahydrate was purchased from CQ Concepts (Ringwood, IL), resazurin sodium salt from Sigma 4 ACS Paragon Plus Environment
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(St. Louis, MO, USA). Sodium hydroxide, sodium silicate and phosphate-buffered saline (PBS)
62
were obtained from Fisher Scientific (Pittsburgh, PA). Zinc oxide (ZnO) 800 was received as a
63
sample from Zinc oxide LLC (Dickson, TN). De-ionized (DI) water was obtained from Nanopure
64
water purifier (Barnstead, model # D11911). Nutrient broth (NB) broth and nutrient agar (NA)
65
were purchased from Fluka (St. Louis, MO, USA) while Brain Heart Infusion (BHI) broth and
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agar were purchased from B-D Chemical Co., Inc. (Greenwood Village, CO). Xanthomonas
67
alfalfae subsp. citrumelonis (ATCC 49120), Pseudomonas syringae pv. syringae (ATCC 19310)
68
and Clavibacter michiganensis subsp. michiganensis (ATCC 10202) was acquired from ATCC
69
(U.S. Department of Agriculture permits P526P-12-04060 and P526P-15-01601).
70 71
ZnO-nCuSi synthesis
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The ZnO-nCuSi was synthesized based on our previous core-shell research with
73
modifications 16. Material synthesis was carried out at room temperature under an overhead stirrer
74
(Arrow 6000, Arrow Engineering) to make large volumes for field trials. Briefly, 1.14 kg of copper
75
sulfate pentahydrate was first dissolved in 5.0 L of deionized water to create a solution in a 5 gallon
76
bucket. After 20 minutes of stirring at 600 rpm, the pH of the solution was raised to ~7 using 1M
77
sodium hydroxide. This solution was allowed to stir for an additional 20 minutes before adding
78
100 mL of 37% sodium silicate. Separately, 285 g of ZnO 800 was dispersed into 5L of deionized
79
and stirred at 600 rpm with an overhead stirrer. The first bucket containing the copper silica
80
solution was allowed to stir for at least 4 hr before the dispersed ZnO solution was completely
81
added to it. The combined solution was raised to a volume of 11.3 L with deionized water and left
82
to stir overnight. The ZnO-nCuSi composite was created with a ratio of metallic Cu:Zn of 1:0.8.
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ZnO-nCuSi was compared against commercial cuprous oxide/ zinc oxide which contains a metallic 5 ACS Paragon Plus Environment
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Cu:Zn of 1:1. The approximate metallic Cu concentration is estimated at 25,675 µg/mL along with
85
a metallic Zn concentration of 20,177 µg/mL. The ZnO-nCuSi was used as synthesized (no
86
purification steps were involved to mimic an industrially-viable process for bulk scale production).
87 88
ZnO-nCuSi characterization
89
The viscosity of ZnO-CuSi was determined using a Fungilab Lead One viscometer with a
90
L1 spindle, while the pH and specific gravity were determined with a Mettler Toledo pH meter
91
and balance respectively. Characterization of ZnO-nCuSi was carried out using Field Emission
92
Scanning Electron Microscopy (SEM; Zeiss Ultra-55 FEG) and High Resolution Transmission
93
Electron Microscopy (HR-TEM; FEI Tecnai F30 TEM) techniques. SEM samples were drop cast
94
onto mica wafers while TEM samples were drop cast onto ultra-thin carbon coated gold TEM
95
grids. Structure and morphology of ZnO-nCuSi materials was studied using SEM and HRTEM
96
imaging techniques. SEM and HRTEM studies were conducted at the Advanced Materials and
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Processing Analysis Center - Materials Characterization Facility (AMPAC-MCF) at the University
98
of Central Florida.
99 100
ZnO-nCuSi antimicrobial studies
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The antimicrobial properties of the ZnO-nCuSi were evaluated by determining the
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Minimum Inhibitory Concentration (MIC). Samples were tested against Gram-negative
103
Xanthomonas alfalfae subsp. citrumelonis strain F1 (ATCC 49120, a citrus canker surrogate),
104
Gram-negative Pseudomonas syringae pv. syringae (ATCC 19310, causative agent of bacterial
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speck in lilac, almond, apricots, peaches and wild beans among others) and Gram-positive
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Clavibacter michiganensis subsp. michiganensis (ATCC 10202, causative agent of bacterial wilt 6 ACS Paragon Plus Environment
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and canker in tomato). X. alfalfae and P. syringae were maintained on NA and NB while C.
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michiganensis was grown with BHI medium. All bacteria were grown at 26 ºC. ZnO-nCuSi was
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compared to copper silica gel, ZnO 800, cuprous oxide/zinc oxide, copper hydroxide (commercial
110
industry standard) and copper sulfate (commercial industry standard).
111 112 113
Minimum Inhibitory Concentration (MIC)
114
The MIC values of ZnO-nCuSi along with controls including copper silica gel, ZnO 800,
115
Copper sulfate and copper hydroxide. MIC was carried out using broth microdilution in
116
accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) 17. The
117
accuracy of the MIC determination was improved by adding 10 μL of resazurin dye (0.0125 %
118
w/v) per 100 μL well volume and observing color changes (blue to pink for live organisms). The
119
suitability of this assay was validated for ZnO-nCuSi containing opaque solutions.
120 121
Phytotoxicity Studies
122
Plant tissue safety as evaluated in a Panasonic Environmental Test Chamber (Model MLR-
123
352H) which allowed for controlled day/night cycling temperatures, light intensity and humidity
124
to simulate summer weather conditions (temperature > 80 °F, humidity 60−80%, biocide
125
application season). Studies conducted on tomato the model fruit plant and Vinca sp. an ornamental
126
plant. Treatments were applied early in the morning and results were assessed after 72 hr as
127
previously published 18.
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128 129
‘Ruby’ grapefruit citrus canker trial
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In 2015 and 2016, a field trial was conducted with 8 and 9-year-old non-bearing ‘Ruby’
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grapefruit trees in Ft. Pierce, St. Lucie County, FL. The ZnO-nCuSi was applied at a foliar spray
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of 0.22 Kg/ha metallic Cu and compared with commercial controls cuprous oxide (1.12 Kg/ha Cu)
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and cuprous oxide/zinc oxide (0.5 and 1.01 Kg/ha Cu). It was found previously by the authors and
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others that a rate of 0.5-1.0 Kg/ha Cu is sufficient to achieve strong disease suppression as higher
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rates such as >2.0 Kg/ha did not significantly reduce canker infection further
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spacing was 3.65 m x 7.62 m (360 trees per ha). The experimental design was a randomized
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complete block design with multiple treatments replicated 5 times in blocks of five contiguous
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trees. The untreated check (UTC) trees received a water-only spray treatment at each foliar spray
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time. Materials were mixed with water and applied as foliar sprays at 3.0 L per tree with a handgun
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sprayer at 1380 kPa of air pressure. During the 2015 trial, treatments were initiated after the spring
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flush and were sprayed at roughly 21 day intervals on 4/14, 5/5, 5/26, 6/16, 7/7, 7/28, 8/18, 9/8,
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9/28 and 10/6. Disease evaluation was on 11/02/15. During the 2016 trial, the same methodology
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for treatment was applied with treatments sprayed on 4/19, 5/10, 5/31, 6/20, 7/11, 8/1, 8/22, 9/12,
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10/10 and 10/24. Disease evaluation was on Nov 14. Disease incidence: the incidence of fruit with
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canker lesions was assessed for 100 fruit per treatment from the middle 3 trees in each plot. Lesions
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were classified as “old” if they were larger than 0.6 mm in diameter, coalescing with surrounding
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lesions, black in color, exuding gum or had a prominent yellow halo; and “young” if lesions were
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smaller than 0.6 cm in diameter, brown in color, and were not coalescing with surrounding lesions.
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Disease severity was rated for each fruit based on the estimated number of old or young lesions:
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1= 0 lesions, 2= 1-5 lesions, 3= 6-20 lesions, 4= 21 or more lesions. Monthly rainfall in 2014 was 8 ACS Paragon Plus Environment
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. The tree
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recorded at University of Florida/ IFAS, Indian River Research and Education Center, Ft. Pierce,
152
FL
153
(http://fawn.ifas.ufl.edu/). The monthly rainfall was compared to the average for the last 10 years.
site,
and
obtained
from
the
Florida
Automated
Weather
Network
website
154 155
Results and Discussion
156
Determination of basic formulation characteristics such as pH, specific gravity and
157
viscosity along with the active ingredient characterization is important for developing a
158
commercially viable product. The pH of ZnO-nCuSi formulation was 9.85, which is comparable
159
to Cu hydroxide based industry standards. The specific gravity and viscosity were determined to
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be 1.14 and ~18.8 mPa/s, respectively (Table 1). The size, morphology and state of dispersion of
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ZnO-nCuSi were observed through electron microscopy. The ZnO core material was irregular,
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mostly rod-like (600-1100 nm) in size and fairly dispersed (Figure 1, Panel A). In contrast, the
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ZnO-nCuSi composite appeared irregular in shape. Particle size was in the multi-micron range.
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Dispersibility of the composite was somewhat compromised due to neutralization of surface
165
charge at pH 7.0 (Figure 1, Panel B). HRTEM images (Figure 1, Panel C and D) further
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supported the ZnO-nCuSi morphology as seen under SEM. The ZnO particles appeared in the
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darker contrast relative to the silica gel matrix. As shown in Figure 1, Panel D, Cu crystallites
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measuring 3-6 nm were seen embedded in the silica gel. Average lattice spacing between crystal
169 170
planes was calculated. Values were determined as ∼2.32,∼1.87, ∼2.47, and ∼3.02 Å to reveal Cu
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The in-vitro antimicrobial efficacy of the ZnO-nCuSi was evaluated by determining the
172
MIC against multiple model phytopathogens. Efficacy of ZnO-nCuSi was significantly higher
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compared to ZnO 800, cuprous oxide/zinc oxide, copper silica gel, copper hydroxide and copper
crystallites of cupric oxide and cuprous oxide 22.
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sulfate (MIC results are shown in Table 2). The superior antimicrobial activity of ZnO-nCuSi over
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cuprous oxide/zinc oxide demonstrates that the metals in ZnO-nCuSi are more bioavailable than
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in the other formulations.
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Phytotoxicity testing was conducted for ZnO-nCuSi compared to copper hydroxide,
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cuprous oxide/ zinc oxide, copper sulfate and copper silica gel. Copper sulfate exhibited severe
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toxicity while the other tested compounds showed no phytotoxicity except cuprous oxide/zinc
180
oxide at 1000 µg/mL Cu (Table 3).
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In the 2015 and 2016 citrus canker trials (conducted in Fort Pierce, FL on ‘Ruby’
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grapefruit), ZnO-nCuSi was more efficacious for control of canker incidence on fruit than cuprous
183
oxide, copper sulfate and cuprous oxide/zinc oxide. The average rainfall was higher in 2016 than
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in 2015, which can explain the increase in disease incidence in the untreated control (UTC)
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between the trials (Table 4). Both years exhibited higher rainfall over the ten year average. During
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2015, ZnO-nCuSi reduced incidence of fruit infection to 25% from 60% in the untreated control
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and compared to cuprous oxide (29%), copper sulfate (25%) and cuprous oxide/ zinc oxide (21-
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23 %). ZnO-nCuSi was more effective at a significantly lower metallic Cu rate than the other
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treatments containing Cu (Table 5). During the 2016 trial, ZnO-nCuSi reduced canker incidence
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from 93% to 58% compared to cuprous oxide (56%) and cuprous oxide/zinc oxide (56%) while
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being applied at a significantly lower metallic Cu rate (Table 5). During the 2015 and 2016 trials,
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ZnO-nCuSi activity against fungal diseases, citrus scab and melanose was also assessed. The
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results indicate that ZnO-nCuSi control was comparable to commercial Cu controls at lower
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metallic rates (Table 6).
195 196
The improved protective ability of ZnO-nCuSi points to the parallel antimicrobial modes of action exhibited by zinc and copper. Zinc oxide induces oxidative stress 10 ACS Paragon Plus Environment
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while cupric and
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cuprous oxide have been shown to cause protein inactivation
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membrane integrity through potential disruption 26. Our results confirm the hypothesis that multi-
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modal approach is effective in maintaining efficacy against canker infection at lower Cu
200
application rate. The present ZnO-nCuSi composite formulation contains 25,450 µg/mL metallic
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Cu along with 20,000 µg/mL metallic Zn and the process is scalable.
24
, DNA damage25 and loss of
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ZnO-nCuSi has the potential to become a competing technology due to its several attractive
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attributes: GRAS, low-cost, commercially-viable synthesis process, EPA acceptable actives and
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inerts, reduced Cu exposure in the environment and most importantly strong field efficacy. Future
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studies will be conducted to ascertain the exact mechanistic effect exerted by the multi-modal
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ZnO-nCuSi upon plant pathogens to elucidate its bactericidal activity.
207 208
Acknowledgment
209
We acknowledge the Materials Characterization Facility (MCF) at the UCF Advanced
210
Materials and Processing Analysis Center (AMPAC) where SEM and TEM analysis were
211
conducted. We acknowledge financial support from the Florida Department of Citrus (Grant 186)
212
and the Citrus Research and Development Foundation, Inc. (Grants 328 and 544).
213 214
References
215 216 217 218 219 220
1. Outten, F. W.; Huffman, D. L.; Hale, J. A.; O'Halloran, T. V., The independent cue and cusSystems confer copper tolerance during aerobic and anaerobic growth inEscherichia coli. J. Biol. Chem. 2001, 276 (33), 30670-30677. 2. Borkow, G.; Gabbay, J., Copper as a Biocidal Tool. Curr. Med. Chem. 2005, 12 (18), 2163-2175. 3. Kiely, T.; Donaldson, D.; Grube, A. Pesticide industry sales and usage: 2000 and 2001 market estimates; U.S EPA: Washington, DC, 2004.
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4. Pavlovic, M., A review of Agribusiness copper Use effects on environment. Bulg J Agric Sci 2011, 17 (4), 491-500. 5. Schaad, N. W.; Postnikova, E.; Lacy, G.; Sechler, A.; Agarkova, I.; Stromberg, P. E.; Stromberg, V. K.; Vidaver, A. K., Emended classification of xanthomonad pathogens on citrus. Syst. Appl. Microbiol. 2006, 29 (8), 690-695. 6. Behlau, F.; Belasque Jr, J.; Bergamin Filho, A.; Graham, J. H.; Leite Jr, R. P.; Gottwald, T. R., Copper sprays and windbreaks for control of citrus canker on young orange trees in southern Brazil. Crop Prot. 2008, 27 (3–5), 807-813. 7. Behlau, F.; Belasque Jr, J.; Graham, J. H.; Leite Jr, R. P., Effect of frequency of copper applications on control of citrus canker and the yield of young bearing sweet orange trees. Crop Prot. 2010, 29 (3), 300-305. 8. Behlau, F.; Canteros, B. I.; Minsavage, G. V.; Jones, J. B.; Graham, J. H., Molecular characterization of copper resistance genes from Xanthomonas citri subsp. citri and Xanthomonas alfalfae subsp. citrumelonis. Appl. Environ. Microbiol. 2011, 77 (12), 4089-4096. 9. Behlau, F.; Hong, J. C.; Jones, J. B.; Graham, J. H., Evidence for acquisition of copper resistance genes from different sources in citrus-associated xanthomonads. Phytopathology 2013, 103 (5), 409418. 10. Young, M.; Santra, S., Copper (Cu)–Silica nanocomposite containing valence-engineered Cu: a new strategy for improving the antimicrobial efficacy of Cu biocides. J. Agric. Food. Chem. 2014, 62 (26), 6043-6052. 11. Khot, L. R.; Sankaran, S.; Maja, J. M.; Ehsani, R.; Schuster, E. W., Applications of nanomaterials in agricultural production and crop protection: a review. Crop Prot. 2012, 35, 64-70. 12. Chen, J.; Peng, H.; Wang, X.; Shao, F.; Yuan, Z.; Han, H., Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation. Nanoscale 2014, 6 (3), 1879-1889. 13. Graham, J.; Johnson, E.; Myers, M.; Young, M.; Rajasekaran, P.; Das, S.; Santra, S., Potential of Nano-Formulated Zinc Oxide for Control of Citrus Canker on Grapefruit Trees. Plant Dis. 2016, 100 (12), 2442-2447. 14. Anderson, A. J.; McLean, J. E.; Jacobson, A. R.; Britt, D. W., CuO and ZnO Nanoparticles Modify Interkingdom Cell Signaling Processes Relevant to Crop Production. J. Agric. Food. Chem. 2017. 15. Zhao, L.; Peralta-Videa, J. R.; Rico, C. M.; Hernandez-Viezcas, J. A.; Sun, Y.; Niu, G.; Servin, A.; Nunez, J. E.; Duarte-Gardea, M.; Gardea-Torresdey, J. L., CeO2 and ZnO Nanoparticles Change the Nutritional Qualities of Cucumber (Cucumis sativus). J. Agric. Food. Chem. 2014, 62 (13), 2752-2759. 16. Maniprasad, P.; Santra, S., Novel copper (Cu) loaded core–shell silica nanoparticles with improved Cu bioavailability: Synthesis, characterization and study of antibacterial properties. J. Biomed. Nanotechnol. 2012, 8 (4), 558-566. 17. CLSI, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically;Approved Standard-Ninth Edition. In CLSI Document M07-A9, Clinical and Laboratory Standards Institute: Wayne, PA, 2012. 18. Rajasekaran, P.; Kannan, H.; Das, S.; Young, M.; Santra, S., Comparative analysis of copper and zinc based agrichemical biocide products: materials characteristics, phytotoxicity and in vitro antimicrobial efficacy. AIMS Environ. Sci. 2016, 3 (3), 439-455. 19. Behlau, F.; Scandelai, L. H. M.; da Silva Junior, G. J.; Lanza, F. E., Soluble and insoluble copper formulations and metallic copper rate for control of citrus canker on sweet orange trees. Crop Prot. 2017, 94, 185-191. 20. Graham, J.; Dewdney, M.; Myers, M. In Streptomycin and copper formulations for control of citrus canker on grapefruit, Proc. Fla. State Hortic. Soc, 2010; pp 92-99.
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21. Graham, J.; Dewdney, M.; Yonce, H. In Comparison of copper formulations for control of citrus canker on ‘Hamlin’orange, Proc. Fla. State Hortic. Soc, 2011; pp 79-84. 22. Data, I. c. f. D., Powder Diffraction File Search Manual. In Inorganic, JCPDS: PA.USA, 1984. 23. Xie, Y.; He, Y.; Irwin, P. L.; Jin, T.; Shi, X., Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni. Appl. Environ. Microbiol. 2011, 77 (7), 2325-2331. 24. Macomber, L.; Imlay, J. A., The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (20), 8344-8349. 25. Aruoma, O. I.; Halliwell, B.; Gajewski, E.; Dizdaroglu, M., Copper-ion-dependent damage to the bases in DNA in the presence of hydrogen peroxide. Biochem. J 1991, 273 (3), 601-604. 26. Santo, C. E.; Quaranta, D.; Grass, G., Antimicrobial metallic copper surfaces kill Staphylococcus haemolyticus via membrane damage. Microbiologyopen 2012, 1 (1), 46-52.
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Figure Captions Figure 1: (A) SEM image of ZnO 800 core material used in the synthesis of ZnO-nCuSi material, displaying irregular was generally rod-like sub-micron shape and size. (B) SEM image of The ZnO-nCuSi after the ZnO 800 has been coated with a copper silica shell. HRTEM images in the low magnification (C) and (D) high magnification of the ZnO-nCuSi. The more electron dense ZnO are seen in the darker contrast to the silica gel in (C). Cu crystallites seen in (D) measured between 3-6 nm.
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Tables pH
9.85
Specific gravity
1.14
Viscosity (mPa/s)
18.78 ± 0.84
Table 1: Properties of ZnO-nCuSi MIC ( µg/mL metallic Cu or Zn) Xanthomonas
Pseudomonas
Clavibacter
alfalfae subsp.
syringae pv.
michiganensis subsp.
citrumelonis
syringae
michiganensis
(ATCC 49120)
(ATCC 19310)
(ATCC 10202)
16-31 (Cu),
31-62 (Cu),
31-62 (Cu),
13-26 (Zn)
26-52 (Zn)
26-52 (Zn)
ZnO 800
125
125
62-125
Copper silica gel
125-250
125-250
125-250
Cuprous oxide/
62-125 (Cu),
125-250 (Cu),
125 (Cu),
zinc oxide
62-125 (Zn)
125-250 (Zn)
125 (Zn)
Copper hydroxide
250-500
250
125-250
Copper sulfate
125-250
250
125-250
Material
ZnO-nCuSi
Table 2: MIC of ZnO-nCuSi demonstrates stronger in-vitro bactericidal activity compared with either ZnO 800 compound or copper silica gel alone, indicating at least an additive effect.
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Material Tested
Vinca sp
Tomato sp
Untreated Control (H2O)
-
-
Copper sulfate at 1000 µg/mL
+++
+++
Copper hydroxide at 1000 µg/mL
-
-
Cuprous oxide/ zinc oxide at 500 µg/mL
-
-
Cuprous oxide/ zinc oxide at 1000 µg/mL
+
+
ZnO 800 at 500 µg/mL
-
-
ZnO 800 at 1000 µg/mL
-
-
Copper silica gel at 500 µg/mL
-
-
Copper silica gel at 1000 µg/mL
-
-
ZnO-nCuSi at 500 µg/mL
-
-
ZnO-nCuSi at 1000 µg/mL
-
-
(+++), (++), (+) – Severe, Moderate or low toxicity. (-) – Zero toxicity Table 3: Phytotoxicity analysis demonstrates both ZnO-nCuSi and its components are not toxic to plant tissue compared to soluble and insoluble copper compounds.
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Journal of Agricultural and Food Chemistry
Monthly rainfall (mm)
March
April
May
June
July
Aug.
Sept.
Oct
2015
41
124
19
239
193
152
254
51
2016
89
66
231
119
109
236
145
102
Ft. Piercez average
64
58
84
168
173
165
150
97
Trial Year
z
Average monthly rainfall from 2000-2010 obtained from FAWN at IRREC
Table 4: Rainfall at Indian River FAWN station, 2016
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Metallic
Incidence
Incidence
Total incidence
Cu (kg/ha)
old lesions (%)
young lesions (%)
(%)
Treatment
2015 Untreated check (UTC)
-
23 a
37 a
60 a
Cuprous oxide
1.12
10 cde z
20 b z
29 c z
Copper sulfate
0.16
16 bc
8.8 c
25 cd
0.5
8.2 cde
13 bc
21 defg
1.01
9.0 cde
14 bc
23 def
0.22
11 cde
14 bc
25 cd
Untreated check (UTC)
-
50 a
43 a
93 a
Cuprous oxide
1.12
28 ef z
28 cdef z
56 def z
0.5
30 def
27 def
56 def
1.01
30 def
27 def
56 def
0.22
29 def
30 bcde
58 de
Cuprous oxide/Zinc oxide Cuprous oxide/Zinc oxide
ZnO-nCuSi 2016
Cuprous oxide/Zinc oxide Cuprous oxide/Zinc oxide
ZnO-nCuSi z
Treatments followed by unlike letters are significantly different at P ≤ 0.05 according to StudentNewman-Keuls multiple range test
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Table 5: Effect of ZnO-nCuSi on incidence of citrus canker-infected fruit with old lesions, young lesions and total incidence of lesions on 8 and 9 year-old ‘Ray Ruby’ grapefruit trees, Ft. Pierce, FL, USA.
Metallic Treatment
Melanose
Burn
Incidence (%)
Incidence (%)
Scab incidence (%) Cu (kg/ha)
2015 Untreated check (UTC)
-
25.4 a
12.2 a
0.0
Cuprous oxide
1.12
2.4 d z
1.0 b z
0.0
Copper sulfate
0.16
3.4 bcd
4.6 b
0.0
0.5
2.2 d
1.0 b
0.0
1.01
2.4 d
1.6 b
0.0
0.22
5.4 b
4.0 b
0.0
Untreated check (UTC)
-
17.92 a
22.08 a
0.0
Cuprous oxide
1.12
5.56 fg
6.04 fgh
0.0
0.5
6 efg
5.08 h
0.0
1.01
5.4 fg
5.2 gh
0.0
0.22
7.84 cdef
7.64 def
0.0
Cuprous oxide/Zinc oxide Cuprous oxide/Zinc oxide
ZnO-nCuSi 2016
Cuprous oxide/Zinc oxide Cuprous oxide/Zinc oxide
ZnO-nCuSi
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z
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Treatments followed by unlike letters are significantly different at P ≤ 0.05 according to StudentNewman-Keuls multiple range test
Table 6: Effect of ZnO-nCuSi on incidence of scab and melanose on fruit of 8 and 9 yr-old ‘Ray Ruby’ grapefruit trees, Ft. Pierce, USA, October 2016 Figure Graphics Figure 1
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TOC Graphic
For Table of Contents Only
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