Fixed-Quat: An Attractive Nonmetal Alternative to Copper Biocides

Dec 4, 2018 - In this paper, we report a nonphytotoxic bactericide and fungicide formulation containing a composite of silica and quaternary ammonium ...
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Fixed-Quat: An Attractive Nonmetal Alternative to Copper Biocides against Plant Pathogens Mikaeel Young,†,‡ Ali Ozcan,‡,§ Parthiban Rajasekaran,‡ Preeti Kumrah,† Monty E. Myers,¶ Evan Johnson,¶ James H. Graham,¶ and Swadeshmukul Santra*,†,‡,§,⊥

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†

Burnett School of Biomedical Sciences, ‡NanoScience Technology Center, §Department of Chemistry, and ⊥Department of Materials Science and Engineering, University of Central Florida, 4353 Scorpius Street, Suite 245, Orlando, Florida 32816, United States ¶ Citrus Research and Education Center, University of Florida, 700 Experiment Station Road, Lake Alfred, Florida 33850, United States ABSTRACT: In this paper, we report a nonphytotoxic bactericide and fungicide formulation containing a composite of silica and quaternary ammonium compound (quat). The composite material was prepared using an acid-catalyzed sol−gel method. Positively charged quat was associated with a negatively charged silica-gel matrix, producing a stable suspension of fixed-quat gel (FQ-G). The morphology of FQ-G and the interaction of quat with silica were characterized using SEM and FTIR, respectively. Silica gel significantly reduced quat phytotoxicity when tested at 500 and 1000 μg/mL foliar-application rates. The in vitro antimicrobial efficacy of FQ-G was evaluated against Xanthomonas alfalfae, Pseudomonas syringae, and Clavibacter michiganensis, showing comparable efficacies to that of quat itself. In field conditions, its efficacy in controlling the bacterial and fungal diseases citrus canker, scab, and melanose on ‘Ray Ruby’ red grapefruit was evaluated. Foliar application rates at 100 and 200 μg/mL provided comparable disease control to those of several copper standards, demonstrating the potential for use as an alternative agricultural biocide. KEYWORDS: fixed-quat, quat, silica gel, agriculture, biocide, copper, phytotoxicity, citrus

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INTRODUCTION Citrus canker is a foliar disease caused by the bacterium Xanthomonas citri subsp. citri. Canker results in unsightly fruit and leaf lesions along with early fruit drop, culminating in a lower crop yield.1 Citrus canker has continued to plague the Florida citrus industry for more than a decade. In Florida, citrus is a signature crop. It is estimated that about 4.5 million pounds of metallic Cu is applied annually to protect citrus crops. Management strategies for citrus canker include planting wind breaks to reduce disease transmission through windblown rain and application of Cu-based pesticides. Aggressive use of these copper biocides for crop protection has led to increased risk of copper toxicity and bacterial copper resistance.1,2 Copper biocides are currently the most commonly used cropprotection product for a wide variety of crops, including citrus, vegetables, stone fruit, legumes, cereals, pome fruit, and berries.3−5 Biocide-application rates and frequency vary per crop and also sometimes depending on the weather from one season to another. For instance, citrus crops receive approximately 5−15 applications per season from March to October,1,2 whereas more frequent applications (several times per month) are required for tomato crops to prevent bacterial and fungal infections.6 Extensive application of Cu biocides in agriculture originates from its strong antimicrobial properties and bioavailability.7 Most commonly used Cu bactericides are insoluble, with low amounts of bioavailable Cu, which include Cu hydroxide, Cu oxychloride, basic Cu sulfate, and Cu oxides, which are formulated to minimize the risk of phytotoxicity and have limited bioavailability. In contrast, Cu(II) chelates such as © XXXX American Chemical Society

Magna-Bon, a copper sulfate based bactericide and fungicide, are water-soluble and produce more bioavailable Cu, but they also possess higher risks of phytotoxicity. In our previous study, we showed that mixed-valence Cu (i.e., a combination of Cu(0), Cu(I), and Cu(II) states) has improved antimicrobial efficacy, because of the enhanced Cu bioavailability, but minimized phytotoxicity.8 Improved efficacy of mixed-valence Cu over traditional Cu biocides has been attributed to multiple mechanisms of action with mixed-valence states of Cu.8 In general, the most prevalent mode of action arises from redox reactions between the Cu(I) and Cu(II) states, leading to the production of reactive oxygen species (ROS). In addition to antimicrobial resistance, persistent use of Cu biocides increases the risk of oxidative stress to crop plants and furthermore to the native microbiome as a result of copper accumulation in the soil.9,10 Studies have shown that some citrus and tomato pathogens such as Xanthomonas spp., including X. citri, Xanthomonas alfalfae, Xanthomonas perforans, and Xanthomonas gardneri among others, have developed varying degrees of Cu tolerance.11,12 Cu soil accumulation also contributes to leaching of Cu into water tables and aquatic ecosystems, potentially increasing the risk of damage to many aquatic species.13 With increasing risks from Cu-based pesticides, the U.S. EPA has recommended reducing the amount of Cu allowed for certain crops14 (Table 1). It is clear Received: August 4, 2018 Revised: November 7, 2018 Accepted: November 19, 2018

A

DOI: 10.1021/acs.jafc.8b04189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Deionized (DI) distilled water was obtained from a Nanopure water purifier (Barnstead, model #D11911). Biofilm studies were conducted using MBEC microtiter plates (catalogue #19121) purchased from Innovotech Inc. (Edmonton, AB, Canada). Nutrient broth (NB) and agar were purchased from Fluka (St. Louis, MO), and brain−heartinfusion (BHI) broth and agar were obtained from BD Chemical Company, Inc. (Greenwood Village, CO). Xanthomonas alfalfae subsp. citrumelonis (ATCC 49120), Pseudomonas syringae pv. syringae (ATCC 19310), and Clavibacter michiganensis subsp. michiganensis (ATCC 10202) cultures were purchased from ATCC (U.S. Department of Agriculture (USDA) permits P526P-12-04060 and P526P-15-01601). Fixed-Quat-Gel Synthesis. Two versions of the fixed-quat gel (FQ-G) were developed over the course of the study (A-I and A-II) using a sol−gel technique based on a published protocol.24 FQ-G A-I synthesis was carried out at room temperature under magnetic-stirring conditions. Briefly, 100 mL of DI water was combined with 1.5 mL of concentrated HCl in 500 mL container. After 5 min, 2.4 mL of 50% DDAC was added to this mixture. Then, after 10 min, 7 mL of TEOS was slowly added. The reaction mixture was stirred for 2 h. Using 1 M sodium hydroxide (∼23 mL), the mixture was adjusted to pH 8. After pH adjustment, the mixture was stirred for 2 h. The DDAC content was calculated to be ∼8066 μg/mL in FQ-G. After an initial field assessment in 2014, the silica source for fixed-quat was changed to a more industrially feasible compound, sodium silicate (37%). For the preparation of the FQ-G A-II gel, 30 mL of DDAC (50% solution) was added to 910 mL of DI water and left to stir at 150 rpm for 1 h; 60 mL of 37% sodium silicate (Fisher Scientific) was added, and the mixture was left to stir for 24 h at 150 rpm. Both FQ-G materials were used as synthesized without any purification steps to mimic an industrially viable, bulk-scale production process. FQ-G Characterization. Characterization of FQ-G was carried out using field-emission scanning electron microscopy (FE-SEM; Ultra-55 FEG, Zeiss, Oberkochen, Germany) and Fourier-transform infrared spectroscopy (FTIR; Spectrum 100, PerkinElmer, Waltham, MA). The chemical interaction between DDAC and the silica gel was investigated using FTIR spectroscopy. A scan range of 4000 to 650 cm−1 was selected. Three scans were done for each measurement with a resolution of 1 cm−1. The structure and morphology of the FQ-G materials were studied using SEM imaging techniques. SEM samples were prepared by placing a drop of sample on a gold grid, followed by coating of the sample with a thin layer of gold (a few atomic layers) using a gold coater. SEM studies were conducted at Advanced Materials and Processing Analysis Center Materials Characterization Facility (AMPAC-MCF) at the University of Central Florida. FQ-G Antimicrobial Studies. The antimicrobial properties of FQ-G were studied using an array of standard microbiological techniques, including determination of the minimum bactericidal concentration (MBC) and the minimum biofilm-eradication concentration (MBEC) and use of a bacterial-viability assay. Samples were tested against Gram-negative Xanthomonas alfalfae subsp. citrumelonis strain F1 (ATCC 49120, the causal agent of citrus bacterial spot and a citrus-canker surrogate), Gram-negative Pseudomonas syringae pv. syringae (ATCC 19310, the causative agent of bacterial speck in lilac, almond, apricots, peaches, and beans, among others), and Grampositive Clavibacter michiganensis subsp. michiganensis (ATCC 10202, the causative agent of bacterial wilt and canker in tomato species). X. alfalfae and P. syringae were maintained with nutrient agar and broth, whereas C. michiganensis was grown with brain−heart-infusion media. All bacteria were grown at 28 °C. FQ-G was compared to DDAC (free quat, positive control) as well as to the industry standards copper hydroxide and copper sulfate. Silica gel was used as a negative control. Minimum Bactericidal Concentration (MBC). The MBC value of FQ-G was determined, along with those for DDAC and the industry standards, copper sulfate and copper hydroxide. MBC-evaluation experiments were carried out using a minimum-inhibitory-concentration (MIC)-broth microdilution method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI).25 A range of concentrations from 4000 to 1 μg/mL active

Table 1. EPA New Recommendation of Cu Use for Specialty Crops in 2017 crop

current use rate (lbs/a)

recommended use rate (lbs/a)

24 8.4 32 6

18 6.3 24 5

6 12 8.2 8

5 8.7 6 4

hazelnut pecans walnut cucurbits (cantaloupe, cucumber, pumpkin, squash, watermelon) onion pepper strawberry tobacco

that the Cu-application rate must be reduced in coming seasons for many specialty crops. Although citrus is not included in the list at this time, it is likely to be listed in the near future. Low-cost Cu alternatives with comparable or better antimicrobial efficacies are therefore desirable. Copper-alternative metals exhibiting antimicrobial properties, such as iron, zinc, silver, and magnesium, have been studied.15−17 Nonmetal alternatives, including plant based extracts and oils as well as antibiotics such as streptomycin and oxytetracycline, have also been evaluated.18 Limited use of these antibiotics is permitted for certain agricultural crops (citrus, apples, and peaches).19 However, long-term use of antibiotics is risky because of the potential for the development of bacterial resistance. Quaternary ammonium compounds (QACs or quats) are a class of positively charged surfactants. Quats are widely used in many household and commercial applications as low-cost detergents, fabric softeners, flocculants, disinfectants, and personal-care products.20 The United States Environmental Protection Agency (U.S. EPA) has classified specific quat compounds for use as “Approved for Food Use” with amounts not exceeding 200 μg/mL active concentration for certain industrial applications. Quats exhibit exceptionally high antimicrobial properties against a wide variety of bacteria and fungi.21 Despite their low cost and high antimicrobial efficacy, to the best of our knowledge, there are no biocide products containing quat active labeled for crop protection in the U.S. Quat causes severe phytotoxicity and ornamental plants are most susceptible.22 Although the biodegradability of quaternary ammonium compounds varies depending on the type, concentration, soil composition, and microbiome, a 2006 study on didecyl dimethylammonium chloride (DDAC) found the compound to be immobile, biodegradable, and environmentally safe.23 In this study, we have designed and developed a nonphytotoxic composite by combining an inexpensive foodgrade quat compound with silica gel. This composite material demonstrates strong crop-protection capabilities in vitro and under field conditions, suggesting that it is a promising candidate for consideration as an alternative to traditional Cu biocides.

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MATERIALS AND METHODS

Materials. All chemicals were reagent-grade and used without any further purification. We purchased tetraethylorthosilicate (TEOS) from Gelest Inc. (Morrisville, PA), didecyldimethylammonium chloride (DDAC) from EMD Millipore (Billerica MA), and resazurin sodium salt from Sigma (St. Louis, MO). Concentrated hydrochloric acid, sodium hydroxide, sodium silicate, and phosphate-buffered saline (PBS) were obtained from Fisher Scientific (Pittsburgh, PA). B

DOI: 10.1021/acs.jafc.8b04189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry was tested for all materials and controls. MBC determination was improved by adding 10 μL of resazurin dye (0.0125%, w/v) per 100 μL of well volume and observing the color changes (blue to pink for live cells). Because of the opaque and turbid nature of the FQ-G, a visual MIC determination was not possible. Minimum Biofilm-Eradication Concentration (MBEC). The MBEC values of FQ-G, DDAC, copper sulfate, and copper hydroxide were determined using broth microdilution in accordance with the guidelines of ASTM E2799-12.26,27 This involved the use of an MBEC microtiter plate with a lid of 96 pegs for biofilm growth. Briefly, 22 mL of a 107 CFU/mL planktonic culture of bacteria was added to the MBEC trough plate and incubated for 48 h at 26 °C while being shaken at 150 rpm, which allowed biofilms to grow on the pegs. After incubation, the MBEC microtiter 96-well lid was washed once by being placed in a 96-well plate filled with 200 μL of 1× PBS for 2 min. After being washed, the lid was transferred to a prepared challenge plate containing serially diluted antimicrobial agents (FQ-G and the controls) and incubated for 48 h at 26 °C while being shaken at 150 rpm. After the challenge, the biofilms were washed twice by being placed in a 96-well plate filled with 200 μL of 1× PBS for 1 min each time. The MBEC microtiter 96-well lid was then placed in a 96well plate filled with 200 μL of fresh broth media, which was then placed in a sonicated bath for 25 min at high power to dislodge the biofilms from the pegs. Survival of the biofilms was confirmed by turbidity after incubation for 24 h at 26 °C with shaking at 150 rpm. The MBEC was taken as the lowest concentration resulting in no turbidity.28 To observe the effect of the FQ-G A-II treatment, a duplicate challenge plate was made, and after challenge incubation, the biofilm was fixed for SEM as briefly described below.29 Briefly, the pegged lid was washed in 1× PBS for 3 min before being placed in a 96-well plate with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C for 20 h. The pegged lid was then washed in 0.1 M cacodylate buffer for 10 min before being dehydrated in 70% ethanol for 20 min. The pegged lid was then air-dried for a minimum of 24 h. After being air-dried, the pegs were broken off the lid, coated with a thin layer of gold, and loaded into a JEOL JSM-6480 SEM. Bacterial-Viability Assay. The bactericidal activities of FQ-G were determined with a colony-forming-unit (CFU) assay, as previously described.30 Treatment of bacteria with materials was carried out following the same procedure as for the MBC assay. After MBC determination, a range of concentrations (0.6−10 μg/mL for the quat-based materials and 31−500 μg/mL for the copper-based materials) were chosen for further quantification. Each sample was serially diluted in 1× PBS and plated on nutrient or BHI agar at 28 °C. After 48 h of incubation, individual colonies were counted, and CFUs per milliliter were quantified. The results were analyzed using GraphPad Prism 7 with ANOVA Dunnet’s multiple-comparison correction, and significant differences were denoted using asterisks (*), indicating P values of 2.0 kg/ha, did not significantly reduce canker infection further.31−33 FQG was applied at 100 and 200 μg/mL quat concentration. Because this compound is not a currently registered active, the concentrations used were chosen on the basis of EPA-approved limits for quat in other applications. Materials were mixed with water and applied as foliar sprays at 3.79 L per tree with a handgun sprayer at 1380 kPa of air pressure. Treatments were initiated after the spring flush and sprayed at roughly 21 day intervals. In 2014, these applications occurred on April 1, April 22, May 12, June 2, June 23, July 14, August 4, August 25, September 15, and October 6. Disease evaluation was on October 20, 2014. During the 2015 trial, treatments were on April 14, May 5, May 26, June 16, July 7, July 28, August 18, September 8, September 28, and October 6. Disease evaluation was on November 2, 2015. During the 2016 trial, the same methodology was applied for treatments sprayed on April 19, May 10, May 31, June 20, July 11, August 1, August 22, September 12, October 10, and October 24. Disease evaluation was on November 14, 2016. Disease Incidence. The incidence of fruit with canker lesions was assessed for 100 fruit per treatment from the middle three trees in each plot. Lesions were classified as “old” if they were larger than 0.6 mm in diameter, coalescing with surrounding lesions, black in color, or exuding gum or had a prominent yellow halo; the lesions were classified as “young” if they were smaller than 0.6 mm in diameter, brown in color, and not coalescing with surrounding lesions. Disease severity was rated for each fruit on the basis of the estimated numbers of old and young lesions: 1, 0 lesions; 2, 1−5 lesions; 3, 6−20 lesions; and 4, 21 or more lesions. Monthly rainfall in 2014−2016 was recorded at the University of Florida Institute of Food and Agricultural Sciences (IFAS), Indian River Research and Education Center, Fort Pierce, FL, and obtained from the Florida Automated Weather Network Web site (http://fawn.ifas.ufl.edu/). The monthly rainfall was compared to the average for the last 10 years. For statistical analysis in each experiment, data were subjected to analysis of variance in PROC GLM (SAS Institute, Cary, NC). Means were separated using the Student−Newman−Keuls multiple-range test at α = 0.05. Evaluation of Plant Safety (Phytotoxicity Study). Quaternary ammonium compounds have not been used as active ingredients previously in agriculture for crop-protection purposes because they cause phytotoxicity. Plant-safety studies of FQ-G and the controls (DDAC, copper hydroxide, and copper sulfate) were carried out to ascertain potential plant-tissue damage. Copper hydroxide was used as a negative control that causes no plant-tissue damage, whereas copper sulfate was used as a positive control that exhibits significant phytotoxicity. Studies were conducted using Vinca sp., an annual ornamental plant purchased from a local Home Depot, and heirloom tomato, a model fruit plant. Ornamental Vinca sp. is highly susceptible to copper-ion phytotoxicity. It is used in industry as a model plant system to evaluate the phytotoxicity of copper bactericide and fungicide product formulations. Phytotoxicity studies were conducted in a Panasonic Environmental Test Chamber (Model MLR-352H, Kadoma, Japan) to control light intensity, humidity, and temperature cycling to simulate summer conditions (85% RH, 34 °C). Formulations were sprayed at concentrations of 500 and 1000 μg/ mL of metallic Cu at 8:00 a.m. before temperatures became too high, and observations were taken at 72 h after spray application.

Table 2. Rainfall at Indian River FAWN Station, 2014 monthly rainfall (in.) trial year

March

April

May

June

July

Aug

Sept

Oct

2014 2015 2016 Fort Pierce averagea

61 41 89 64

56 124 66 58

76 19 231 84

231 239 119 168

172 193 109 173

147 152 236 165

292 254 145 150

38 51 102 97

a

Average monthly rainfall from 2000 to 2010, obtained from FAWN at IRREC. C

DOI: 10.1021/acs.jafc.8b04189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. SEM images of the fixed-quat A-I gel (A) and the fixed-quat A-II gel (B), displaying a micrometer-sized irregular porous composite.

Figure 2. (A) FTIR of FQ-G demonstrating the presence of silica and DDAC within FQ-G. Silica was identified by the Si−O−Si stretching (∼1630 cm−1), SiO−H stretching (∼3390 cm−1), and Si−O bending (800−950 cm−1) found in the silica gel (SiG) and FQ-G. The presence of DDAC was confirmed by identification of C−H stretching (2800−3000 cm−1) and C−H bending (1375−1450 cm−1 in FQ-G and DDAC). (B) Table exhibiting the exact wavenumbers of the peaks corresponding to the groups identifying silica and DDAC within FQ-G.

Table 3. MBC and MBEC of Fixed-Quat Gel Compared to Quat (DDAC) Alone and Cu Controlsa Xanthomonas alfalfae subsp. citrumelonis (ATCC 49120) fixed-quat gel A-I fixed-quat gel A-II quat (DDAC) copper hydroxide copper sulfate

Pseudomonas syringae pv. syringae (ATCC 19310)

Clavibacter michiganensis subsp. michiganensis(ATCC 10202)

MBC (μg/mL)

MBEC (μg/mL)

MBC (μg/mL)

MBEC (μg/mL)

MBC (μg/mL)

MBEC (μg/mL)

4 4 4 250 250

125 125 250 1000 500

4 4 4 250 250

62 62 125 1500 1000

4 4 4 250 125

16 16 32 1500 1000

The fixed-quat gel demonstrated no loss of efficacy after DDAC was combined with silica.

a

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RESULTS AND DISCUSSION

that positively charged quat molecules can be immobilized in a silica-gel matrix. We have intentionally performed hydrolysis and co-condensation of TEOS in the presence of quat. During this synthesis process, quat molecules were electrostatically captured by the silica (Si−O−) matrix. As demonstrated by its

In this study, we show that quat has the potential to be an effective alternative to Cu bactericides and fungicides. Foliar application of antimicrobial quat is a high-risk proposition because of its inherent phytotoxic properties. We have shown D

DOI: 10.1021/acs.jafc.8b04189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 5. Bacterial viability of C. michiganensis after treatment with SiG, FQ-G, DDAC, and Cu controls. FQ-G exhibited complete microbiocidal activity at 2.5 μg/mL quant concentration, and DDAC killed completely at 5 μg/mL, demonstrating that antimicrobial activity was improved when DDAC was fixed into silica gel. Copper hydroxide and CuSO4 completely killed at 250 μg/mL Cu. A classic antibiotic, kanamycin (50 μg/mL), was used as a control for bacterial killing. No significant differences are denoted with ns, whereas the asterisks (*) mark data significantly different from the growth control at P < 0.05, as determined using one-way ANOVA with Dunnett’s correction in GraphPad Prism 7.0.

Figure 3. Bacterial viability of X. alfalfae after treatment with SiG, FQG, DDAC, and Cu controls. DDAC and FQ-G exhibited complete microbiocidal activity at 5 μg/mL quat concentrations, demonstrating that antimicrobial activity was not compromised when DDAC was fixed into silica gel. Copper hydroxide and CuSO4 completely killed at 250 μg/mL Cu. A classic antibiotic, kanamycin (50 μg/mL), was used as a control for bacterial killing. No significant differences are denoted with ns, whereas the asterisks (*) mark data significantly different from the growth control at P < 0.05, as determined using one-way ANOVA with Dunnett’s correction in GraphPad Prism 7.0.

high potency (MBC < 5 ppm for tested pathogens), the antimicrobial efficacy of fixed-quat was not compromised, suggesting that quat molecules were released from the gel matrix. In the fixed-quat composite, quat phytotoxicity was drastically reduced, and in some cases, we have seen that the effect is negligible. It is suggested that local concentrations of free quat at the interface of the silica−quat (fixed-quat)-gel material and the plant tissue are low enough to not cause any significant damage. The role of silica gel is that of a slowrelease quat-delivery system. In observing the morphology of the FQ-G composite material, SEM images revealed multimicrometer-sized particulate structures with no defined shape (Figure 1A,B). FQ-G particles appear to be aggregated, which is typical for TEOSderived silica sol−gel matrixes.8 FTIR (Figure 2) showed characteristic silica peaks, Si−O−Si stretching (∼1630 cm−1), SiO−H stretching (∼3390 cm−1), and Si−O bending (800− 950 cm−1) in both the silica-gel (SiG) control and the FQ-G composite. Association of DDAC with the SiG matrix was confirmed by FTIR through identification of C−H stretching (2800−3000 cm−1) and C−H bending (1375−1450 cm−1 in FQ-G and DDAC) peaks. The antimicrobial efficacy of FQ-G was compared to that of the controls, DDAC, copper hydroxide, and copper sulfate, using MBC, MBEC, and a bacterial-viability assay. Results have been compiled in Table 3 and Figures 3−5. The MBC values of FQ-G and DDAC were 4, 4, and 4 μg/mL for X. alfalfae, P. syringae, and C. michiganensis, respectively. The MBC values of copper hydroxide and copper sulfate were both 250 μg/mL for X. alfalfae and P. syringae. For C. michiganensis, copper hydroxide had an MBC value of 125 μg/mL, whereas copper sulfate had an MBC value of 250 μg/mL. The bacterial

Figure 4. Bacterial viability of P. syringae after treatment with SiG, FQ-G, DDAC and Cu controls. FQ-G exhibited complete microbiocidal activity at 2.5 μg/mL quat concentration, and DDAC killed completely at 5 μg/mL, demonstrating that antimicrobial activity was improved when DDAC was fixed into silica gel. Copper hydroxide and CuSO4 completely killed at 250 μg/mL Cu. A classic antibiotic, kanamycin (50 μg/mL), was used as a control for bacterial killing. No significant differences are denoted with ns, whereas the asterisks (*) mark data significantly different from the growth control at P < 0.05, as determined using one-way ANOVA with Dunnett’s correction in GraphPad Prism 7.0.

E

DOI: 10.1021/acs.jafc.8b04189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 6. SEM images of untreated X. alfalfae biofilms (A) grown on polystyrene pegs and X. alfalfae biofilms treated with 125 μg/mL fixed-quat AII (B) and 250 μg/mL fixed-quat A-II (C).

Table 4. Effect of Formulations on Incidence of CankerInfected Fruit with Old Lesions, Young Lesions, and Total Incidence of Lesions on 7 and 8 Year Old ‘Ray Ruby’ Grapefruit Trees, Fort Pierce, FL, October 2014 and 2015a treatment untreated check (UTC) copper hydroxide copper hydroxide cuprous oxide cuprous oxide copper sulfate fixed-quat A-I gel (100 μg/mL) fixed-quat A-I gel (200 μg/mL) untreated check (UTC) cuprous oxide copper sulfate fixed-quat A-II gel (200 μg/mL) untreated check (UTC) cuprous oxide fixed-quat A-II gel (200 μg/mL)

incidence of young lesions (%)b

total incidence (%)b

metallic Cu (kg/ha)

incidence of old lesions (%)b



2014 45.0 a

17.8 a

62.8 a

1.57

11.8 bc

3.8 b

15.6 cde

1.01

11.8 bc

4.6 b

16.4 cde

1.12 2.24 0.16 

16.8 b 10.2 bc 12.4 bc 18.0 b

4.4 5.8 6.0 5.6

21.2 bcd 16.0 cde 18.4 bcd 23.6 bc

b b b b



12.6 bc



2015 23 a

37 a

60 a

1.12 0.16 

10 cde 16 bc 14 b

20 b 8.8 c 12 bc

29 c 25 cd 26 cd



2016 50 a

43 a

93 a

1.12 

28 ef 28 ef

28 cdef 27 def

56 def 55 efg

2.6 b

Table 5. Effect of Copper Formulations on Incidence of Scab and Melanose on Fruit of 7 Year Old ‘Ray Ruby’ Grapefruit Trees, Fort Pierce, October 2014a treatment untreated check (UTC) copper hydroxide copper hydroxide cuprous oxide cuprous oxide copper sulfate fixed-quat A-I gel (100 μg/mL) fixed-quat A-I gel (200 μg/mL) untreated check (UTC) cuprous oxide copper sulfate fixed-quat A-II gel (200 μg/mL)

15.2 de

untreated check (UTC) cuprous oxide fixed-quat A-II gel (200 μg/mL)

melanose incidence (%)b

burn incidence (%)

2014 18.4 a

18.0 a

0.0

1.57 1.01 1.12 2.24 0.16 

4.4 bc 3.8 bc 3.0 bc 4.6 bc 4.6 bc 5.6 b

0.6 c 0.4 c 1.8 bc 0.4 c 1.6 bc 3.0 bc

0.0 0.0 0.0 0.0 0.0 0.0



3.4 bc

1.4 bc

0.0



2015 25.4 a

12.2 a

0.0

1.12 0.16 

2.4 d 3.4 bcd 5.4 b

1.0 b 4.6 b 3.0 b

0.0 0.0 0.0



2016 17.92 a

22.08 a

0.0

1.12 

5.56 fg 10.84 b

6.04 fgh 11.48 b

0.0 0.0

metallic Cu (kg/ ha) 

scab incidence (%)b

a The nonmetal fixed-quat gel provided comparable protection to that of the Cu-based biocides. bTreatments followed by different letters are significantly different at P ≤ 0.05 according to the Student− Newman−Keuls multiple-range test.

a The nonmetal fixed-quat gel provided comparable protection to that of the Cu-based biocides. bTreatments followed by different letters are significantly different at P ≤ 0.05 according to the Student− Newman−Keuls multiple-range test.

was fixed within the silica gel in the FQ-G composite material. Complete killing was observed for Kocide 3000 and CuSO4 at 250 μg/mL metallic Cu content. The MBEC assay was included to check biofilm eradication potential, as bacterial biofilms play an important role in infection and in the survival of bacteria in complex natural environments, including in plant systems. The MBEC for FQ-G was determined to be 125, 62, and 16 μg/mL for X. alfalfae, P. syringae, and C. michiganensis, respectively. Higher MBEC values were obtained for DDAC (250, 125, and 32 μg/mL), copper hydroxide (1000, 1000, and 1500 μg/mL), and copper sulfate (500, 1000, and 1000 μg/ mL) for X. alfalfae, P. syringae, and C. michiganensis, respectively. The MBEC was visually checked by fixing the biofilms of X. alfalfae and observing them in a JEOL SEM

viability of X. alfalfae (Figure 3), P. syringae (Figure 4), and C. michiganensis (Figure 5) after treatment with SiG, FQ-G, DDAC, copper hydroxide, and copper sulfate were examined using CFU analysis. FQ-G exhibited complete microbiocidal activity at a DDAC concentration of 5 μg/mL for X. alfalfae and at 2.5 μg/mL for P. syringae and C. michiganensis. The MBC and bacteria-viability values were the same or improved compared to those of DDAC alone, demonstrating that antimicrobial efficacy was not compromised when DDAC F

DOI: 10.1021/acs.jafc.8b04189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

These results were comparable to the reduction in infection displayed by copper hydroxide (15.6%) and cuprous oxide (16%), the industry standards. During the 2015 and 2016 trials, only the 200 μg/mL application rate was used because of limited space availability in the citrus grove. The reductions in infection were 60−26% (2015) and 93−55% (2016), whereas cuprous oxide reduced the infection rates to 29% in 2015 and 56% in 2016. Fungal infections resulting in scab and melanose were also assessed during all field trials, with the FQ-G material displaying an ability to reduce these infections as well. No sign of spray phytotoxicity was observed on the ‘Ray Ruby’ grapefruit during and after foliar treatment with the FQ-G material (Table 5). To confirm the hypothesis that combining quat with silica will reduce its toxicity toward plant tissues of multiple species, phytotoxicity studies were conducted with ornamental Vinca sp. and tomato species (Table 6 and Figure 7). A comparative visual rating was used to estimate the phytotoxicity level (−, no phytotoxicity; +, mild phytotoxicity; ++, medium phytotoxicity; +++, severe phytotoxicity) in comparison with those of the controls, DDAC, copper sulfate (soluble Cu), and copper hydroxide (insoluble copper). This phytotoxicity-rating method is often practiced in the agrichemical industry. As expected, both DDAC and copper sulfate exhibited phytotoxicity, whereas copper hydroxide was not phytotoxic. FQ-G and the controls were tested on Vinca sp. and tomato for 72 h. It was observed that the FQ-G material was less phytotoxic than DDAC alone when applied at the same rate. Copper sulfate resulted in plant-tissue damage on both Vinca sp. and tomato at the application rate of 1000 μg/mL Cu. Copper hydroxide demonstrated no plant-tissue damage on either species at 1000 μg/mL Cu. FQ-G and DDAC were tested at 500 and 1000 μg/ mL. DDAC resulted in severe plant-tissue damage on both Vinca sp. and tomato at 500 and 1000 μg/mL, whereas both FQ-G A-I and FQ-G A-II only showed minimal damage at 1000 μg/mL on Vinca sp. Fixed-quat A-I and A-II were

Table 6. Phytotoxicity Studies of Fixed-Quat Gel, Quat (DDAC), and Cu Controlsa material tested

Vinca sp.b

tomatob

untreated control (H2O) copper sulfate at 1000 μg/mL copper hydroxide at 1000 μg/mL fixed-quat A-I gel at 500 μg/mL fixed-quat A-I gel at 1000 μg/mL fixed-quat A-II gel at 500 μg/mL fixed-quat A-II gel at 1000 μg/mL quat (DDAC) at 500 μg/mL quat (DDAC) at 1000 μg/mL

− +++ − − + − + + ++

− +++ − − − − − + ++

a The fixed-quat gel was seen to cause no plant-tissue damage at concentrations as high as 500 μg/mL for Vinca sp. and 1000 μg/mL for the tomato species. These values are above the suggested application rate of 100−200 μg/mL. b+++, severe toxicity; ++, moderate toxicity; +, low toxicity; −, zero toxicity.

(Figure 6). It was seen that FQ-G A-II treatment at the MBEC value and above began to disrupt the biofilm structure and eradicate the biofilm. A thick, full biofilm can be seen in Figure 6A for the untreated biofilm, whereas a highly disrupted biofilm can be seen from the samples treated with 125 μg/mL (Figure 6B), and no biofilm formation is seen after treatment with 250 μg/mL (Figure 6C). The protective capability of the FQ-G material to reduce infections of Xanthomonas citri in a commercial grove was tested with ‘Ray Ruby’ grapefruit in Fort Pierce, FL, from 2014 to 2016 (Table 4). The field efficacy of the FQ-G material was evaluated against both bacterial (citrus canker) and fungal (citrus melanose and scab) diseases. Citrus-canker infections in the 2014 field trial were quantified and determined to be 62.8% for untreated trees, whereas FQ-G reduced the prevalence of infection to 23.6% with the 100 μg/mL application rate and 15.2% with the 200 μg/mL application rate. This demonstrates that the efficacy of FQ-G is rate-dependent in field conditions.

Figure 7. Phytotoxicity studies of fixed-quat gel, quat (DDAC), and the Cu controls. Materials were applied to Vinca sp. (A−E) and tomato (F−J) at 1000 μg/mL: untreated control (A,F), copper sulfate (B,G), copper hydroxide (C,H), DDAC (D,I), and fixed-quat gel A-II (E,J). G

DOI: 10.1021/acs.jafc.8b04189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

perforans and tomato bacterial spot. Phytopathology 2018, 108, 196− 205. (7) 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, 30670−30677. (8) 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, 6043−6052. (9) Holmgren, G.; Meyer, M.; Chaney, R.; Daniels, R. Cadmium, lead, zinc, copper, and nickel in agricultural soils of the United States of America. J. Environ. Qual. 1993, 22, 335−348. (10) Borkert, C.; Cox, F.; Tucker, M. Zinc and copper toxicity in peanut, soybean, rice, and corn in soil mixtures. Commun. Soil Sci. Plant Anal. 1998, 29, 2991−3005. (11) 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, 4089−4096. (12) Behlau, F.; Hong, J. C.; Jones, J. B.; Graham, J. H. Evidence for acquisition of copper resistance genes from different sources in citrusassociated xanthomonads. Phytopathology 2013, 103, 409−418. (13) Kiaune, L.; Singhasemanon, N. Pesticidal copper (I) oxide: environmental fate and aquatic toxicity. In Reviews of Environmental Contamination and Toxicology; Springer: New York, NY, 2011; Vol. 213, pp 1−26. (14) Proposed Interim Registration Review Decision; Case Nos. 0636, 0649, 4025, 4026; United States Environment Protection Agency, 2017. (15) 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, 2442−2447. (16) Young, M.; Ozcan, A.; Myers, M. E.; Johnson, E. G.; Graham, J. H.; Santra, S. Multimodal generally recognized as safe ZnO/ nanocopper composite: A novel antimicrobial material for the management of citrus phytopathogens. J. Agric. Food Chem. 2018, 66, 6604−6608. (17) 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. (18) McManus, P. S.; Stockwell, V. O.; Sundin, G. W.; Jones, A. L. Antibiotic use in plant agriculture. Annu. Rev. Phytopathol. 2002, 40, 443−465. (19) Stockwell, V.; Duffy, B. Use of antibiotics in plant agriculture. Rev. Sci. Technol. 2012, 31, 199−210. (20) Rutala, W. A.; Weber, D. J.; Healthcare Infection Control Practices Advisory Committee (HICPAC). Guideline for disinfection and sterilization in healthcare facilities; Center for Disease Control, 2008. (21) Gerba, C. P. Quaternary ammonium biocides: efficacy in application. Appl. Environ. Microbiol. 2015, 81, 464−469. (22) Ferk, F.; Mišík, M.; Hoelzl, C.; Uhl, M.; Fuerhacker, M.; Grillitsch, B.; Parzefall, W.; Nersesyan, A.; Mičieta, K.; Grummt, T.; et al. Benzalkonium chloride (BAC) and dimethyldioctadecylammonium bromide (DDAB), two common quaternary ammonium compounds, cause genotoxic effects in mammalian and plant cells at environmentally relevant concentrations. Mutagenesis 2007, 22, 363− 370. (23) Didecyl Dimethyl Ammonium Chloride (DDAC) Risk Assessment; DP Barcode 069149; Office of Pesticide Programs, Antimicrobials Division, United States Environmental Protection Agency: Arlington, VA, 2006. (24) Santra, S.; Bazata, J.; Young, M. Core-shell quaternary ammonium nanomaterials, methods and applications. Patent US20140308330A1, 2014. (25) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, Approved Standard, 9th ed.; CLSI

determined to be safe on tomato at tested concentrations up to 1000 μg/mL. In summary, we have demonstrated for the first time the potential use of quat as a crop-protectant alternative to traditional Cu bactericides and fungicides. The FQ-G formulation is industrially viable and cost-effective. The antimicrobial activity of quat is not compromised when combined with the SiG matrix. In field conditions, FQ-G demonstrated comparable efficacy to that of the copper standards in controlling both bacterial (citrus canker) and fungal (citrus scab and melanose) diseases. The environmental impact is expected to be low, as the quat field-application rate is below 200 μg/mL, which is within the EPA tolerance limit. The release rate of quat from the FQ-G material is expected to be low and therefore should not exhibit acute toxicity to nontarget species such as honey bees and small fish. Furthermore, quat degrades over time in the environment, which should reduce the risk of accumulation.23 Future studies will include an FQ-G shelf-life evaluation and an investigation of its environmental fate on nontarget species.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 407-882-2848. Fax: 407-882-2819. E-mail: [email protected]. ORCID

Ali Ozcan: 0000-0001-6781-535X Swadeshmukul Santra: 0000-0001-5929-5323 Funding

We acknowledge financial support from the Citrus Research and Development Foundation, Inc. (Grant #759) and CDRE grant #2016-70016-24828/project accession #1008984 from the USDA National Institute of Food and Agriculture. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Materials Characterization Facility (MCF) at the UCF Advanced Materials and Processing Analysis Center (AMPAC), where SEM analysis was conducted. The authors acknowledge technical support from the UCF Materials Innovation for Sustainable Agriculture (MISA) center.

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REFERENCES

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DOI: 10.1021/acs.jafc.8b04189 J. Agric. Food Chem. XXXX, XXX, XXX−XXX