Patent Application
20200120937
Plant diseases contribute to 10-16% of global harvest loss thus limiting food supply.1 Viruses, bacteria, and fungi are the three major infectious factors that cause crop diseases, severely affecting food crop productivity and quality.2 Copper (Cu) based bactericides/fungicides are extensively applied to control a broad spectrum of crop diseases. However, prolonged use of Cu contributes to development of Cu resistance in certain plant pathogens.3,4 As a result, multiple treatments are often needed to achieve proper crop protection5,6 Also, Cu-based disease control program experience very limited success in treating tomato bacterial spot disease due to development of resistance. Recently, United States Environmental Protection Agency (EPA) has proposed to reduce the maximum Cu annual applications rate to certain crops such as hazelnut, pecans, walnut, onion, strawberry and pepper (EPA-HQ-OPP-2010-0212). To minimize the usage of Cu products in agriculture, there is a strong need to develop Cu alternatives.
Metal oxide materials, such as Titanium Dioxide (TiO2), Calcium Oxide (CaO), Magnesium Oxide (MgO), Copper Oxide (CuO) and Zinc Oxide (ZnO) have been extensively studied as antimicrobial agents.7-14 However, the antimicrobial properties of metal hydroxide particles have not been studied thoroughly. Low-cost and environmentally friendly magnesium hydroxide (Mg(OH)2) has been broadly applied for acidic water neutralization, fire retardants and fertilizers.15-17 Mg is a plant macronutrient and particularly important for plant enzyme activation, i.e. ATPases, RNA polymerase and protein kinases.18 Mg also participates in numerous plant physiological processes such as photosynthesis and photosynthetic carbon metabolism.19 At the same time, recent studies suggest that Mg in the form of Mg(OH)2 appears to possess antimicrobial activity at certain concentrations,20,21 thus positioning Mg(OH)2 as a potential Cu alternative biocide that is made up of plant macronutrients.
Table 1. XRD data collection for as-synthesized Mg(OH)2 particles and commercial bulk Mg(OH)2. The possible crystallization orientation preference was deducted by the peak intensity ratio of (001) and (101) plane.
Table 2. Zeta potential values of as-synthesized Mg(OH)2 particles at specific pH. The Zeta potential values were given as mean±SD (standard deviation) of triplicate samples.
Table 3. MIC value of as-synthesized Mg(OH)2 NPs and selected controls. The numbers in the table represent metallic magnesium/copper concentration in g/ml. “W” in the sample labels means sample was washed with DI water for three times.
Table 4. Phytotoxicity rating scale: Tomato plants were treated with different concentrations of as-synthesized Mg(OH)2 particles or selected controls. Phytotoxicity was rated 72 hours post treatment.
Tomato plant phytotoxicity rating on a scale of “−” non leaf damage, “+” slightly leaf damage, and “++” moderately leaf damage.
Table 5. Minimum inhibitor concentration (MIC) and Minimum Bactericidal Concentration (MBC) of P. syringae. The respective concentrations of Mg2+, Zn2+ and H2O2 are shown.
Table 6. Minimum inhibitor concentration (MIC) and Minimum Bactericidal Concentration (MBC) of P. syringae. The respective concentrations of Mg2+, Zn2+ and Cu2+ are shown.
Table 7. Zeta potential of NPs: Mg-Sol, Mg-Sol (25% Cu), and Mg-Sol (25% Cu)+0.25 TC+0.25 NAC.
Table 8. Minimum inhibitor concentration (MIC) and Minimum Bactericidal Concentration (MBC) of X. alfalfae. The respective concentrations of Mg2+ and Cu2+ are shown.
Disclosed herein are Magnesium (Mg) hydroxide particles composition embodiments and method embodiments of making antimicrobial non-phytotoxic Magnesium (Mg) hydroxide particles for use as an environmentally-friendly alternative to Copper (Cu) bactericide/fungicide. Particle size varies from a few nanometer (nm, a billionth of a meter) to hundreds of nanometer. Also disclosed is a method of making charged (both positive and negative) particles using food grade chemicals. Therefore, by changing the relative percentage of oppositely charged chemicals, overall particle surface charge can be tuned from negative to null (zero) to positive. This property improves customizable rainfastness property of Mg hydroxide particles for crop protection. Mg is a plant nutrient and therefore the material is expected to improve overall plant health.
There are many ways to synthesize Mg(OH)2, such as hydration of MgO,22 alkaline precipitation of Mg salt precursor,23 and electrolysis of aqueous Mg salt solution.24 The first two methods are heavily applied in industrial scale Mg(OH)2 manufacturing.25 In specific examples presented herein, Mg(OH)2 NPs were synthesized through alkaline precipitation method using magnesium chloride hexahydrate (MgCl2.6H2O) as the Mg precursor. In addition, positively charged or negatively charged capping agents or both were used to coat the Mg(OH)2 particles.
Xanthomonas alfalfae, Pseudomonas syringae and Escherichia coli were used for assessing the antimicrobial properties of as-synthesized Mg(OH)2 NPs. A comprehensive set of characterization studies were performed to assess the size, particle morphology, surface charge, cytotoxicity and phytotoxicity of as-synthesized Mg(OH)2 NPs.
According to other embodiments, Mg(OH)2_Citrate NPs were synthesized in the presence of H2O2, and optionally in the further presence of Zinc. H2O2 added Mg(OH)2_Citrate NPs had higher bacteriocidal efficacy compared to Mg(OH)2_Citrate NPs. The Zinc added Mg(OH)2_Citrate NPs further increased the bacteriocidal efficacy of the NPs.
In alternative embodiments, Mg(OH)2_Citrate NPs were synthesized in the presence of a metal Cu or Zn, or both. Bacteria viability or CFU assay results suggest that Cu added Mg(OH)2_Citrate exhibit similar bacteriostatic & bactericidal efficacy compare to Mg(OH)2_Citrate. However, Cu added Mg(OH)2_Citrate exhibit higher bactericidal efficacy (80 ppm metallic Cu) when compare with KOCIDE 3000 (320 ppm metallic Cu). The antimicrobial test result for Zn & Cu added Mg(OH)2_Citrate suggest enhanced bacteriostatic & bactericidal efficacy compare Mg(OH)2_Citrate and KOCIDE® 3000. and n-acetyl cysteine (NAC). In these alternative compositions, a high bactericidal efficacy is achieved with reduced amounts of copper compared to copper based bactericides like KOCIDE® 3000. The alternative embodiments can also include NPs synthesized in the presence of H2O2, which enables production of smaller nanoparticles.
Embodiments of NP compositions described herein can be used to treat infected plants or protect plants from infection. Plants that may be treated or protected include vegetable or crop plants, fruit plants, beverage plants, ornamental plants, nut plants, or herb/spice plants.
The term “plant(s)” includes the following:
The terms “protect” or “protecting” as used herein with respect to magnesium hydroxide nanoparticle containing compositions means delaying progession or reducing rate of infection on or in target plants.
The term “plant disease” is a disease caused by infection by a plant pathogen, including but not limited to bacterial, viral, fungal, nematode, phytomyxean, protozoan, algal and parasite plant pathogens.
The term “target plants” are plants to which MgOH2 NP containing compositions (as described herein) have been applied.
The term “plant pathogen” as used herein refers to an organism that causes a plant disease or reduces the health of a plant. Examples of plant pathogens include bacteria, viruses, fungi, nematodes, phytomyxea, protozoa, algae and parasites. Typically, harmful micro-organisms are carried by biological vectors such as insects. Specific examples of plant pathogens include but are not limited those related to or cause the following plant diseases: Algal leaf spot, Alternaria, Anthracnose, Bacterial spot, Bacterial blast, Bacterial blight, Brown rots, Botrytis(gray mold), Citrus canker, Downy mildew, Early blight, Fire blight, Late blight, Melanose, Powdery mildew, Leaf curl, Leaf spots, Scab, Shot hole, and Walnut blight.
As used herein, the term “about” modifying the quantity of an ingredient in the compositions of the invention or employed in the methods of the invention refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term about also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities. In a specific embodiment, the term “about” refers to an amount that is 5, 7, or 10 percent greater or lesser than the specified amount.
As used herein, a composition or combination “consisting essentially” of certain ingredients refers to a composition including those ingredients and lacking any ingredient that materially affects the basic and novel characteristics of the composition or method. The phrase “consisting essentially of” excludes from the claimed compositions and methods additional antimicrobial agents; unless such an ingredient is specifically listed after the phrase.
The term “applying,” “application,” “administering,” “administration,” and all their cognates, as used herein, refers to any method for contacting the plant with the Mg(OH)2 NPs discussed herein.
Administration generally is achieved by application of the compounds in a vehicle compatible with the plant to be treated (i.e., a botanically compatible vehicle or carrier), such as an aqueous vehicle, to the plant or to the soil surrounding the plant or by injection into the plant. Any application means can be used, however preferred application methods include trunk injection and foliar spraying as described herein. Other methods include application to the soil surrounding the plant, by injection, soaking or spraying, so that the applied compounds can come into contact with the plant roots and can be taken up by the roots.
The term “botanically acceptable carrier/vehicle” or “botanically compatible carrier/vehicle,” as used herein, refers to any non-naturally occurring vehicle, in liquid, solid or gaseous form which is compatible with use on a living plant and is convenient to contain a substance or substances for application of the substance or substances to the plant, its leaves or root system, its seeds, the soil surrounding the plant, or for injection into the trunk, or any known method of application of a compound to a living plant, preferably a crop plant, for example a citrus tree.
Useful vehicles can include any known in the art, for example liquid vehicles, including aqueous vehicles, such as water, solid vehicles such as powders, granules or dusts, or gaseous vehicles such as air or vapor. Any vehicle which can be used with known devices for soaking, drenching, injecting into the soil or the plant, spraying, dusting, or any known method for applying a compound to a plant, is contemplated for use with embodiments of the invention. Typical carriers and vehicles contain inert ingredients such as fillers, bulking agents, buffers, solvents, preservatives, anti-caking agents, pH modifiers, surfactants, soil wetting agents, adjuvants, and the like. Suitable carriers and vehicles within this definition also can contain additional active ingredients such as plant defense inducer compounds, nutritional elements, fertilizers, pesticides, and the like.
The term “effective amount” or “therapeutically effective amount,” as used herein, means any amount of the compound or composition which serves its purpose, for example, treating plant disease, improving the ability of plants to defend against disease, reducing disease symptoms, treating HLB disease, minimizing crop yield decreases due to plant disease, improving crop productivity, and increasing crop quality.
The term “improved ability to defend against disease,” as used herein, refers to a measurable increase in plant defense against a disease. This can be measured in terms of a measurable decrease in disease symptoms, pathogen titer, or loss of crop yield and/or quality, or a measurable increase in growth, crop quantity or quality.
The term “improved crop productivity,” as used herein, refers to a measurable increase in the quantity of a crop in a plant or a population of plants, in terms of numbers, size, or weight of crop seeds, fruits, vegetable matter, fiber, grain, and the like.
The term “improved crop quality,” as used herein, refers to a measurable increase in the quality of a crop, in terms of numbers, size, or weight of crop seeds, fruits, vegetable matter, fiber, grain, and the like, or in terms of sugar content, juice content, unblemished appearance, color, or taste.
The term “improved resistance to disease,” as used herein, refers to an increase of plant defense in a healthy plant or a decrease in disease severity of a plant or a population of plants, or in the number of diseased plants in a plant population.
The term “plant disease symptoms,” as used herein, refers to any symptom of disease, including the detectable presence of a known plant pathogen, or the presence of rot, mottling, galls, discoloration such as yellowing or browning, fruit greening, stunted growth, plant death, cellular death, cell wall breakdown, the presence of spots, the presence of lesions, dieback, wilting, dwarfing, knots, and Witch's broom.
The term “population of plants,” as used herein, refers to a group of plants, all of the same species, that inhabit a particular area at the same time. Therefore, the plants in a nursery, a grove, a farm, and the like are considered a population.
The term “reduction of disease symptoms,” as used herein, refers to a measurable decrease in the number or severity of disease symptoms.
Material.
All reagents used for synthesis and studies of Mg(OH)2 NPs were purchased from commercial vendors without any purification: magnesium chloride hexahydrate (Fisher Scientific, USA), magnesium hydroxide (Acros), sodium hydroxide (Fisher Scientific, USA), betaine anhydrous (Acros), trisodium citrate dihydrate (Trademark Nitrogen, FL, USA), KOCIDE® 3000 (Citrus Research and Education Center, Lake Alfred, Fla., USA; gift from Dr. James H. Graham), copper (II) sulfate pentahydrate (CQ Concepts, Ringwood, Ill., USA), copper (II) chloride anhydrous (Acros), deionized (DI) water (Nanopure; Barnstead model D11911), phosphate-buffered saline (PBS) (Fisher Scientific, USA), Tryptic soy broth (TSB), nutrient broth (NB) and agar for solid media preparation were purchased from Fluka (St. Louis, Mo., USA). All the bacterial strains, Xanthomonas alfalfae subsp. citrumelonis (ATCC 49120), Pseudomonas syringae pv. syringae (ATCC 19310) and Escherichia coli (ATCC 8739) were purchased from ATCC (Manassas, Va., USA). E. coli was grown in TSB, X. alfalfae and P. syringae were grown in NB. (U.S. Department of Agriculture (USDA) permits P526P-12-04060 and P526P-15-01601).
Synthesis of Mg(OH)2 NPs.
Mg(OH)2 NPs in solution phase was prepared by following procedure: (a) Co-precipitation of 5.016 g of MgCl2.6H2O, 1.46 g of betaine and 5M sodium hydroxide (NaOH) in deionized water on magnetic stirring at room temperature (hereafter referred as Mg(OH)2_Betaine). (b) Co-precipitation of 5.016 g MgCl2.6H2O, 3.68 g of trisodium citrate dihydrate and 5M NaOH in deionized water under magnetic stirring at room temperature (hereafter referred as Mg(OH)2_Citrate). (c) Co-precipitation of 5.016 g MgCl2.6H2O and 5M NaOH in deionized water under magnetic stirring at room temperature (hereafter named as Mg(OH)2_Bare). After 24 hours of mechanical stirring at room temperature, the as-synthesized Mg(OH)2 NPs were then washed three times with deionized water by centrifuging at 11000 rotations per minute (RPM) for 5 min to wash off extra chemicals in Eppendorf centrifuge 5810R. The pH values of all synthesized Mg(OH)2 NPs (20000 μg/ml of metallic Mg) are around 11.
Characterization of Mg(OH)2 NPs.
The crystal structure of as-synthesized Mg(OH)2 NPs was identified by X-ray diffraction (PANalytical Empyrean); by applying Cu Kc radiation with wavelength equal to 1.5406 Å and two-dimensional area detector. The average crystallite size was calculated by the Ebye-Scherrer equation. The hydrodynamic size of the as-synthesized Mg(OH)2 NPs was measured by dynamic light scattering technique (PDDLS/Cool/Batch 40T Precision Detector). The interaction between betaine or citrate with Mg(OH)2 NPs was measured by Fourier-transform infrared spectroscopy (FTIR, Perkin Elmer Spectrum 100 Series). The surface charge of as-synthesized Mg(OH)2 NPs were measured by ZetaPlus Zeta Potential Analyzer (Malvern Instruments). For each sample, 750 μl of undiluted solution was slowly transferred into the folded capillary zeta cell (Malvern Instruments) without any air bubbles. The morphology and size of as-synthesized Mg(OH)2 NPs were observed through Scanning Electron Microscopy (Zeiss ULTRA-55 FEG SEM) and Transmission Electron Microscopy (FEI Tecnai F30 TEM).
Microplate Alamar Blue Assay (MABA).
To determine the minimum inhibitory concentration (MIC) of the synthesized Mg(OH)2 NPs, a microdilution assay was performed.26 The MABA assay was used as a variant of broth microdilution assay to determine the MIC considering the interference of light absorbance by test materials. Briefly, triplicates (20 l/well) of as-synthesized Mg(OH)2 NPs and relevant controls were added to a 96 well plate. As described by clinical and laboratory standards institute (CLSI), all the tested bacterial species (180 l/well) were added such that the final concentration was 5×105 CFU/ml.
In case of E. coli, the 96-well plate was incubated at 37° C. and for X. alfalfae and P. syringae, the plates were incubated at 27° C. under shaking (150 RPM). After 24 hours of incubation, 10 μl of alamar blue dye (Molecular probes, Eugene, Oreg., USA) was added to each well. The plate was then kept back in the incubator for one more hour before the absorbance was measured at both 570 and 600 nm for each well. The reduction of the dye in percentage value was calculated by using the formula as suggested by the manufacturer.
Bacterial Killing/Colony Forming Unit (CFU) Assay:
To determine the absolute values of reduction in bacterial numbers after Mg(OH)2 NPs treatment, CFU assay was performed. The protocol used in the MABA assay was followed for this assay for treatment of bacteria with samples, but at the end of 24 hours incubation, serial dilutions of the bacteria from each well were made in respective bacterial growth media and plated on corresponding agar plates. The colonies were counted after overnight (for E. coli) and 48 hours (for X. alfalfae and P. syringae) incubation and expressed in logarithmic scale. The three concentrations of Mg(OH)2 NPs were chosen by picking the MIC value and a dilution higher and one lower than that of MIC values derived from the MABA assay.
Cytotoxicity assay. The potential cytotoxic effects of as-synthesized Mg(OH)2 NPs were tested against alveolar epithelial cells (A549). Various concentrations of as-synthesized Mg(OH)2 NPs were incubated with alveolar epithelial cells in 96-well plate for 24 hours at 37° C. in the presence of 5% CO2. After the exposure time, the cells were washed with complete fresh media [DMEM (Corning, 10-090-CV) with 10% fetal calf serum and 1% antibiotic and antimycotic]. After 3 hours incubation with 20 μl of alamar blue reagent, the contents of each well were transferred to black 96-well plate (Costar 3916, Corning life Sciences) for fluorescence (590 nm) measurement. Each concentration has three replications and the growth control was wells with only macrophages and media.
Phytotoxicity Studies.
The potential phytotoxicity of as-synthesized Mg(OH)2 NPs and selected controls were tested on S. lycopersicum (tomato) plant (purchased from local Home Depot). Tomato plants were purchased and then placed in plant growth chamber (Panasonic MLR-352H-PA) 24 hours prior to materials spraying (programmed to simulate summer conditions, maximum temperature set at 31° C.). All as-synthesized Mg(OH)2 NPs, DI water, CuSO4 and KOCIDE® 3000 were foliar sprayed by using hand-operated pump mist sprayer at 1000 and 2000 μg/ml of metallic Mg or Cu. Visual observations were conducted at 24, 48 and 72 hours post-spray application.
Seed germination test. Tomato seeds (purchased from local Home Depot) were sterilized by 10% sodium hypochlorite solution for 10 min, then washed three times with DI water to ensure the removal of extra sterilizing solution remaining on the seed surface. After sterilization, all the tomato seeds were soaked in the following samples: DI water, as-synthesized Mg(OH)2 NPs, commercial bulk Mg(OH)2, KOCIDE® 3000, CuCl2 or MgCl2 at selected concentration (250, 125. 62.5, 32, 16 μg/ml of metallic Mg or Cu) for 2 hours. After soaking, the seeds were washed three times with DI water.27 5 ml of test material was added to each petri dish (85 mm diameter) with one piece of filter paper. 10 seeds were transferred onto the filter paper and each seed has at least 1 cm distance from the neighboring seed.28 Three replicates of each treatment were prepared. Seeds were incubated at dark and controlled environment for six days (25° C. and 50% Relative Humidity). Seeds were considered as germinated when the coleoptiles were longer than 2 mm in length.29 Seed germination rate and root length was recorded.
Statistical Analysis.
For all experiments, each treatment was conducted in triplicates, and the results were presented as mean±SD (standard deviation). The experimental data was analyzed by PASW Statistics 18 (IBM SPSS) software. Statistical data was considered significant when the experimental value compares with its corresponding control at a significance level of P less than 0.05.
Characterization of Mg(OH)2 NPs.
To identify the crystal structure of the as-synthesized materials, XRD technique was adopted.
The hydrodynamic size of as-synthesized Mg(OH)2 NPs was measured by DLS. The average size for Mg(OH)2_Betaine, Mg(OH)2_Citrate and Mg(OH)2_Bare in aqueous solution was around 240 nm, 228 nm and 351 nm, respectively (
The morphology of as-synthesized Mg(OH)2 NPs were first observed under SEM.
The morphology and crystallinity of as-synthesized Mg(OH)2 NPs were further studied by HRTEM.
Mg(OH)2_Bataine (
Antimicrobial Studies:
Bacterial viability or CFU assay results suggest that the as-synthesized Mg(OH)2 NPs (at or above MIC) exhibit bacterial killing effect within the first four hours of contact with the bacteria (
Similarly, the capping agent betaine and citrate by themselves did not appear to exhibit any significant bacterial killing ability. However, the as-synthesized Mg(OH)2 NPs with betaine or citrate capped exhibited bacterial killing activity suggesting the bacterial killing efficacy of as-synthesized Mg(OH)2 NPs may not directly due to the capping agent. For these studies, commercially available copper bactericide (KOCIDE® 3000), a current standard for biocides in plant protection was used as a control. For all bacteria except X. alfalfae, that was screened in this experiment, KOCIDE® 3000 appeared to exhibit significant killing activity at 24 hours when used at the concentrations identified as MIC values from MABA assay. It is worthwhile to mention here that the MIC values obtained in MABA assay for X. alfalfae for KOCIDE® 3000 did not have significant levels of bacterial killing when screened by CFU assay. The inherent fluorescence of KOCIDE® 3000 could have influenced the MIC value determination in the MABA assay. Since X. alfalfae is comparatively more resistant to killing, a higher concentration of KOCIDE® 3000 could produce statistically significant bacterial growth inhibition as observed by M. Young et al.14 All the synthesized Mg(OH)2 NPs, with both the capping agents exhibited comparable killing efficacy suggesting that these particles can be potential alternatives to copper-based biocides. A commercially available bulk Mg(OH)2 used as control failed to exhibit similar antibacterial properties. Furthermore, the concentration of the active Mg that was required to exhibit killing effect differed with each bacterium suggesting differential surface effect and differential activity of Mg in the physiology of these bacteria. Since there was no killing observed in case of X. alfalfae at 4 hour time point, the possible mechanism of killing may not involve active cell wall lysis upon contact in that bacterium. The net positive charge of Mg(OH)2_Betaine and Mg(OH)2_Bare might in turn facilitate greater attachment of particles to the negatively charged cell surface of bacteria. However, the net negative charged Mg(OH)2_Citrate showed similar bacterial killing efficacy as the ones with net positive charged Mg(OH)2 NPs suggesting that surface charge of the particles has a minimal role to play in bacterial killing. Since understanding the mechanism of killing is beyond the scope of this current manuscript, we can speculate that Mg(OH)2 NPs exhibit bacterial killing by raising the intracellular Mg concentrations.
The impact of unbound chemicals (not part of the particulate) on the bacterial killing ability was assessed by screening washed materials alongside as-synthesized unwashed materials (
In another experiment, the effect of Mg(OH)2 NPs on eukaryotic cell lines was tested using A549 alveolar epithelial cells (
Phytotoxicity Assay.
The potential plant tissue damage on the leaves of as-synthesized Mg(OH)2 NPs with controls was tested on tomato plant through foliar spray. After three days incubation, potential phytotoxicity symptoms were evaluated by visual observation (
Seed Germination Assay:
The potential phytotoxicity of as-synthesized Mg(OH)2 NPs were further tested on tomato seed germination and root elongation.
In summary, Mg(OH)2 NPs were synthesized with different shapes, size and surface charges using betaine or trisodium citrate as capping agents. As-synthesized Mg(OH)2 NPs showed comparable bacterial killing efficacy and reduced phytotoxicity when compared with commercial Cu-based products. These results suggest that as-synthesized Mg(OH)2 NPs have greater potential in crop protection as alternatives for copper-based biocides.
Formulations of Mg(OH)2 NP were prepared. Hydrogen peroxide (H2O2), Copper (Cu), Zinc (Zn), N-acetylcysteine (NAC) were added during the synthesis Mg(OH)2_Citrate. Antimicrobial studies were performed to test the possibility of enhanced bacteriostatic/bactericidal properties.
1. Mg(OH)2_Citrate with Hydrogen Peroxide (H2O2)
Mg(OH)2_Citrate with H2O2 was synthesized following the same protocol for Mg(OH)2_Citrate in Example 1, except for adding H2O2 during the synthesis process (molar ratio: Mg2+:H2O2=1:1). The hydrodynamic size of H2O2 added Mg(OH)2_Citrate was around 14 nm (See
2. Mg(OH)2_Citrate with Copper (Cu) and Zinc (Zn)
Mg(OH)2_Citrate with H2O2 was synthesized following the same protocol for Mg(OH)2_Citrate in Example 1, except for adding Cu or Zn during the synthesis process (molar ratio: Mg2+:Zn2+:Cu2+=3:3:1). The hydrodynamic size of Cu added Mg(OH)2_Citrate was around 220 nm, and the hydrodynamic size of Cu & Zn added was around 142 nm (see
3. Mg(OH)2_Citrate with Double Coating
Cu added Mg(OH)2 with double coating was synthesized following the same protocol for Mg(OH)2_Citrate in Example 1, except for adding Cu and NAC during the synthesis process (molar ratio: Mg2+:NAC=1:0.25:0.25, metallic concentration ratio: Mg2+ (75%): Cu2+ (25%)). The hydrodynamic size of Cu added Mg(OH)2 double coated was around 342 nm (
E. coli
P. syringae
X. alfalfa
This application is related to U.S. Provisional Application No. 62/747,937 filed Oct. 19, 2018 to which priority is claimed under 35 USC 119. The teachings of this provisional are incorporated herein in their entirety by this reference.
| Number | Date | Country | |
|---|---|---|---|
| 62747937 | Oct 2018 | US |
