Patent Application
20220079147
Bacterial pathogens have devastated the Florida citrus production in recent years. Citrus canker, is a harmful foliar disease affecting most citrus varieties, caused by the bacterium Xanthomonas citri subsp. citri. This disease results in the loss of fruit production through unsightly blemishes, premature fruit drop, and defoliation of the tree. The canker-causing bacteria are spread by windblown rain which can deposit the pathogens directly in the plant stomata. Film-forming copper based bactericides such as copper hydroxide and copper oxychloride are extensively used to control the spread of the disease. These Cu compounds form a lasting protective copper film on the leaf surface due to their lower solubility product. However, frequent applications (up to 15 sprays per season) of these Cu compounds is needed for disease control. Aggressive use of copper contributes to soil Cu and increases the risks of toxic Cu leaching through water runoff. This could damage the aquatic ecosystems, potentially harming many aquatic lives. Although Cu is still a standard bactericide/fungicide, frequent applications increase the risks of development of Cu-tolerant bacteria and can be also harmful to the soil microbiome.
Recently, antibiotics streptomycin and oxytetracycline, originally developed for human use, have been labeled for controlling citrus disease in Florida including Huanglongbing (HLB, also known as citrus greening). These antibiotics are used to control bacterial diseases in pom fruit, such as fire blight in apple and pear. Although they are effective for contact killing of bacteria, unlike Cu, antibiotics do not offer long-term protection, which is particularly important for controlling citrus canker disease. The ability of a material to resist wash-off, also known as rainfastness, is poor for highly water-soluble antibiotics such as streptomycin sulfate. A lasting antimicrobial residue on the leaf surface is critical to defend the plant against bacterial invasion through windblown rain. This is especially important for citrus production areas in Florida and Costa Rica that experience frequent rainfall. Thus, the development of suitable adjuvant to improve the rainfastness of antibiotics such as streptomycin sulfate, is important to improve performance and minimize negative impacts on non-target organisms.
This section will be completed once the claims are finalized.
These and other features, aspects, and advantages of various embodiments will become better understood with reference to the following description, Figures, and claims.
Many aspects of this disclosure can be better understood with reference to the following Figures.
The various embodiments are not limited to the examples illustrated in the Figures.
Various embodiments may be understood more readily by reference to the following detailed description. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The term “mol percent” or “mole percent” generally refers to the percentage that the moles of a particular component are of the total moles that are in a mixture. The sum of the mole fractions for each component in a solution is equal to 1.
It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
All numeric values are herein assumed to be modified by the term “about,” whether or not explicitly indicated. The term “about” generally refers to a range of numbers that one of skill in the art would consider equivalent to the recited value (i.e., having the same function or result). In many instances, the term “about” may include numbers that are rounded to the nearest significant Figure.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit (unless the context clearly dictates otherwise), between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
Unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.
As used herein the term “therapeutic benefit” is used to refer to suppression of one or more plant pathogens. A therapeutic benefit may be preventative or curative. For example, a therapeutic benefit may suppress a plant pathogen by strengthening a plant's resistance to the pathogen and/or by reducing the extent of a pre-existing infection of the plant pathogen.
As used herein the term “suppress or suppression” is used to mean changing the amount or rate of spread of a plant pathogen infection or reducing the extent of a plant pathogen infection.
As used herein the term “plant pathogen” relates to an organism (e.g. insect, bacteria, fungus, or virus) that infects a plant resulting in an adverse consequence in the plant and/or causes a plant disease.
As used herein, the term “rainfastness” refers to the degree to which a surface treatment resists being washed off of a surface by rain.
The following abbreviations are used throughout: NP, nanoparticle; NAC, N-acetylcysteine; ZnS:Mn, manganese doped zinc sulfide; ZnNP-NAC, ZnS:Mn coated with NAC; ZnNP-Strep, ZnNP-NAC conjugated with streptomycin; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; CFU, colony forming unit; DLS, dynamic light scattering; HRTEM, high resolution transmission electron microscopy. All the features disclosed in this specification (including any accompanying claims, abstract, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure.
Composition Comprising Cargo Delivery Particle(s) and Cargo(s)
Cargo Delivery Particle(s)
The cargo delivery particle 101 may deliver the one or more cargos 104 to previously unreachable or difficult to access areas of a plant, including but not limited to via components of a plant's vasculature. Embodiments delivering cargos via a plant's vasculature may provide systemic delivery of the cargos 104. Such systemic delivery may provide a systemic effect, meaning that even if locally administered, one or more cargo delivery particles 101 and cargos 104 may ultimately be found in multiple tissues of the plant to which the composition 100 is administered. For example, according to various embodiments, despite being administered at a single location or at a limited number of locations of the plant, cargo delivery particles 100 and/or cargos 104 may ultimately be found in many tissues of the plant or even in every tissue of the plant. The cargos 104 may provide one or more therapeutic benefits to the plant. Additionally, according to various embodiments, the core 102 and/or the shell 103 may provide one or more therapeutic and/or nutritional benefits to the plant.
The compositions described herein provide significant advantages compared to administering the cargo alone. When administered alone, some cargoes may be bound to a plant enzyme and subsequently absorbed. When associated with a cargo delivery particle 101, according to various embodiments, however, the cargo is not 100% available immediately upon injection. The composition 100 may, therefore, provide improved systemic effect via a slow-release mechanism, whereby the cargo only becomes available for absorption after it is transported through the plant's vasculature. According to some embodiments, the dissolution rate of composition 100 may vary based on the plant type, on the in planta environment, as well as on various environmental conditions. Performance of compositions, may, therefore, vary based on geographical locations and plant type.
Prior to various embodiments described herein, efficient delivery of cargos to a plant's vasculature system, in particular to phloem tissue, was challenging. According to various embodiments, the cargo delivery particles 101 may be “protein-sized” as defined hereinafter. The cargo delivery particles 101 may, therefore, have a unique in planta mobility, enabling them to freely move to phloem tissue by passing through cambiums. Each of the cargos 104 may also be “protein-sized” as defined hereinafter. According to various embodiments, a “protein-sized” particle may have a size of less than about 10 nm or from about 2 nm to about 6 nm, or of less than about 5.4 nm. Each range described herein is intended to include all numerical values encompassed by the range. Furthermore, additional ranges may be formed from any lower limits and/or upper limits described herein. For example, a “protein-sized” particle may have a size within a range having a lower limit and/or an upper limit. The range may include or exclude the lower limit and/or the upper limit. By way of example and not limitation, a lower limit and/or an upper limit may be selected from 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.1, 5.2, 5.3, 5.4 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5 and 20 nm. A range formed from a single lower limit includes at least the lower limit and all numerical values greater than the lower limit regardless of whether the values are explicitly recited in this disclosure. A range formed from a single upper limit includes at least the upper limit and all numerical values less than the upper limit regardless of whether the values are explicitly recited in this disclosure. A range formed from a combination of a lower limit and an upper limit includes at least the lower limit, the upper limit, and all numerical values there between regardless of whether the values are explicitly recited in this disclosure. For example, based on the set of exemplary upper limits and lower limits explicitly recited above, a protein-sized particle may have a size of: about 0.5 to about 20 nm, less than about 0.5 nm, greater than about 0.5 nm, less than about 20 nm, or greater than about 20 nm, etc. All such ranges are contemplated and are intended to be explicitly disclosed and recited. Each value recited is intended to be modified by the term “about.”
Core of the Cargo Delivery Particle(s)
Still referring to
The core 102 may further include one or more inorganic compounds. Any or all of the one or more inorganic compounds may have one or more of a variety of properties, including but not limited to antibacterial efficacy and fluorescence. For example, the one or more inorganic compounds may include fluorescent material. The one or more inorganic compounds may additionally or alternatively include an antibacterial material. One example of an inorganic compound that may be present in the core is Zinc sulfide (ZnS).
The materials that comprise the core 102 may be in the form of one or more nanostructures. As used herein, the term “nanostructures” refers to materials having at least one dimension between about 1 to about 100 nm. A variety of nanostructures may be employed, including, for example, nanowires, nanorods, tetrapods, nanobelts, nanoflowers, and nanoparticles. A person having ordinary skill in the art will readily appreciate the morphologies of various nanostructures as well as how to produce them. As a specific example, the core 102 may include a nanoparticle comprising at least one metal micronutrient and ZnS, or in a variety of morphologies including, but not limited to those listed above.
The “protein-sized” cargo delivery particles 101 having cores 102 comprising metal micronutrients, such as ZnO or ZnS, may eventually degrade to metal ions, such as Zn ions, and become a part of the plant's micronutrient pool, contributing to productivity improvement. The cargo delivery particles 101, according to various embodiments, may be more soluble than fertilizer-grade bulk metal oxides, such as ZnO due to increased surface area. The U.S. Food and Drug Administration (US FDA) listed ZnO as being generally recognized as safe (GRAS) if it is used as food additive. The U.S. Environmental Protection Agency (US EPA) has recently exempted ZnO (CAS Reg. No. 1314-13-2) from the requirement of tolerance when is used as an additive in an amount of up to 15% (wt/wt) in bactericide/fungicide formulations. A beneficial effect of ZnO (having a size of about 10 nm) on cucumber fruit (Cucumis sativus) was reported in greenhouse settings. As reported, soil application of ZnO at rates of 400 and 800 mg/kg did not demonstrate any negative effect on carbohydrate and antioxidant contents, but increased starch and protein content in comparison to untreated controls. According to various embodiments, ZnO may also provide various antimicrobial properties. For example, ZnO may provide effective antibacterial activity against gram-negative bacteria Escherichia coli, Xanthomonas alfalfa, Pseudomonas aeruginosa, and gram-positive bacteria Staphylococcus aureus. Detailed mechanisms of antimicrobial activity of ZnO have not been fully understood yet. Suggested mechanisms of killing are linked to release of antimicrobial zinc ions and production of reactive oxygen species.
The core 102 may additionally or alternatively comprise carbon-based nanomaterials such as carbon nanotubes, or nano-onions, which may further contribute to productivity increases, presumably due to their activity as plant stimulant.
According to various embodiments, the core may contain the metal micronutrient in an amount of from about 1 to about 5% by weight. Each range described herein is intended to include all numerical values encompassed by the range. Furthermore, additional ranges may be formed from any lower limits and/or upper limits described herein. For example, according to various embodiments, the core may contain the metal micronutrient in an amount within a range having a lower limit and/or an upper limit. The range may include or exclude the lower limit and/or the upper limit. By way of example and not limitation, a lower limit and/or an upper limit may be selected from 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10% by weight. A range formed from a single lower limit includes at least the lower limit and all numerical values greater than the lower limit regardless of whether the values are explicitly recited in this disclosure. A range formed from a single upper limit includes at least the upper limit and all numerical values less than the upper limit regardless of whether the values are explicitly recited in this disclosure. A range formed from a combination of a lower limit and an upper limit includes at least the lower limit, the upper limit, and all numerical values therebetween regardless of whether the values are explicitly recited in this disclosure. For example, based on the set of exemplary upper limits and lower limits explicitly recited above, according to various embodiments, core may contain the metal micronutrient in an amount of: about 0.5 to about 10% by weight, less than about 0.5% by weight, greater than about 0.5% by weight, less than about 10% by weight, or greater than about 10% by weight, etc. All such ranges are contemplated and are intended to be explicitly disclosed and recited. Each value recited is intended to be modified by the term “about.” As an example, according to various embodiments, the core may comprise Zn in an amount of from about 1 to about 5% by weight, or in an amount of about 2.5% by weight (25,000 ppm). Various embodiments include amounts of a metal micronutrient in the core that are non-phytotoxic for the particular plant. For example, up to 1000 ppm of metallic Zn has been tested against ornamental vinca and found to be non-phytotoxic.
An advantage of various embodiments is that the core 102 may eventually degrade to metal ions, which may then be metabolized by the plant, leaving behind no nano-residue in the plant.
Shell of the Cargo Delivery Particle(s)
Still referring to
According to various embodiments, the shell may be made of a coating material that may interact with the cargo. According to various embodiments, the interaction between the coating material and the cargo(s) may include a covalent bond, a non-covalent interaction, an intermolecular interaction, or any combination thereof. As used herein, a covalent bond refers to a chemical bond that involves the sharing of electron pairs between atoms. Covalent bonding includes many kinds of interactions, including at least σ-bonding, π-bonding, metal-to-metal bonding, agostic interactions, bent bonds, and three-center two-electron bonds. As used herein, a non-covalent interaction differs from a covalent bond in that it does not involve the sharing of electrons, but rather involves more dispersed variations of electromagnetic interactions between molecules or within a molecule. Non-covalent interactions include at least electrostatic interactions, Van der Waals forces, 7-effects, hydrophobic effects. Electrostatic interactions may include at least ionic interactions, hydrogen bonding, and halogen bonding. Van der Waals forces may include at least dipole-dipole interactions, dipole-induced dipole interactions, and London dispersion forces. 7-effects may include at least π-π interactions, cation-π interactions, anion-π interactions, and polar-π interactions. As used herein, intermolecular interactions or forces are the forces which mediate interaction between molecules, including forces of attraction or repulsion which act between molecules and other types of neighboring particles, e.g. atoms or ions. Intermolecular interactions include at least ion-dipole interactions, dipole-dipole interactions, dipole-induced dipole interactions, and induced dipole-induced dipole interactions. Any of these types of interactions or any combination of these types of interactions may provide a mechanism by which the coating material may interact with the cargo(s) disposed thereon or associated therewith.
The coating material may be any material suitable for coating the core and providing a desirable interaction with a particular cargo to be delivered. According to various embodiments, the coating material may comprise N-acetylcysteine (NAC), salicylate, caffeine, or any combination thereof. For example, various embodiments provide a composition and a method of making “protein-sized” zinc sulfide (ZnS) nanoparticles coated with N-acetylcysteine (NAC). Zinc sulfide nanoparticles coated with N-acetylcysteine (NAC-ZnS) is an example of a systemic cargo delivery system according to various embodiments. A wide range of cargos can be systemically delivered using NAC-ZnS, as long as NAC interacts with the cargos of interest. For various applications, the coating material, such as a NAC coating, may be useful to improve dispersibility (solubility) of the core nanoparticles, such as ZnS, in an aqueous medium, as well as, to prevent agglomeration of the nanoparticles. Various tested embodiments demonstrate that NAC-ZnS is capable of systemic delivery of a cargo comprising an antimicrobial composition to a vascular plant.
An have the advantage that NAC and Zn are generally recognized as safe (GRAS), requiring minimal regulatory requirements. Both Zn and NAC should be considered favorably by Organic Materials Review Institute (OMRI) for organic farming. In fact, both NAC and Zn are nutritional supplements for humans. According to various embodiments the ratio of Zn to NAC may be in a range of from about 1:1 to about 1:0.25. Less NAC results in smaller particles. More NAC allows more S to S binding causing agglomeration, which may be a problem when the composition is systemically administered to a vascular plant, but may be useful in spraying application, because the agglomeration allows for film formation on various surfaces of the plant.
Some Exemplary Cargo(s)
Various embodiments provide systemic delivery of a wide variety of cargos. For convenience and clarity, some examples of various cargos are grouped into categories hereinafter, but it should be understood than any cargo or combination of cargos may be employed. Cargos may include therapeutic molecules for controlling plant disease or any type of plant deficiencies, such as antimicrobial compounds. Cargos may include agrichemical active ingredients, such as insecticides, bactericides, fungicides, or combinations thereof.
For example, according to various embodiments, a cargo may comprise mancozeb. Mancozeb is registered as a general use pesticide by the U.S. Environmental Protection Agency. Mancozeb is a dithiocarbamate non-systemic agricultural fungicide with multi-site, protective action on contact. More specifically, mancozeb is a combination of two other dithiocarbamates: maneb and zineb. The mixture controls many fungal diseases in a wide range of field crops, fruits, nuts, vegetables, and ornamentals.
Cargos may include metal micronutrients, as described above with respect to possible components of the core. Examples of metal micronutrients include, but are not limited to iron (Fe), copper (Cu), zinc (Zn), manganese (Mn), molybdenum (Mo), nickel (Ni), and various combinations thereof. Cargos may include biomolecules, such as proteins, peptides, nucleic acids, Systemic Acquired Resistance (SAR) chemicals, antioxidants, or combinations thereof. For example, according to various embodiments, a cargo may comprise double-stranded RNA.
Other exemplary cargos may include one or more agricultural antibiotics. According to various embodiments, the agricultural antibiotic may be positively charged to facilitate adhesion to the shell. The positively-charged agricultural antibiotic may be, for example, streptomycin. Compositions according to various embodiments may, therefore, be antimicrobial treatment compositions for suppressing or preventing a plant pathogenic bacteria. According to various embodiments, the plant pathogenic bacteria may include X. alfalfae and/or P. syringae.
Antibiotic streptomycin has been extensively used for the treatment of bacterial infection in human. Streptomycin is also used in agriculture to protect pome fruits from fire blight, a destructive bacterial disease. Recently, streptomycin sulfate (65.8% A.I.) has been labeled for suppression of Huanglongbing in Florida, USA. The A.I. is water soluble and therefore suffers from poor rainfastness after foliar application. Due to high polarity, uptake and mobility of streptomycin sulfate through phloem vascular system is limited. Furthermore, streptomycin is prone to UV degradation when exposed to direct sunlight. Various embodiments provide an efficient adjuvant to improve rainfastness, UV-stability and vascular mobility. Various embodiments provide a non-phytotoxic zinc sulfide (ZnS) nanoparticle based adjuvant, demonstrating improved rainfastness and sustained release of streptomycin. Antibacterial efficacy of streptomycin and the ZnS-streptomycin conjugate was found comparable when tested against model plant pathogenic bacteria (E. coli, X. alfalfae, and P. syringae).
According to various embodiments, rainfastness of streptomycin foliar treatments may be improved by utilizing a nanoparticle (NP) as the sticker. NPs, due to their small size, possess a high surface-to-volume ratio which can help them stick to surfaces such as plant foliage. According to various embodiments, the rainfastness property of antibiotics may be improved if the antibiotics are coated with the NP sticker. Protein size fluorescent (<10 nm) manganese doped zinc sulfide NPs (ZnNPs) may be used as an antibiotic sticker (as well as carrier) due to their ultra-small size, low toxicity profile and plant micronutrient (Mn, S, Zn) based composition. The ZnNPs may be synthesized via a scalable and environmentally-friendly precipitation reaction. Dopant Mn ions within the semiconductor zinc sulfide (ZnS) crystal lattice serves as the fluorescent center that exhibits characteristic 590 nm emission. Unlike other fluorescent semiconductor NPs, these are free of toxic heavy-metals (such as cadmium and lead). Potential use of these NPs as biomedical imaging agent has been evaluated due to their ultra-small size, fluorescent property and low toxicity. However, applicability of micronutrient-rich ZnNPs in plant system has not been extensively evaluated. Sustained release of essential micronutrient Zn2+ ions may be achieved, according to various embodiments, as the ZnNP goes through a degradation process, improving plant growth as a nutritional supplement. Various embodiments relate to N-acetylcysteine (NAC) coated ZnNPs as an antibiotic sticker.
Methods of Administration
The pith 205 is a tissue layer that provides partially undifferentiated cells for plant growth. It is found in the area between the two types of transport tissue in vascular plants, xylem 206 and phloem 204. Particles having sizes, larger than about 10 nm are often phloem-restricted, having limited accessibility to phloem tissue, although they can populate xylem vasculature easily after their root uptake. Various embodiments provide efficient, systemic delivery of cargos to plant vasculature system, in particular to phloem tissue. As used herein, the term “phloem tissue” or “phloem” has its usual meaning and refers generally to the living tissue in vascular plants that transports the soluble organic compounds made during photosynthesis. In trees, the phloem is the innermost layer of the bark. Particles having sizes larger than 10 nm often have limited accessibility to phloem tissue although they can populate xylem vasculature easily after their root uptake. As used herein, the term “size” as it relates to particles generally refers to an average diameter of a plurality of particles. For particles that are not perfectly spherical, the term “diameter” generally refers to the average of cross-sectional diameters in a plurality of directions such that the “diameter” is a reasonable approximation of the average dimensions of the particles. As such, all specific numerical values associated with particle size as used herein should be understood to be modified by the term “about.” For example, a particle having a size larger than 10 nm may generally refer to a particle that would be likely to be captured by a filter having 10 nm gaps. Similarly, a plurality of particles having a size larger than 10 nm would indicate that on average the particles would be likely to be captured by a filter having 10 nm gaps.
Various embodiments provide a systemic cargo delivery system for plants that may also be applied with traditional application methods (foliar, soil-drench), no such cargo delivery system currently exists for treating plants. Various embodiments of the composition may also be useful in seed/fertilizer coatings.
Methods of Producing the Composition(s)
Various embodiments provided a straight-forward one-pot synthesis process that can be easily scaled-up. For example, a method of producing the compositions may include dispersing a salt of a metal micronutrient, such as zinc nitrate in a solution with a coating material, such as N-acetylcysteine. The coating material may form a complex by chelating the metal ions. The pH of the solution may then be raised to prompt formation of the cargo delivery particles comprising the metal micronutrient coated with the coating material. Subsequently the cargo may be added to the solution and may interact with the shells of the cargo delivery particles. Various embodiments of the production method are very cost-effective as agricultural-grade metal micronutrients like zinc and food-grade coating materials like N-acetylcysteine may be easily sourced as commodity products.
The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and how to make and to use the compositions and compounds disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. The purpose of this example is not to limit the scope of the various embodiments, but merely to provide an example illustrating specific embodiments of the composition, the method of making the composition, and the method of administering the composition.
Materials and Methods
Materials
Manganese (II) acetate tetrahydrate was purchased from Acros Organics. Anhydrous sodium sulfide (Na2S), N-acetylcysteine (NAC), zinc acetate dihydrate, streptomycin sulfate, and rezazurin sodium salt were purchased from Sigma-Aldrich. Ethanol (95%), hydrochloric acid (HCl), sodium hydroxide (NaOH), and Falcon® 96-well standard tissue culture (TC)-treated Culture Microplate were purchased from Fisher Scientific (Cat. #08-772-2C). Deionized water was obtained from a Barnstead purifier, model D11911. Xanthomonas alfalfae subsp. citrumelonis (X. alfalfae, ATCC 49120), Escherichia coli (E. coli, ATCC 35218), Pseudomonas syringae (P. syringae, ATCC 19310) were obtained from ATCC. All materials were used as received at room temperature and without further purification. All solvents used were reagent grade and used as received.
NAC-ZnNP Synthesis and Streptomycin Coating
NAC-ZnNP were synthesized via a precipitation method. First a solution of 0.22 M Zn2+ with 1% (mol) Mn2+ was made in deionized (DI) water. A separate sulfide stock solution was prepared by dissolving Na2S in DI water to make a 0.66 M solution. A third stock solution of NAC was prepared in DI water with a NAC concentration of 0.26 M. Next, 540 μL of the NAC and 540 μL of the sulfide solution were mixed in a separate vial. This mixture was added dropwise to 720 μL zinc-manganese solution under magnetic stirring to produce NAC-ZnNP. The NAC-ZnNP solution was then filtered with a 0.22 μm syringe filter (Thermo Scientific Nalgene). The filtrate containing the NAC-ZnNP was centrifuged at 11,000 RCF (Relative Centrifugal Force) and subsequently washed with 95% ethanol (v/v) for five times. Each washing step involved brief vortexing of the solution followed by the centrifugation at 11,000 RCF for 5 minutes. The solution was then redispersed in DI water and lyophilized (FreeZone 4.5L Freeze Dry System, Labconco) to obtain NAC-ZnNP dry powder for further investigation.
High Resolution Transmission Electron Microscopy (HRTEM)
One drop of NAC-ZnNP solution in DI water was placed on carbon-coated gold TEM grid and allowed to air dry for 24 hours. The NPs were imaged using a FEI Tecnai F30 TEM operated at 300 kV. Images were processed with ImageJ software with particle size assessed from particles with clearly defined crystal lattices.
Fourier Transform Infrared Spectroscopy (FTIR)
NAC-ZnNP and ZnNP-Strep samples were washed five times with ethanol and lyophilized 48 h prior to FTIR measurement. The FTIR spectra were collected with a Perkin Elmer Spectrum 100 Series ATR FT-IR Spectrometer as an average of three scans with a resolution of 1 cm−1.
Solution Spectroscopy
UV-visible spectroscopy was carried out using a Cary 300 UV-Vis Spectrophotometer. Fluorescence emission was collected using a NanoLog Fluorescence Spectrophotometer (SPEX, Jobin Yvon Horiba). Spectra were collected in DI water using 1 cm path length quartz cuvette.
Hydrodynamic Size and Zeta Potential
The hydrodynamic size was characterized by dynamic light scattering (DLS) and zeta potential measurements were carried out using electrophoretic light scattering on a Malvern ZetaSizer ZS90 (Malvern Instruments Inc. UK). Samples were analyzed by DLS in DI water with a 1 cm quartz cuvette. Zeta potential measurements were carried out in DI water using a clear disposable folded capillary zeta cell (Malvern Instruments Inc., UK). The pH of all samples was adjusted to 8.0 with either dilute NaOH or HCl. Samples were briefly sonicated prior to each measurement.
Streptomycin Loading and Release Studies
The loading of streptomycin on the NAC-ZnNPs was determined by filtering the as-synthesized unwashed ZnNP-Strep with a centrifuge filter (3 kDa cutoff Amicon ultra centrifugal filter unit) for 10 minutes at 11000 RCF. The unbound streptomycin that passed through the filter was diluted and quantified via LCMS using an Agilent 1260 Infinity Series LC with 6820 TOF MS equipped with an Agilent Zorbax C-18 column. Flow rate was 0.5 mL per minute isocratic flow of DI water with 0.5% formic acid for 3 minutes. The source was set to a gas temperature of 325° C. at 8 l/min flow rate and a nebulizer pressure of 35 psig. The fragmentor was set to 175° C. and acquisition scan set to 100-3000 m/z. A 6-point calibration curve was built with a linear range of 1 to 50 μg/mL streptomycin and analyzed with Agilent TOF Quantitative Analysis software (
Loaded streptomycin (%)=Concentration streptomycin (initial)−Concentration streptomycin (filtrate)/Concentration streptomycin (initial)
The ZnNP-Strep conjugate was washed multiple times by centrifuging in ethanol to remove any unbound streptomycin. The ZnNP-Strep samples were digested by diluting them with 1% HCl followed by sonication. The zinc content of the solutions analyzed by atomic absorption spectroscopy (AAS) with a Perkin Elmer Analyst flame AAS and compared to a 6-point zinc calibration curve built from zinc standard solution (Fluka) diluted in 1% HCl (
To determine the rate of release of streptomycin from the ZnNPs, equal volumes of ZnNP-Strep and streptomycin sulfate both at the same concentration of streptomycin were sealed in separate dialysis bags (3.5 kDa MW cutoff, SPECTRA/POR®) and dialyzed against equal volumes DI water in two separate containers. Without changing the water, samples were collected from the dialysate at specific timepoints. The samples were filtered with a 0.22 μm syringe filter and the streptomycin concentration was analyzed by LCMS using the method described previously.
Antimicrobial Assays
The antimicrobial efficacy of the ZnNP-Strep and controls were evaluated by the antimicrobial assay methods as previously described. Briefly, Microplate Alamar Blue Assay (MABA) was performed to initially screen the antimicrobial efficacy of the NPs. Then, the minimum inhibitory concentration (MIC) was determined by broth microdilution method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI). As per the CLSI requirements, from a starter culture of 0.5 McFarland Standard, 5×105 CFU/well was added to all the wells. After 24 h of incubation with ZnNP-Strep and controls, the bacterial growth inhibition was ascertained using a spectrophotometer (570 and 600 nm). The accuracy of the MIC determination was improved by adding 10 μL of resazurin dye (0.0125%, w/v) per 100 μL well volume and observing color changes (from blue to pink for live organisms).
The antimicrobial efficacy of ZnNP-Strep was also studied after dialysis of the particles. The particles were dialyzed to remove any unbound streptomycin and simulate the effect of rainfall on the particles in field conditions. ZnNP-Strep was dialyzed against DI water with a 3.5 kDa MW cutoff dialysis membrane. The particles were dialyzed for 0, 1, 3, and 12 h and the remaining streptomycin loaded on the particles for each timepoint was quantified by LCMS by diluting the particles with 1% HCl and analyzing them with the previously described method. After streptomycin quantification the particles were diluted to 500 ppm streptomycin and their antimicrobial efficacy was assessed via MIC assay described above. The minimum bactericidal concentration (MBC) of the particles was also assessed by performing colony forming unit (CFU) plating. After 24 h incubation of bacteria with the samples, 100 μL of the treated bacteria was then transferred to nutrient agar plates. They were then spread dry using sterile spreaders (Fisher Scientific) and incubated for 48 h to check for bacterial growth at each concentration tested.
Phytotoxicity
The phytotoxicity of the ZnNP adjuvant with and without streptomycin was tested on tomato plants (Solanum lycopersicum, model fruit plant, 11 cm tall) obtained from Home Depot. The ZnNP treatments were applied directly on plant leaves (foliar application) using a hand-held sprayer until excess liquid starts dripping. Foliar application was conducted at 8:00 a.m. in a greenhouse setting to simulate field conditions. Treated plants were then placed in a growth chamber, Panasonic environmental test chamber (model MLR-352H) with summer settings (34° C., 60-80% RH). Phytotoxicity was evaluated 72 h post application. Digital photographs of treated leaves were taken for the visual assessment (phenotypic) plant tissue damage. Parallel treatments were done with copper sulfate at 1000 μg/mL (positive control) and DI water (untreated control, UTC) to compare phytotoxicity level with the ZnNP treatments. The appearance of brown necrotic spots, chlorosis, and necrosis on the leaves is indicative of plant tissue damage (i.e. phytotoxicity). Phytotoxicity was rated as follows: No toxicity (−), symptoms appear on less than 5% of the leaves was rated as minimal (+), 5 to 50% was rated as moderate (++), and above 50% was rated as severe (+++). Phytotoxicity studies were carried out in triplicate for each treatment and concentration.
Rainfastness Assay
Rainfastness of ZnNP-Strep and streptomycin was compared using leaves collected from 8-12-month-old citrus seedlings (Cleopatra; Citrus reshni). First, similar sized leaves were freshly cut from seedlings. The leaves were then copiously rinsed with DI water and patted dry. Next, 20 drops (2 μL per drop) of sample (streptomycin concentration of 500 μg/mL) was applied to each leaf and allowed to dry. To simulate rainfall, the leaves were slowly submerged 30 seconds into a glass beaker filled with DI water. The leaves were then placed on a paper towel and allowed to air dry for 1 hour. This process was then repeated to simulate a second rainfall. After each simulated rainfall, the remaining streptomycin on the leaf was removed and quantified using the following procedure. The leaves were placed in 5 mL polypropylene screw cap vials with 1 to 3 mL PBS (pH 7.4) and sonicated for 10 minutes using a bath sonicator. Vortex is intermittently used to ensure uniform mixing of the contents during sonication. The streptomycin removed from the leaves was filtered with a 0.22 μm syringe filter and quantified by LCMS analysis. Data was reported as a percentage of streptomycin removed from the leaf after washing compared to the total amount of streptomycin initially added.
Results and Discussion
Particle Size and Size Distribution
HRTEM analysis of NAC-ZnNPs showed the formation of monodisperse spherical particles with an average core diameter of 3.5 nm.
Next the possibility of creating a conjugate between the NAC and streptomycin to reduce its release rate in water and improve rainfastness was investigated. There are two ways one can immobilize streptomycin onto NAC-ZnNPs, electrostatic interactions and covalent coupling. The latter method will require additional coupling chemistry to conjugate carboxyl group of the NAC-ZnNPs with the anime groups of streptomycin. Another disadvantage of this method is that it forms a stable amide bond between streptomycin and NAC molecules, limiting availability of streptomycin it its original form. Streptomycin sulfate in water produces positively charged streptomycin cation where amine groups are protonated (
Several characterization techniques were used to determine solubility, surface charge, optical properties, and colloidal stability of the ZnNP-Strep.
Fluorescence emission maxima of NAC-ZnNP and ZnNP-Strep were observed at 590 nm under UV excitation (320 nm) due to the presence of Mn2+ dopant.
The loading of streptomycin on the particles was assessed by separating the ZnNP-Strep conjugates from unbound streptomycin by centrifugal filtration and it was found that 77.2% of the streptomycin was loaded on the particles. For agricultural field applications it may be beneficial that not all the streptomycin is loaded to the particle as the initial unbound streptomycin can be used for contact killing and the loaded streptomycin would be slowly released to provide a barrier against incoming bacteria. LCMS analysis showed that 1 mole of NAC-ZnNP could associate with a maximum of 26±5 mole of streptomycin.
Antimicrobial Studies
The antibacterial efficacy of ZnNP-Strep was tested against E. coli, P. syringae and X. alfalfae and compared to streptomycin sulfate, NAC, and NAC-ZnNP (Table 5). X. alfalfae was chosen as it is a surrogate for citrus canker causing pathogen Xanthomonas citri subsp. citri. It was found that the NAC-ZnNP and NAC by themselves had no growth inhibitory effect even at the highest concentration tested, i.e. 1000 μg/mL. This is due to the low solubility of ZnS which limits antimicrobial Zn ion release from the particles. The antimicrobial activity of ZnNP-Strep was compared to streptomycin sulfate. The purpose was to check if there is any loss of efficacy of streptomycin when it is associated with the ZnNP. Our results showed fairly comparable bactericidal activity of ZnNP-Strep with the streptomycin alone in inhibiting growth of P. syringae, X. alfalfa, and E. coli (Table 1). To test the rate of release of streptomycin from the ZnNP-Strep, the material was dialyzed against water to simulate the conditions of rainfall on treated leaves.
E. coli
Table 5 shows MIC (concentration active) of streptomycin sulfate, ZnNP-Strep, NAC-ZnNP, and NAC only on X. alfalfae, P. syringae, and E. coli.
Phytotoxicity Assay
The phytotoxicity of ZnNP-Strep was tested at different concentrations and the results were compared with controls, streptomycin, NAC-ZnNP and copper sulfate using tomato plants. Tomato plants were selected for this study as they are more susceptible to phytotoxicity than citrus plant due to lack the proactive waxy coating on the leaves. Table 6 compiles the phytotoxicity data after 72 hrs of foliar application. No signs of phytotoxicity was observed for any treatments and controls except Cu sulfate at 1000 μg/mL metallic Cu (positive control). As expected, tomato plants exhibited many brown spots due to the free copper ion phytotoxicity.
Table 6 shows Phytotoxicity of Streptomycin, NAC-ZnNP, ZnNP-Strep, and copper sulfate on tomato. In Table 6, the following abbreviations are used: a+++, severe toxicity; ++, moderate toxicity; +, low toxicity; −, zero toxicity.
Rainfastness Assay
Rainfastness experiments were conducted to determine if the ZnNPs improve the retention of streptomycin on citrus leaves. The extraction efficiency of streptomycin from the leaf surface was assessed by adding and then removing streptomycin from unwashed leaves. The amount of streptomycin removed from the leaf was compared to the amount of streptomycin applied to the leaf to determine the extraction efficiency. This was done to ensure what percentage of streptomycin was being removed from the leaf surface. The extraction efficiency for streptomycin sulfate was 98% and for ZnNP-Strep was 85% due to some streptomycin retained on the NAC-ZnNP after sample preparation. The extraction efficiency for FireWall™ 50 WP (a commercial product containing streptomycin sulfate as the active ingredient) was greater than 100% which can be attributed to the inhomogeneous and insoluble nature of the raw material. After the materials were dried the leaves were submerged in water to simulate the effects of heavy rainfall and remaining streptomycin on the leaves removed and quantified by LCMS. Around 50% of the streptomycin sulfate was removed after the first wash with only 30% remaining after the second wash.
An investigation was conducted to determine whether ZnNP-Strep would retain antimicrobial efficacy after rainfall. To simulate this, MIC testing was carried out on ZnNP-Strep using dialysis technique that simulates removal of treatment due to rainfall. Table 7 shows the antibacterial efficacy of the ZnNP-Strep particles with respect to streptomycin concentration after dialysis from 1 to 12 h. It was found that the antimicrobial efficacy was not compromised with the dialysis time. Our rainfastness study shows that streptomycin content is reduced with the increase of dialysis time.
This suggests that antimicrobial potency of streptomycin might have increased in association with ZnNP.
Table 7 shows Antibacterial efficacy of ZnNP-Strep after dialysis on X. alfalfae. In Table 7, MIC and MBC were determined relative to concentration of streptomycin in the sample.
In summary, various embodiments provide a ZnNP based adjuvant for improving streptomycin rainfastness. The ZnNPs were synthesized via a one-step one-pot precipitation reaction and coated with streptomycin. The ZnNP-Strep was shown to retain more streptomycin on the leaf surface after simulated rainfall than the commercially formulated streptomycin. This particulate adjuvant was shown to allow controlled the release of streptomycin, needed for long-lasting antimicrobial protection and therefore disease control. Based on the MIC study of the dialyzed ZnNP-Strep samples, it appears that the antimicrobial potency of streptomycin might have been increased when it is associated with ZnNP.
This invention was made with government support under USDA-NIFA CDRE grant #2015-70016-23010. The government has certain rights in this invention.
