WO2025199353A1 - Nanoparticulate formulations and foliar administration thereof in the treatment and prevention of citrus greening disease - Google Patents

Abstract

A nanoparticulate formulation comprising (i) a core comprising (a) at least one antibiotic, which is effective in treating Candidatus Liberibacter asiaticus (CLas), alone or in further combination with, (b) (i') at least one hydrophobic counterion, (ii') at least one hydrophobic molecule, or (iii') a combination of (i') and (ii'); and (ii) a shell encapsulating the core and comprising a stabilizer; and a method of preventing or treating infection of a citrus plant with Candidatus Liberibacter asiaticus (CLas) comprising administering an effective amount of the nanoparticulate formulation to the foliage of the citrus plant.

Description

NANOPARTICULATE FORMULATIONS AND FOLIAR ADMINISTRATION THEREOF IN THE TREATMENT AND PREVENTION OF CITRUS GREENING DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application no. 63/568,308, which was filed March 21, 2024, and which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under 2022-70029-38668 awarded by the U.S. Department of Agriculture (USDA). The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The present disclosure relates to nanoparticulate formulations, such as formulations comprising at least one antibiotic, alone or in further combination with at least one hydrophobic counterion/molecule, and a method of administering the formulation to the foliage of a citrus plant in the treatment or prevention of infection with Candidatus Liberibacter asiaticus (CLas).

BACKGROUND

[0004] Huanglongbing (HLB) disease or citrus greening disease is a devastating bacterial infection, which is thought to have originated in Asia and was first described in the early 1900s. It was described in Brazil in 2004. HLB has severely reduced the output of the Florida citrus industry since it was detected in 2005. HLB is now endemic to Florida, and the economic loss due to the disease is over $1 billion annually. Detected in California and Texas in 2012, HLB has been reported in residential areas in southern California and is eventually expected to affect commercial groves. The projected incidence of HLB in Texas was 80% in 2022. HLB is also known as citrus vein phloem degeneration (CVPD), yellow shoot disease, leaf mottle yellow (Philippines), libukin (Taiwan), and citrus dieback (India).

[0005] Candidatus Liberibacter asiaticus (CLas), the primary bacterium that causes HLB, is a gram-negative bacterium in the Rhizobiaceae family that lives in plant phloem vasculature and roots. Trees affected by HLB display low yield with high fruit drop and decreased fruit quality, due to the build-up of acidic compounds, which lead to a bitter, salty-tasting flesh or juice, making it unpalatable. Other signs include thin canopies, small blotchy leaves, branch dieback symptoms, and significant root collapse. Carbon sequestration in above-ground tissues leads to root carbohydrate starvation and poor below-ground translocation of photo-assimilates due to phloem sieve tube occlusion from CLas infection.

[0006] Particularly sensitive citrus plants include Citrus halimii, Nules’ clementine mandarin, Valencia sweet orange, ‘Madam Vinous’ sweet orange, ‘Duncan’ grapefruit, ‘Ruby’ red grapefruit, and ‘Mineola’ tangelo.

[0007] The pathogen is vectored by an invasive insect, Diaphorina citri, which is also known as the Asian citrus psyllid. Insect populations have been mainly controlled by removal of infected trees and intensive regimens of synthetic insecticide applications, such as aldicarb and imidacloprid. Routes of administration include systemic and contact. The efficacy and activity of these methods is less than satisfactory, particularly given that newly infected trees show signs of infection after a latency period of 6-12 months. The use of cyclic ketoenols against CLas is described in IntT Pat. App. Pub. No. WO 2011/029536. Other treatments include administration of nutrients, soil conditioners, and small molecules.

[0008] In heavily affected orchards, trees are also treated with antibiotics, such as penicillin, oxytetracycline and streptomycin, to reduce levels of pathogen inoculum reservoirs. The only effective treatment currently available is a formulation of oxytetracycline (Invaio) administered by injection into the trunks of infected trees, which is time- and labor-intensive. No formulation delivered as a foliar spray has demonstrated efficacy at reducing the disease burden on trees, since most agrochemicals, including antibiotics that are effective against the causal agent for HLB, are not taken up by leaves and translocated systemically throughout the plant.

[0009] Application of antibiotics, however, has raised environmental concerns. Unintended consequences include reduced biodiversity and selection for resistance in bacterial and insect populations.

[0010] In view of the above, it is an object of the present disclosure to provide an antibiotic, such as streptomycin, encapsulated in a nanocarrier, which can be administered to foliage and are taken up by leaves and translocated to the roots. This and other objects and advantages, as well as inventive features, will be apparent from the detailed description provided herein. SUMMARY

[0011] Provided is a nanoparticulate formulation comprising (i) a core comprising (a) at least one antibiotic, which is effective in treating Candidatus Liberibacter asiaticus (CLas), alone or in further combination with, (b) (i’) at least one hydrophobic counterion, (ii’) at least one hydrophobic molecule, or (iii’) a combination of (i’) and (ii’); and (ii) a shell encapsulating the core and comprising a stabilizer. The at least one antibiotic can be selected from the group consisting of streptomycin, oxy tetracycline, polymyxin B, gentamycin, vancomycin, mastoparan 7, tobramycin, kanamycin, amikacin, and a combination of two or more of the foregoing. The at least one hydrophobic counterion can have a logP value > 2. The at least one hydrophobic counterion can have one, two or more ionizable functional groups. The at least one hydrophobic counterion can be selected from the group consisting of oleic acid (or a sodium salt thereof), sodium dodecyl sulfate, sodium dodecyl benzenesulfonate, and a combination of two or more of the foregoing. The at least one hydrophobic molecule can be selected from the group consisting of a tocopherol, a tocopherol acetate, astaxanthin, P-carotene, lycopene, lutein, zeaxanthin, P- cryptoxanthin, a-carotene, xanthophyll, retinol, retinal, canthaxanthin, violaxanthin, phytoene, phytofluene, neoxanthin, echinenone, resveratrol, pterostilbene, and a combination of two or more of the foregoing. The stabilizer can be selected from the group consisting of an amphiphilic molecule, a non-block copolymer, a block copolymer, and a combination of two or more of the foregoing. The amphiphilic molecule can be selected from the group consisting of lecithin (e.g., soybean lecithin), gelatin, zein, casein, sucrose stearate, sucrose distearate, lauryl glucoside, decyl glucoside, octyl glucoside, alkyl glucoside, and combinations thereof. The nonblock copolymer can be selected from the group consisting of hydroxypropyl methylcellulose acetate succinate and tocopherol polyethylene glycol succinate. The block copolymer can comprise (a) at least one hydrophobic block polymer selected from the group consisting of polycaprolactone, poly(lactic-co-glycolic acid), polystyrene, and polylactic acid and (b) at least one hydrophilic block polymer selected from the group consisting of polyethylene glycol, polyacrylic acid, and poly [2(dimethylamino)ethyl methacrylate].

[0012] Also provided is a method of preventing or treating infection of a citrus plant with Candidatus Liberibacter asiaticus (CLas). The method comprises administering an effective amount of an above-described nanoparticulate formulation to the foliage of the citrus plant. In certain embodiments, the administration can reduce or eliminate the titer of CLas bacteria on or in the citrus plant. Tn other embodiments, the administration can prevent colonization of the citrus plant with CLas. The method can increase the number (e.g., the average number) of fruits set per tree. The method can increase the number (e.g., the average number) of fruits harvested per tree. The method can increase the fruit yield, i.e., the number (e.g., the average number) of boxes harvested per acre.

DETAILED DESCRIPTION

[0013] The present disclosure is predicated on the discovery that a formulation of streptomycin encapsulated into a nanocarrier is taken up by leaves of citrus plants and translocated throughout the plant. Application of the formulation to citrus foliage effectively reduces Candidatus Liberibacter asiaticus (CLas), the primary cause of Huanglongbing (HLB) disease or citrus greening disease.

[0014] “Citrus” refers to any plant of the genus Citrus, family Rutaceae. Citrus includes, but is not limited to, Citrus maxima, Citrus medica, Citrus micrantha, Citrus reticulata, Citrus trifolata, Citrus japonica, Citrus australasica, Citrus australis, Citrus glauca, Citrus garrawayae, Citrus gracilis, Citrus inodora, Citrus warburgiana, Citrus wintersii, Citrus halimii, Citrus indica, Citrus macroptera, and Citrus latipes. Citrus also includes hybrids such as Citrus x aurantiifolia, Citrus x aurantium, Citrus x latifolia, Citrus x limon, Citrus x limonia, Citrus x paradisi, Citrus x sinensis, Citrus x tangerina, Poncirus trifoliata x C. senensis.

Common names of citrus include, but are not limited to, Imperial lemon, tangelo, orangelo, tangor, kinnow, kiyomi, Minneola tangelo, oroblanco, sweet orange, ugli, Buddha’s hand, citron, lemon, orange, bergamot orange, bitter orange, blood orange, calamondin, clementine, grapefruit, Meyer lemon, Rangpur, tangerine, and yuzu.

[0015] In view of the above, provided is a nanoparticulate formulation comprising (i) a core comprising (a) at least one antibiotic, which is effective in treating Candidatus Liberibacter asiaticus (CLas), alone or in further combination with, (b) (i’) at least one hydrophobic counterion, (ii’) at least one hydrophobic molecule, or (iii ’ ) a combination of (i’) and (ii’); and (ii) a shell encapsulating the core and comprising a stabilizer.

[0016] Any active agent, such as an antibiotic, that is effective against CLas can be used. The at least one active agent/antibiotic can be selected from the group consisting of streptomycin, oxytetracycline, polymyxin B, gentamycin, vancomycin, mastoparan 7, tobramycin, kanamycin, amikacin, and a combination of two or more of the foregoing. Other examples of antibiotics include blasticidin S, kasugamycin, validamycin, aurcofungin, cyclohcximidc, griscofulvin, moroxydine, natamycin, polyoxin, polyoxorim, chlortetracycline, demeclocycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, doxycycline, tigecycline, eravacycline, sarecycline, oromadacycline, kanamycin A, tobramycin, dibekacin, sisomicin, netilmicin, neomycin B, neomycin C, neomycin E, tetracycline, an aminoglycoside, colistin, 0- cyfluthrin, zeta-cypermethrin, and plaomicin. Other examples of active agents/antibiotics include, but are not limited to, chlorantraniliprole, cyantraniliprole, propiconazole, propamocarb, prothioconazole, azoxystrobin, fludioxonil, benzovindiflupyr, boscalid, methoxyfenozide, sarmentine, spliceostatin C, spermine, spermidine, jasmonic acid, farnesene, squalene, diploptene, phytane, cycloartenol, stigmasterol, labdane, phytol, betulinic acid, abietane, cembrene A, cadinene, humulene, zingiberene, longifolene, caryophyllene, farnesol vetivazulene, guaiazulene, germacrene A, germacrene D, linalyl butyrate, copaene, geranyl butyrate, beta- Ocimene, P-cymene, patchoulol, geranyl propionate, viridiflorol, geranyl acetate, estragole, limonene, a-terpinyl acetate, thymol, carvacrol, beta-pinene, neral, geraniol, citral, methyl eugenol, a-terpinene, linalool, perillic acid, perillaldehyde, methyl cinnamate, eucalyptol, eugenol, acetyleugenol, thujone, 4-carvomenthenol, camphor, guaianolide, cinnamaldehyde, bisacurone, rishitin, cyphenothrin, esfenvalerate, etofenprox, fenpropathrin, fenvalerate, flucythrinate, flumethrin, cyhalothrin, phenothrin, resmethrin, silafluofen, fluvalinate, cyfluthrin, and permethrin.

[0017] Any suitable hydrophobic counterion can be used. The at least one hydrophobic counterion can have a logP value > 2. The counterions that cause precipitation can have logP values of 2 or greater at pH=7. The at least one hydrophobic counterion can have one, two, three or more ionizable functional groups. Anionic counterions can have pKa values of -2 to 5. Cationic counterions can have pKb values above 2 or may be quaternized cationic species that are permanently cationic. The ionic sites may be carboxylic acids, sulfates, sulfonates, sulfates, or amines. The at least one hydrophobic counterion can be selected from the group consisting of oleic acid (or a sodium salt thereof), sodium dodecyl sulfate, sodium dodecyl benzenesulfonate, and a combination of two or more of the foregoing. Other examples of anionic, hydrophobic counterions include, but are not limited to, (lR)-(-)-10-camphorsulfonic acid, 1,2-ethanesulfonic acid, 11-eicosenoate, 1 -decanesulfonic acid, 1 -heptanesulfonic acid, 1 -octanesulfonic acid, 2- naphthalenesulfonic acid, 3-hydroxy-2-naphthoic acid, 4-octylbenzenesulfonic acid, a-linolenic acid, alpha- tocopherol phosphonic acid, alpha-tocopherol succinic acid, bcnzcncsulfonic acid, capric acid, cholesterol sulfonic acid, decanoic acid, decyl sulfonic acid, deoxycholic acid, dioctyl sulfosuccinic acid (docusate), dodecyl sulfonic acid, dodecylbenzene sulfonic acid, elaidic acid, hexanoic acid, lauric acid, linoelaidic acid, Linoleic acid, linolelaidic acid, myristatic acid, myristoleic acid, n-dodecylphosphonic acid, n-octadecylphosphonic acid, nonanoic acid, oleic acid, palmitic acid, palmitoleic acid, pamoic acid, petroselinic acid, ricinelaidic acid, ricinoleic acid, sebacic acid, stearic acid, taurodeoxycholic acid, tetracosanoic acid, vaccenic acid, or salts (e.g., sodium or other) thereof. Other examples of cationic, hydrophobic counterions include, but are not limited to, salts (e.g., bromide, chloride, stearate, or others) of hexyltrimethylammonium, octyltrimethylammonium, decyltrimethylammonium, dodecyltrimethylammonium, trimethylhexadecylammonium, trimethyloctadecylammonium, didodecyldimethylammonium, dimethylditetradecylammonium, dimethyldihexadecylammonium, dimethyldioctadecylammonium, tetraoctylammonium, tetrakis(decyl)ammonium, tetradodecylammonium, tetrahexadecylammonium, tetraoctadecylammonium, and dioctyldimethylammonium.

[0018] Any suitable hydrophobic molecule can be used. The at least one hydrophobic molecule can be selected from the group consisting of a tocopherol, a tocopherol acetate, astaxanthin, 0- carotene, lycopene, lutein, zeaxanthin, 0-cryptoxanthin, a-carotene, xanthophyll, retinol, retinal, canthaxanthin, violaxanthin, phytoene, phytofluene, neoxanthin, echinenone, resveratrol, pterostilbene, and a combination of two or more of the foregoing.

[0019] Any suitable stabilizer can be used. The stabilizer can be selected from the group consisting of an amphiphilic molecule, a non-block copolymer, a block copolymer, and a combination of two or more of the foregoing.

[0020] Amphiphilic molecules are chemical compounds that have both polar and nonpolar regions, giving them both hydrophilic (water-loving) and lipophilic (fat-loving) properties. Any suitable amphiphilic molecule, as well-known in the art, can be used. Examples of amphiphilic molecules include, but are not limited to, lecithin (e.g., soybean lecithin), gelatin, zein, casein, sucrose stearate, sucrose distearate, lauryl glucoside, decyl glucoside, octyl glucoside, alkyl glucoside, and combinations thereof. In some embodiments, the amphiphilic molecule is sucrose stearate, sucrose distearate, or a combination thereof. [0021] Any suitable non-block copolymer can be used. The non-block copolymer can be selected from the group consisting of hydroxypropyl mcthylccllulosc acetate succinate (HPMCAS) and tocopherol polyethylene glycol succinate.

[0022] Any suitable block copolymer can be used. The block copolymer can comprise (a) at least one hydrophobic block polymer selected from the group consisting of polycaprolactone, poly(lactic-co-glycolic acid), polystyrene, and polylactic acid and (b) at least one hydrophilic block polymer selected from the group consisting of polyethylene glycol, polyacrylic acid, and poly [2(dimethylamino)ethyl methacrylate] .

[0023] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising a bactericide, a fungicide, an acaricide, a nematicide, an herbicide, and/or an insecticide. Additionally or alternatively, the composition can further comprise, or can be used in combination with, micronutrients, safeners, surfactants (e.g., wetting/spreading agents), lipochito-oligosaccharide compounds (see, e.g., U.S. Pat. Nos. 5,549,718; 5,646,018; 5, 175,149; and 5,321,011), and/or products that reduce stress (e.g., Myconate). Chelated micronutrient compositions described, for example, in USPAPN 2022/0394981 (published December 15, 2022) also can be used in combination with the disclosed composition, whether in the same composition or a separate composition, which is administered simultaneously or sequentially (in either order).

[0024] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising a host defense inducer, such as isotianil, acibenzolar-S-methyl, probenazole, and tiadinil or combinations thereof (see, e.g., USPAPN 2015/0011394, published January 8, 2015). Isotianil can be combined with fosetyl AL. Other host defense inducers include P- aminobutyric acid, 2-deoxy-D-glucose, salicylic acid, and oxalic acid (see, e.g., USPAPN 2016/0227774, published August 11, 2016).

[0025] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising a gibberellin, such as Al, A3, A4 and/or A7. A3, which is gibberellic acid, can be preferred. See, e.g., Wegler, Chemistry of plant protection and pesticide agents, vol. 2, Springer Verlag, Berlin-Heidelberg-New York, 1970, pp. 401-412.

[0026] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising a compound that interrupts the ATP-hydrolysis process in a Sec system and disrupts pre-protein translocation, such as a SecA inhibitor (see, e.g., compounds (e.g., C16) described in USPAPN 2015/0087512, published March 26, 2015).

[0027] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising a cladosporol compound, a radicinin compound, and/or an epicoccamide compound (see, e.g., USPAPN 2023/0102379, published March 30, 2023).

[0028] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising a polyphenol, such as a proanthocyanidin (PAC), benzoic acid, quinic acid, and/or xyloglucan, such as a composition comprising cranberry solids (see, e.g., USPAPN 2019/0059392, which was published February 28, 2019). Compositions containing cranberry solids can inhibit CLas bacteria growing in the gut of psyllids.

[0029] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising an oak leaf extract, such as an aqueous oak leaf extract from Quercus hemisphaerica (see, e.g., USPAPN 2021/0186031, which was published June 24, 2021).

[0030] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising kaolin clay and a dye, such as a red dye (see, e.g., USPAPN 2021/0337803, which was published November 4, 2021).

[0031] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising aminocaproic acid, carbinoxamine maleate, chloroxylenol, chlorpropamide, cinoxacin, duartin, and/or cyclopentolate hydrochloride (see, e.g., USPAPN 2021/0368795, which was published December 2, 2021).

[0032] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising an antimicrobial peptide comprising a first amphipathic helical peptide and a second amphipathic helical peptide connected by a peptide linker comprising 2-15 amino acids to form a helix-turn-helix structure as described in USPAPN 2022/0053773, which was published on February 24, 2022.

[0033] The composition can further comprise or be simultaneously or sequentially administered (in either order) with a composition comprising RejuAgro A, the compound of formula I as described in USPAPN 2022/0104487, which was published on April 7, 2022. [0034] The nanoparticulate formulation can be prepared using methods known to those of ordinary skill in the art. Preferred methods arc exemplified herein. Briefly, the nanocarricr is prepared by flash nano-precipitation with a surface- stabilizing material (e.g., an amphiphilic lipid, such as soy lecithin, a small molecule surfactant, such as sucrose stearate or sucrose distearate, a polymer, such as HPMCAS, or a combination of two or more thereof). The nanocarrier encapsulates an antibiotic, such as streptomycin, by hydrophobic ion pairing. A hydrophobic small molecule, such as a-tocopherol or a-tocopherol acetate, which acts as an antioxidant, can be encapsulated in the core with the antibiotic to help the tree, such as a tree with intraluminal callose, recover from HLB. The colloidal suspension of the nanoparticulate formulation can be subsequently processed into a dry powder via spray-drying. The nanoparticulate formulation can be customized based on the ratio of agents used in the core and shell and particle size. The dosage amount and delivery approach can be optimized based on the nanoparticulate formulation delivered and the plant treated.

[0035] The size of the nanoparticle can be about 20 nm to about 300 nm, such as about 20 nm to 300 nm, or 20 nm to about 300 nm, or 20 nm to 300 nm. In some embodiments, the size of the nanoparticle is about 25 nm to about 250 nm. In some embodiments, the size of the nanoparticle is about 50 nm to about 200 nm. In some embodiments, the size of the nanoparticle is about 100 nm to about 200 nm. In some embodiments, the size of the nanoparticle is about 120 nm to about 250 nm. In some embodiments, the size of the nanoparticle is about 150 nm to about 200 nm. [0036] Also provided is a method of preventing or treating infection of a citrus plant with Candidatus Liberibacter asiaticus (CLas). The method comprises administering an effective amount of an above-described nanoparticulate formulation to the foliage of the citrus plant. In certain embodiments, the administration can reduce or eliminate the titer of CLas bacteria on or in the citrus plant. Any reduction in the titer of CLas bacteria is beneficial. Desirably, however, the administration results in the elimination of the titer of CLas bacteria. In other embodiments, the administration can prevent colonization of the citrus plant with CLas. The method can increase the number (e.g., the average number) of fruits set per tree. The method can increase the number (e.g., the average number) of fruits harvested per tree. The method can increase the fruit yield, i.e., the number (e.g., the average number) of boxes harvested per acre.

[0037] The composition desirably is applied to the foliage of a citrus tree in a manner and/or at a time to minimize, if not eliminate, any possible damage to the foliage as a result of the administration. Typically, the product is applied to the foliage by spraying until run-off. The application rate of the formulation can vary substantially. By way of example, a composition comprising an active ingredient at a concentration of about 0.05 mM to about 2 mM, such as about 0.1 mM to about 1.5 mM, such as about 0.2 mM to about 1 mM can be administered to the foliage of a citrus plant in an amount of about 0.1 gallon to about 0.8 gallon per tree, such as about 0.2 gallon to about 0.6 gallon per tree, such as about 0.25 gallon to about 0.5 gallon per tree. Alternatively, when a composition is sprayed, the amount of active ingredient can be 10- 10,000 g/ha such as 25-5,000 g/ha.

[0038] An effective amount or therapeutically effective amount is an amount of the composition, which improves the health, growth or productivity of the citrus plant, which reduces the effect, titer or signs/symptoms of HLB disease, and/or which prevents worsening of HLB or signs/symptoms thereof. An improvement or a reduction of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% are indicative of the amount of the composition being an effective or therapeutically effective amount.

[0039] When the composition is used with one or more other active agents, such as one or more compositions comprising active agents, the compositions can be administered simultaneously or sequentially by the same or different routes. Other routes of administration include, but are not limited to, dusting, sprinkling, spraying, brushing, dipping, smearing, impregnating, injection into the vascular system, and/or application to the root system (e.g., soil injection or soil drenching).

[0040] Treatment can slow down the population growth in planta of CLas, reduce severity of HLB, increase fruit yield, improve fruit quality, improve juice quality, and/or improve juice quantity. Results will depend on various factors, such as the age and severity of HLB at the time of treatment.

EXPERIMENTAL SECTION

[0041] This section serves to illustrate the present disclosure. This section is not intended to limit the scope of the claimed invention in any way. Preparation of nanoparticulate formulations

[0042] The process was carried out in a confined impinging jets mixer (CH) or multi-inlet vortex mixer (MIVM).

[0043] The antibiotic (streptomycin) (concentration of 1.25 to 10 mg/mL) was dissolved in deionized water (DI water) to obtain an aqueous solvent feed stream.

[0044] The stabilizer (Hypromellose acetate succinate [HPMCAS], lecithin, PCL-P-PEG, PS-P- PDMAEMA, PS-P-PAA, sucrose stearate, or sucrose distearate; concentration of 1.25 to 20 mg/mL) and a co-core (a tocopherol acetate [vitamin e acetate]; concentration of 0 to 10 mg/mL) were dissolved in tetrahydrofuran (THF) to obtain an organic solvent feed stream.

[0045] The counterion (sodium oleate, sodium dodecyl sulfate, or sodium dodecyl benzenesulfonate) was dissolved in methanol (sodium oleate) or dimethyl sulfoxide (DMSO) (sodium dodecyl sulfate or sodium dodecyl benzenesulfonate) to obtain a second organic solvent feed stream.

[0046] Alternately, the counterion (sodium oleate, sodium dodecyl sulfate, or sodium dodecyl benzenesulfonate) was dissolved in methanol (sodium oleate) or DMSO (sodium dodecyl sulfate or sodium dodecyl benzenesulfonate) and mixed with the THF stream to obtain a combined organic solvent feed stream.

[0047] Alternately, the counterion (sodium oleate, sodium dodecyl sulfate, or sodium dodecyl benzenesulfonate) was dissolved in water to obtain a second aqueous feed stream.

[0048] The feed streams were rapidly introduced into the mixer, resulting in rapid homogeneous mixing, which led to the rapid precipitation of the components. The sudden change in solvent quality led to particle assembly on the scale of tens of milliseconds. The resulting nanoparticles were formed within a range of sizes from 50 nm to approximately 350 nm, depending on the formulation.

Table I. Example formulations.

STP - Streptomycin sulfate; HPMCAS - Hydroxypropyl methylcellulose acetate succinate; LEC

- Lecithin; SS - Sucrose stearate; SD - Sucrose distearate; PCL-PEG - Polycaprolactone-P- polyethyleneglycol; PS-PDMAEMA Polystyrene-P-polydimethylaminoethylmethacrylate; PS- PAA - Polystyrene-P-polyacrylic acid; OL - Sodium oleate; SDS - Sodium dodecyl sulfate; SDBS

- Sodium dodecyl benzenesulfonate; VitEAc - a tocopherol acetate; N/A - not applicable.

Rescue of HLB-infected citrus tress by foliar application of anti-Clas streptomycin nanoparticles

[0049] Two field trials were conducted at the University of Florida Citrus Research and Education Center using five-year-old Hamlin and OLL orange trees (10’x20’ spacing). For each of four treatments, five replicate plots (7 trees per plot) were sprayed to runoff, approximately 0.57 gal/tree. The four treatments were; (1) negative control (water + 0.1% Silwet L-77); (2) positive conventional control (non-encapsulated streptomycin 100 mg/L in water with 0.1% Silwet L-77); (3) test case (nanocarrier-encapsulated streptomycin (100 mg/L) in water with 0.1 % Silwet L-77); and (4) positive oxytetracycline control (oxytetracycline trunk injection with commercially labeled dose and methods).

[0050] For Hamlin trees, the average number of fruits set per tree were (1) 168.1 for negative control; (2) 175.1 for positive conventional control of non-encapsulated streptomycin; (3) 223.0 for test case of nanocarrier-encapsulated streptomycin; and (4) 253.5 for positive oxytetracycline control, p= 0.015. Therefore, the administration of non-encapsulated streptomycin increased the number of fruits set per tree by seven (175.1 - 168.1 = 7). The administration of nanoencapsulated streptomycin increased the number of fruits set per tree by 54.9 (223.0 - 168.1 = 54.9). This is a 7.8-fold higher increase in the number of fruits set per tree compared to that of non-encapsulated streptomycin (54.9 / 7 = 7.8).

[0051] For Hamlin trees, the average number of fruits harvested per tree were (1) 69.1 for negative control; (2) 80.3 for positive conventional control of non-encapsulated streptomycin; (3) 110.0 for test case of nanocarrier-encapsulated streptomycin; and (4) 127.0 for positive oxytetracycline control, p= 0.035. Therefore, the administration of non-encapsulated streptomycin increased the number of fruits harvested per tree by 11.2 (80.3 - 69.1 = 11.2). The administration of nanocarrier-encapsulated streptomycin increased the number of fruits harvested per tree by 40.9 (110.0 - 69.1 = 40.9). This is a 3.65-fold higher increase in the number of fruits harvested per tree compared to that of non-encapsulated streptomycin (40.9 / 11.2 = 3.65).

[0052] For Hamlin trees, the fruit yield (boxes of fruit) per acre were (1) 50.1 for negative control; (2) 56.2 for positive conventional control of non-encapsulated streptomycin; (3) 68.8 for test case of nanocarrier-encapsulated streptomycin; and (4) 83.6 for positive oxytetracycline control, p= 0.114. Therefore, the administration of non-encapsulated streptomycin increased the boxes of fruit per acre by 5.1 boxes (56.2 - 50.1 = 5.1). The administration of nanocarrier- encapsulated streptomycin increased the boxes of fruit per acre by 18.7 boxes (68.8 - 50.1 = 18.7). This is a 3.66-fold higher increase in the number of boxes per acre compared to that of non-encapsulated streptomycin (18.7 / 5.1 = 3.66).

[0053] By all three of the above metrics, the nanocarrier-encapsulated streptomycin demonstrated a significant improvement over non-encapsulated streptomycin. This corroborates data from greenhouse trials of nanocarrier-encapsulated streptomycin translocation, which showed up to 15% translocation of nanocarrier-encapsulated streptomycin to phloem, compared to <1% translocation of non-encapsulated streptomycin to phloem. [0054] For Hamlin trees, the fruit diameter (mm) for the treatments were (1) 68. 15 [no treatment], (2) 67.07 [conventional streptomycin], (3) 66.5 [nanocarricr streptomycin], and (4) 67.99 [oxytetracycline trunk injection], p= 0.691. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect. [0055] For Hamlin trees, the fruit juice percent for the treatments were (1) 32.5 [no treatment], (2) 34.4 [conventional streptomycin], (3) 33.7 [nanocarrier streptomycin], and (4) 37 [oxytetracycline trunk injection], p= 0.376. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect.

[0056] For Hamlin trees, the fruit Brix score for the treatments were (1) 7.34 [no treatment], (2) 7.48 [conventional streptomycin], (3) 7.54 [nanocarrier streptomycin], and (4) 7.9 [oxytetracycline trunk injection], p= 0.376. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect.

[0057] For Hamlin trees, the fruit titratable acid percent for the treatments were (1) 0.6060 [no treatment], (2) 0.6200 [conventional streptomycin], (3) 0.6200 [nanocarrier streptomycin], and (4) 0.6280 [oxytetracycline trunk injection], p= 0.706. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect.

[0058] For Hamlin trees, the total soluble solids (Ib/box) for the treatments were (1) 2.160 [no treatment], (2) 2.320 [conventional streptomycin], (3) 2.260 [nanocarrier streptomycin], and (4) 2.660 [oxytetracycline trunk injection], p= 0.159. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect.

[0059] For OLL trees, the average number of fruits set per tree were (1) 57.1 [no treatment], (2) 64.5 [conventional streptomycin], (3) 87.1 [nanocarrier streptomycin], and (4) 91.9 [oxytetracycline trunk injection], p= 0.156. The effect of non-encapsulated streptomycin was an improvement of (64.5 - 57.1 =) 7.4 fruits per tree. The effect of nano-encapsulated streptomycin was an improvement of (87.1 - 57.1 =) 20 fruits per tree, which is (20 / 7.4 =) 2.7-fold higher than the effect from non-encapsulated streptomycin.

[0060] For OLL trees, the average number of fruits harvested per tree were (1) 64.8 [no treatment], (2) 76.6 [conventional streptomycin], (3) 102.9 [nanocarrier streptomycin], and (4) 111.0 [oxytetracycline trunk injection], p= 0.137. Therefore, the effect of non-encapsulated streptomycin was an improvement of (76.6 - 64.8 =) 11.8 fruits per tree. The effect of nano- encapsulated streptomycin was an improvement of (102.9 - 64.8 =) 38. 1 fruits per tree, which is (38.1 I 11.8 =) 3.23-fold higher than the effect from non-cncapsulatcd streptomycin.

[0061] By both of the above metrics, the nanocarrier streptomycin demonstrated a significant improvement over unencapsulated streptomycin. This corroborates data from greenhouse trials of NC translocation, which showed up to 15% translocation of NCs to phloem, compared against <1% translocation of unencapsulated streptomycin.

[0062] For OLL trees, the fruit diameter (mm) for the treatments were (1) 70.86 [no treatment], (2) 71.22 [conventional streptomycin], (3) 69.96 [nanocarrier streptomycin], and (4) 70.51 [oxytetracycline trunk injection], p= 0.351. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect.

[0063] For OLL trees, the fruit juice percent for the treatments were (1) 48.75 [no treatment], (2) 51.87 [conventional streptomycin], (3) 48.84 [nanocarrier streptomycin], and (4) 53.09 [oxytetracycline trunk injection]. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect.

[0064] For OLL trees, the fruit Brix score for the treatments were (1) 7.920 [no treatment], (2) 7.900 [conventional streptomycin], (3) 7.640 [nanocarrier streptomycin], and (4) 8.060 [oxytetracycline trunk injection], p= 0.376. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect.

[0065] For OLL trees, the fruit titratable acid percent for the treatments were (1) 0.6520 [no treatment], (2) 0.6770 [conventional streptomycin], (3) 0.6650 [nanocarrier streptomycin], and (4) 0.6660 [oxytetracycline trunk injection], p= 0.385. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect.

[0066] For OLL trees, the total soluble solids (Ib/box) for the treatments were (1) 3.475 [no treatment], (2) 3.686 [conventional streptomycin], (3) 3.363 [nanocarrier streptomycin], and (4) 3.853 [oxytetracycline trunk injection], p= 0.060. No significant differences were observed among treatments, so the streptomycin NPs had neither a beneficial nor a detrimental effect.

Measurements of fruit drop, fruit weight, color analysis, diameter measurement, juice extraction, juice content, and brix

[0067] During the fruit maturation phase prior to harvest, fruit drop is monitored by hand raking, removal, and counting dropped fruit every week. At harvest time, fruit is picked and weighed per net plot, subsampled for photography and color analysis (internal and external: CTE L*a*b*), diameter measurement, juice extraction, juice content (%), and brix (total dissolved solids) with a refractometer and acid content by titration.

Measurements of plant nutrients, oxytetracycline and streptomycin residues, and CLas titer

[0068] Leaf samples are collected from each plot at the end of each quarter in year 2 (approximately March, June, September, and December). Samples are partitioned so that one- third is analyzed for plant nutrients (P, K, Ca, Mg, Fe, Mn, Zn, B, and Cu) using acid digestion and ICP-MS, or dry combustion methods (N, S), one-third is analyzed for oxytetracycline and streptomycin residues using ELISA kits, and one-third is analyzed by q-PCR for CLas titer. Before analysis, fresh leaves are scanned on a flatbed scanner so that leaf symptoms can be digitally analyzed. The colors of leaves are determined by semantic segmentation using DeepLabV3+ and the CIE L*a*b* color coordinate system. HLB symptoms are scored with a deep learning citrus diagnostic system developed at UF (http://www[dot]makecitrusgreatagain[dot]com/SmartphoneApp.htm). The leaf tissue results are analyzed with the Diagnosis and Recommendation Integrated System (DRIS) system (http://www[dot]makecitrusgreatagain[dot]com/cgi-bin/driscgiprojectl) to identify the most limiting nutrient deficiencies and excesses.

ENUMERATED EMBODIMENTS

[0069] The following list of enumerated embodiments presents claims with multiply dependent claims depending from multiply dependent claims for presentation in those jurisdictions where such dependencies are allowed as well as additional claims, which may be pursued during the examination of the application or a divisional thereof.

EEL A nanoparticulate formulation comprising:

(i) a core comprising (a) at least one antibiotic, which is effective in treating Candidatus Liberibacter asiaticus (CLas), alone or in further combination with, (b) (i’ ) at least one hydrophobic counterion, (ii’ ) at least one hydrophobic molecule, or (iii’ ) a combination of (i’) and (ii’); and (ii) a shell encapsulating the core and comprising a stabilizer.

EE2. The nanoparticulate formulation of EE1, wherein the at least one antibiotic is selected from the group consisting of streptomycin, oxy tetracycline, polymyxin B, gentamycin, vancomycin, mastoparan 7, tobramycin, kanamycin, amikacin, and a combination of two or more of the foregoing.

EE3. The nanoparticulate formulation of EE1 or EE2, wherein the at least one hydrophobic counterion has a logP value > 2.

EE4. The nanoparticulate formulation of any one of EE1-EE3, wherein the at least one hydrophobic counterion has one, two, three or more ionizable functional groups.

EE5. The nanoparticulate formulation of any one of EE1-EE4, wherein the at least one hydrophobic counterion is selected from the group consisting of oleic acid (or a sodium salt thereof), sodium dodecyl sulfate, sodium dodecyl benzenesulfonate, and a combination of two or more of the foregoing.

EE6. The nanoparticulate formulation of any one of EE1-EE5, wherein the at least one hydrophobic molecule is selected from the group consisting of a tocopherol, a tocopherol acetate, astaxanthin, P-carotene, lycopene, lutein, zeaxanthin, -cryptoxanthin, a-carotene, xanthophyll, retinol, retinal, canthaxanthin, violaxanthin, phytoene, phytofluene, neoxanthin, echinenone, resveratrol, pterostilbene, and a combination of two or more of the foregoing.

EE7. The nanoparticulate formulation of any one of EE1-EE6, wherein the stabilizer is selected from the group consisting of an amphiphilic molecule, a non-block copolymer, a block copolymer, and a combination of two or more of the foregoing.

EE8. The nanoparticulate formulation of EE7, wherein the amphiphilic molecule is selected from the group consisting of lecithin (e.g., soybean lecithin), gelatin, zein, casein, sucrose stearate, sucrose distearate, lauryl glucoside, decyl glucoside, octyl glucoside, alkyl glucoside, and combinations thereof.

EE9. The nanoparticulate formulation of EE7, wherein the amphiphilic molecule is sucrose stearate, sucrose distearate, or a combination thereof.

EE 10. The nanoparticulate formulation of any one of EE7-EE9, wherein the non-block copolymer is selected from the group consisting of hydroxypropyl methylcellulose acetate succinate and tocopherol polyethylene glycol succinate.

EE11. The nanoparticulate formulation of any one of EE7-EE10, wherein the block copolymer comprises (a) at least one hydrophobic block polymer selected from the group consisting of polycaprolactone, poly(lactic-co-glycolic acid), polystyrene, and polylactic acid and (b) at least one hydrophilic block polymer selected from the group consisting of polyethylene glycol, polyacrylic acid, and poly[2(dimethylamino)ethyl methacrylate].

EE 12. A method of preventing or treating infection of a citrus plant with Candidatus Liberibacter asiaticus (CLas), which method comprises administering an effective amount of a nanoparticulate formulation of any one of EE 1 -EE 11 to the foliage of the citrus plant, whereupon infection of the citrus plant with CLas is prevented or treated.

EE 13. The method of EE 12, wherein the titer of CLas bacteria on or in the citrus plant is reduced or eliminated.

EE 14. The method of EE 12, wherein colonization of the citrus plant with CLas is prevented.

EE15. The method of EE 12, wherein the number (e.g., the average number) of fruits set per tree is increased. EE 16. The method of EE 12, wherein the number (e.g., the average number) of fruits harvested per tree is increased.

EE17. The method of EE12, wherein the fruit yield, i.e., the number (e.g., the average number) of boxes harvested per acre, is increased

[0070] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range.

[0071] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1 % to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0072] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation.

[0073] Any use of section headings and subheadings is solely for ease of reference and is not intended to limit any disclosure made in one section to that section only; rather, any disclosure made under one section heading or subheading is intended to constitute a disclosure under each and every other section heading or subheading.

[0074] Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

[0075] The terms and expressions, which have been employed, are used as terms of description and not of limitation. In this regard, where certain terms are defined and are described or discussed elsewhere, the definitions and all descriptions and discussions are intended to be attributed to such terms. There also is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof.

[0076] Further, all publications and patents mentioned herein are incorporated by reference in their entireties for all purposes. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

Claims

WHAT IS CLAIMED IS:

1. A nanoparticulate formulation comprising:

(i) a core comprising (a) at least one antibiotic, which is effective in treating Candidatus Liberibacter asiaticus (CLas), alone or in further combination with, (b) (i’) at least one hydrophobic counterion, (ii’) at least one hydrophobic molecule, or (iii’) a combination of (i’) and (ii’); and

(ii) a shell encapsulating the core and comprising a stabilizer.

2. The nanoparticulate formulation of claim 1, wherein the at least one antibiotic is selected from the group consisting of streptomycin, oxy tetracycline, polymyxin B, gentamycin, vancomycin, mastoparan 7, tobramycin, kanamycin, amikacin, and a combination of two or more of the foregoing.

3. The nanoparticulate formulation of claim 1, wherein the at least one hydrophobic counterion has a logP value > 2.

4. The nanoparticulate formulation of claim 1, wherein the at least one hydrophobic counterion has one, two, three or more ionizable functional groups.

5. The nanoparticulate formulation of claim 1, wherein the at least one hydrophobic counterion is selected from the group consisting of oleic acid (or a sodium salt thereof), sodium dodecyl sulfate, sodium dodecyl benzenesulfonate, and a combination of two or more of the foregoing.

6. The nanoparticulate formulation of claim 1, wherein the at least one hydrophobic molecule is selected from the group consisting of a tocopherol, a tocopherol acetate, astaxanthin, -carotene, lycopene, lutein, zeaxanthin, P-cryptoxanthin, a-carotene, xanthophyll, retinol, retinal, canthaxanthin, violaxanthin, phytoene, phytofluene, neoxanthin, echinenone, resveratrol, pterostilbene, and a combination of two or more of the foregoing.

7. The nanoparticulate formulation of claim 1, wherein the stabilizer is selected from the group consisting of an amphiphilic molecule, a non-block copolymer, a block copolymer, and a combination of two or more of the foregoing.

8. The nanoparticulate formulation of claim 7, wherein the amphiphilic molecule is selected from the group consisting of lecithin (e.g., soybean lecithin), gelatin, zein, casein, sucrose stearate, sucrose distearate, lauryl glucoside, decyl glucoside, octyl glucoside, alkyl glucoside, and combinations thereof.

9. The nanoparticulate formulation of claim 7, wherein the amphiphilic molecule is sucrose stearate, sucrose distearate, or a combination thereof.

10. The nanoparticulate formulation of claim 7, wherein the non-block copolymer is selected from the group consisting of hydroxypropyl methylcellulose acetate succinate and tocopherol polyethylene glycol succinate.

11. The nanoparticulate formulation of claim 7, wherein the block copolymer comprises (a) at least one hydrophobic block polymer selected from the group consisting of polycaprolactone, poly(lactic-co-glycolic acid), polystyrene, and polylactic acid and (b) at least one hydrophilic block polymer selected from the group consisting of polyethylene glycol, polyacrylic acid, and poly [2(dimethylamino)ethyl methacrylate].

12. A method of preventing or treating infection of a citrus plant with Candidatus Liberibacter asiaticus (CLas), which method comprises administering an effective amount of a nanoparticulate formulation comprising:

(i) a core comprising (a) at least one antibiotic, which is effective in treating Candidatus Liberibacter asiaticus (CLas), alone or in further combination with, (b) (i’) at least one hydrophobic counterion, (ii’) at least one hydrophobic molecule, or (iii’) a combination of (i’) and (ii’); and

(ii) a shell encapsulating the core and comprising a stabilizer, to the foliage of the citrus plant, whereupon infection of the citrus plant with CLas is prevented or treated.

13. The method of claim 12, wherein the at least one antibiotic is selected from the group consisting of streptomycin, oxy tetracycline, polymyxin B, gcntamycin, vancomycin, mastoparan 7, tobramycin, kanamycin, amikacin, and a combination of two or more of the foregoing.

14. The method of claim 12, wherein the at least one hydrophobic counterion has a logP value > 2.

15. The method of claim 12, wherein the at least one hydrophobic counterion has one, two, three or more ionizable functional groups.

16. The method of claim 12, wherein the at least one hydrophobic counterion is selected from the group consisting of oleic acid (or a sodium salt thereof), sodium dodecyl sulfate, sodium dodecyl benzenesulfonate, and a combination of two or more of the foregoing.

17. The method of claim 12, wherein the at least one hydrophobic molecule is selected from the group consisting of a tocopherol, a tocopherol acetate, astaxanthin, P-carotene, lycopene, lutein, zeaxanthin, -cryptoxanthin, a-carotene, xanthophyll, retinol, retinal, canthaxanthin, violaxanthin, phytoene, phytofluene, neoxanthin, echinenone, resveratrol, pterostilbene, and a combination of two or more of the foregoing.

18. The method of claim 12, wherein the stabilizer is selected from the group consisting of an amphiphilic molecule, a non-block copolymer, a block copolymer, and a combination of two or more of the foregoing.

19. The method of claim 18, wherein the amphiphilic molecule is selected from the group consisting of lecithin (e.g., soybean lecithin), gelatin, zein, casein, sucrose stearate, sucrose distearate, lauryl glucoside, decyl glucoside, octyl glucoside, alkyl glucoside, and combinations thereof.

20. The method of claim 18, wherein the amphiphilic molecule is sucrose stearate, sucrose distearate, or a combination thereof.

21. The method of claim 18, wherein the non-block copolymer is selected from the group consisting of hydroxypropyl methylcellulose acetate succinate and tocopherol polyethylene glycol succinate.

22. The method of claim 18, the block copolymer comprises (a) at least one hydrophobic block polymer selected from the group consisting of polycaprolactone, poly(lactic-co-glycolic acid), polystyrene, and polylactic acid and (b) at least one hydrophilic block polymer selected from the group consisting of polyethylene glycol, polyacrylic acid, and poly [2(dimethylamino)ethyl methacrylate] .