Use of oxytetracycline to control citrus green fruit disease in citrus plants

Abstract

The present disclosure relates generally to methods and systems for controlling citrus green fruit disease in citrus plants, such as, for example, orange trees.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 350,826 filed on June 9, 2022 and U.S. Provisional Patent Application No. 63 / 459,925 filed on April 17, 2023, each of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates generally to methods of controlling citrus greening disease in citrus plants, and more particularly to such methods of precisely delivering oxytetracycline to the active vasculature of citrus plants using a trunk or stem injection system. Background Art

[0004] Citrus Huanglongbing (HLB), also known as citrus greening disease, is one of the most destructive diseases of citrus worldwide. Citrus greening disease is a bacterial disease that attacks the vascular system of citrus trees and is usually caused by phloem-colonizing bacteria, such as "Candidalus Liberibacter asiaticus" (CLas). The bacteria are mainly carried and spread by the insect vector, the Asian citrus psyllid. See the Florida Department of Agriculture and Consumer Services "Huanglongbing (HLB) / Citrus Greening Disease Information". Three forms of greening disease have been described. The African form is spread by the African citrus psyllid Trioza erytreae and symptoms appear under cold conditions. The Asian and American forms are spread by the Asian citrus psyllid Diaphorina citri and symptoms appear under warmer conditions. For example, since 2005, HLB has spread to Florida's citrus production areas, reducing citrus yields by 75% while doubling production costs. To date, commercially available treatments have been ineffective in combating the disease. For example, once infected, trees carry the disease for life. Continuous reinfection with the bacteria that carry the psyllid increases the disease burden on already infected trees. Abandoned orchards without psyllid management are problematic, placing a higher burden on any adjacent production plots.

[0005] The bacteria that causes citrus greening can infect most citrus cultivars. For example, newly infected trees develop spots on their leaves. Long-term infected trees have smaller leaves and display asymmetrical spots. Fruit from infected trees tends to be small and of poor quality. The juice from such fruit is usually low in soluble solids and has a sour, bitter taste.

[0006] HLB affects many aspects of tree physiology. For example, citrus greening can cause weakening of the tree's root system, especially the absorbing roots that inhibit the tree's water absorption, and 30-50% root loss in the early stages of the disease. When symptoms appear in the crown, root losses are as high as 70%. See Diepenbrock et al. (eds.) "2022-2023 Florida Citrus Production Guide". University of Florida, Institute of Food and Agricultural Sciences Extension. Evidence also suggests that HLB thickens the xylem cell walls, affecting water flow in trees; Hamido et al. Plants 2019, 8, 298. These effects, in turn, increase the plant's sensitivity to secondary stresses. Disease symptoms include leaf mottling, stunted development, softened veins, and root decline. See United States Department of Agriculture. “Citrus Greening.” https: / / www.aphis.usda.gov / aphis / ourfocus / planthealth / plant-pest-and-disease-programs / pests-and-diseases / citrus / citrus-greening. The disease can also be detrimental to fruit production, causing abnormal fruit coloring, smaller fruit, premature fruit drop, reduced quality, salty and bitter flavors, and overall reduced yield.

[0007] HLB has particularly impacted Florida's citrus industry. Since it was first discovered in Florida in 1998, HLB has spread to every citrus producing region in Florida. Florida is currently under a statewide citrus greening quarantine. Since 1998, HLB has caused an approximately 80% decrease in orange production and an approximately 70% decrease in yield per acre.

[0008] There is currently no practical or commercially available cure for the disease. Therefore, to prevent the spread of citrus greening disease, trees often need to be quickly removed. Therefore, there is a need for commercially viable treatment options for controlling citrus greening disease. Summary of the invention

[0009] In one aspect, a method for controlling citrus greening disease of citrus plants (such as citrus trees or citrus shrubs) is provided. In some embodiments, the method comprises injecting the infected citrus plants with an injection formulation comprising oxytetracycline (OTC). In some embodiments, the injection formulation is injected using an injection system comprising an injection tool. In some embodiments, the injection tool is operably connected to a fluid delivery unit. In some embodiments, the fluid delivery unit is configured to deliver the injection formulation.

[0010] In some embodiments, the injection of the injection formulation comprises piercing the trunk or stem of the citrus plant using an injection tool of the injection system. In some embodiments, the injection of the injection formulation comprises delivering at least a portion of the injection formulation from the fluid delivery unit through the injection tool into the active vasculature of the citrus plant without exceeding the active vasculature.

[0011] In some embodiments, the citrus plant suffers from citrus greening disease. In some embodiments, the injection formulation is distributed in the trunk or stem and other parts of the citrus plant, such as leaves and / or fruits. In some variations, the injection formulation is precisely injected into the young shoots of the citrus plant. In some embodiments, the citrus plant is at least about 1 year old, at least about 2 years old, at least about 3 years old, at least about 4 years old, at least about 5 years old, at least about 10 years old, at least about 15 years old, at least about 20 years old, at least about 30 years old, at least about 40 years old, or at least about 50 years old.

[0012] In some embodiments, the delivery unit is a spring-loaded fluid delivery unit. In some embodiments, the delivery unit includes a pressurized formulation cartridge. In some embodiments, the method includes replacing the fluid delivery unit with a second fluid delivery unit. In some embodiments, the method includes delivering at least a portion of the injection formulation from the second fluid delivery unit through an injection tool into the active vasculature of the citrus plant without exceeding the active vasculature.

[0013] In some embodiments, the injection tool remains in the trunk or stem (including young shoots) of the citrus plant for at least one growing season. In some embodiments, the injection tool remains in the trunk or stem (including young shoots) of the citrus plant after multiple re-injections. In some embodiments, the trunk or stem of the citrus plant has bark. In some embodiments, the method includes removing at least a portion of the bark before piercing the trunk. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present application may be understood by referring to the following description in conjunction with the accompanying drawings.

[0015] Figures 1A-1C An exemplary injection tool is shown.

[0016] Figures 2A-2D An exemplary multi-port injection tool is shown.

[0017] Figure 3 An exemplary spring loaded fluid delivery unit is shown.

[0018] Figure 4A An exemplary chassis is shown.

[0019] Figure 4B An exemplary tank is shown.

[0020] Figure 5 Depicted are the harvest weights of trees treated with OTC hydrochloride (OTC-HCl) compared to untreated trees after one growing season.

[0021] Figure 6 Shown are the harvest weights of trees treated with OTC-HCl compared to untreated trees after the second growing season.

[0022] Figure 7 Shown are the number of fruits in trees treated with OTC-HCl compared to untreated trees after two growing seasons.

[0023] Figure 8 Shown is the percentage of fruit drop in trees treated with OTC-HCl compared to untreated trees after two growing seasons.

[0024] Fig. 9 Shown are the Brix values ​​of trees treated with OTC-HCl compared to untreated trees.

[0025] Fig.10 Shown are the Brix / acid ratios of trees treated with OTC-HCl compared to untreated trees.

[0026] Fig.11 It was shown that the yield of trees treated with OTC-HCl increased after one year compared to trees treated with the control.

[0027] Fig.12 Shown are the average harvest weights of trees treated with OTC-HCl compared to trees treated with controls.

[0028] Fig.13 Shown are the average yield increases for trees in different locations after one growing season of treatment with OTC-HCl.

[0029] Fig.14 It was shown that the average yield of trees at different locations increased after one growing season of treatment with OTC-HCl compared to trees treated with the control.

[0030] Figure 15-21Shown are the harvest weights of trees at a particular site after one growing season of treatment with OTC-HCl compared to trees treated with controls.

[0031] Fig. 22 Shown are the number of fruits in trees at different locations treated with OTC-HCl compared to trees treated with control trees after one growing season.

[0032] Fig.23 Shown is the percentage of fruit drop in trees at different locations treated with OTC-HCl compared to trees treated with control trees after one growing season.

[0033] Figure 24-27 The total fruit residues were shown to decrease for various treatments of trees with OTC-HCl.

[0034] Fig.28 Shown are the average total residues in the fruit at different days after the last application of OTC-HCl.

[0035] Fig.29 Shown are the average total fruit residues in the fruits at different days after the last application of OTC-HCl.

[0036] Fig.30 Depicted are graphs showing the effect of OTC-HCl treatment on yield increase compared to water treatment.

[0037] Fig.31 Depicted are graphs showing the effect of OTC-HCl treatment on yield increase compared to untreated controls.

[0038] Fig.32 The effect of OTC-HCl treatment on reducing citrus greening disease and leaf mottling was compared with untreated controls. DETAILED DESCRIPTION

[0039] The following description is for those skilled in the art to be able to make and use various embodiments. The description of specific devices, techniques and applications is provided as examples only. Those of ordinary skill in the art will readily appreciate the various modifications of the examples described herein, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the examples described and shown herein, but should conform to the scope consistent with the claims.

[0040] In some aspects, provided herein are methods for controlling citrus greening disease in citrus plants. In some embodiments, citrus greening disease in these citrus plants is controlled by precisely injecting a liquid formulation comprising oxytetracycline or a salt thereof into the active vasculature of the plant. In some variations, the liquid formulation is injected no further than the active vasculature of the plant. The system used in the methods described herein minimizes the amount of oxytetracycline leaking into the surrounding environment.

[0041] Injectable preparations

[0042] In one aspect, an injectable formulation suitable for controlling citrus greening disease in citrus plants is provided. In certain embodiments, the injectable formulation is water soluble. In some embodiments, the injectable formulation comprises oxytetracycline or a salt thereof. In certain variations, the injectable formulation further comprises nutrients. In one variation, the injectable formulation comprises micronutrients.

[0043] In some embodiments, the injection preparation comprises a reservoir preparation (stock formulation) or a commercial preparation. In some embodiments, the injection preparation comprises a reservoir preparation diluted with water or other solvents or preparations. In some embodiments, the injection preparation comprises a commercial preparation diluted with water or other solvents or preparations. In some embodiments, the reservoir preparation comprises a commercial preparation. In some variations, commercial preparations or reservoir preparations can be diluted and / or further formulated for use in the methods described herein.

[0044] In some embodiments, the concentration of OTC or its salt in the injection preparation is about 1.25mg / mL-about 1.75mg / mL. In some embodiments, the concentration of OTC or its salt in the injection preparation is about 0.1mg / mL-about 10mg / mL, about 0.1mg / mL-about 5mg / mL, about 0.25mg / mL-about 5mg / mL, about 0.25mg / mL-about 2.5mg / mL, about 0.5mg / mL-about 2.5mg / mL, about 0.75mg / mL-about 2.5mg / mL, about 1mg / mL-about 2mg / mL or about 1.25mg / mL-about 1.75mg / mL. In some embodiments, OTC or its salt is present in the injection preparation at about 1.6mg / mL. In some embodiments, the concentration of OTC or a salt thereof in the injection formulation is about 0.25, 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10 mg / mL.

[0045] In some embodiments, the injection preparation comprises OTC or a salt thereof. In some embodiments, the injection preparation comprises the hydrochloride salt (OTC-HCl) of OTC. In some embodiments, the amount of OTC-HCl in the injection preparation is about 0.625 mg / mL-about 1.125 mg / mL. In some embodiments, the concentration of OTC-HCl in the injection formulation is about 0.25 mg / mL, about 0.5 mg / mL, about 0.75 mg / mL, about 1 mg / mL, about 1.1 mg / mL, about 1.2 mg / mL, about 1.3 mg / mL, about 1.4 mg / mL, about 1.5 mg / mL, about 1.6 mg / mL, about 1.7 mg / mL, about 1.8 mg / mL, about 1.9 mg / mL, about 2 mg / mL, about 2.25 mg / mL, about 2.5 mg / mL, about 2.75 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL or about 10 mg / mL.

[0046] In some embodiments, the injection preparation comprises OTC or a salt thereof. In some embodiments, the amount of OTC or a salt thereof in the injection preparation is about 35 mg-about 150 mg. In some embodiments, the amount of OTC or a salt thereof in the injection preparation is about 37.5 mg-about 100 mg. In some embodiments, the amount of OTC or a salt thereof in the injection preparation is about 75 mg-about 150 mg. In some embodiments, the amount of OTC or a salt thereof in the injection preparation is about 37.5 mg-about 125 mg, about 37.5 mg-about 100 mg, about 37.5 mg-about 75 mg, or about 37.5 mg-about 50 mg. In some embodiments, the amount of OTC or a salt thereof in the injection preparation is about 37.5 mg-about 150 mg, about 50 mg-about 150 mg, about 75 mg-about 150 mg, about 100 mg-about 150 mg or about 125 mg-about 50 mg. In some embodiments, the amount of OTC or a salt thereof in the injection formulation is about 37.5 mg, about 50 mg, about 75 mg, about 100 mg, about 125 mg, or about 150 mg.

[0047] In some variations of the foregoing, the injection formulation comprises an aqueous solution of an OTC or a salt thereof. In some variations of the foregoing, the injection formulation comprises a salt of an OTC. In some variations, the salt is a hydrochloride. Any suitable OTC formulation can be used in the methods and systems herein, including commercially available OTC formulations. For example, a suitable example of an OTC formulation that can be used with the injection system herein is ArborBiotech TM, which is a systemic water-soluble injectable antibiotic used to control or inhibit Huanglongbing (HLB, citrus greening disease) caused by Asian citrus Huanglongbing bacteria (Clas) in orange trees (crop subgroup 10-10A). ArborBiotech TM Contains OTC hydrochloride salt accounting for 39.60% of the formulation (equivalent to 36.7% OTC) and other ingredients accounting for the remaining 60.40% of the formulation.

[0048] In some embodiments, the injection preparation comprises an aqueous solution of OTC or its salt. In some embodiments, the volume of the aqueous solution of OTC or its salt of the injection preparation is about 30mL-about 240mL. In some embodiments, the volume of the aqueous solution of OTC or its salt of the injection preparation is about 30mL-about 60mL, about 60mL-about 120mL, or about 120mL-about 240mL.

[0049] In some embodiments, the amount of OTC or its salt in the injection preparation is determined by the diameter of the trunk (e.g., young shoot) at about 10 cm above the graft of the tree receiving the injection preparation. In some embodiments, the diameter of the trunk is about 2 cm-about 6 cm, and the injection preparation contains about 37.5 mg of OTC or its salt. In some embodiments, the diameter of the trunk receiving the injection preparation is greater than about 6 cm and the injection preparation contains about 75 mg of OTC or its salt. In some embodiments, the diameter of the trunk receiving the injection preparation is greater than about 6 cm and the injection preparation contains about 150 mg of OTC or its salt.

[0050] In some embodiments, the maximum amount of OTC or its salt injected into a tree in one growing season is determined by the diameter of the trunk (e.g., young shoot) about 10 cm above the graft of the tree receiving the injection formulation. In some embodiments, the diameter of the trunk is about 2 cm to about 6 cm, and the maximum amount of OTC or its salt injected into the tree in one growing season is about 75 mg. In some embodiments, the trunk diameter is greater than about 6 cm, and the maximum amount of OTC or its salt injected into the tree in one growing season is about 150 mg.

[0051] In some embodiments, the injection preparation comprises an aqueous solution of OTC or its salt, and the volume of the aqueous solution is determined by the diameter of the stem (e.g., bud) at about 10 cm above the graft of the tree receiving the injection preparation. In some embodiments, the trunk diameter is about 2 cm-about 6 cm, and the volume of the aqueous solution of OTC or its salt is about 30 mL-about 60 mL. In certain embodiments, the trunk diameter is about 2 cm-about 6 cm, the volume of the aqueous solution of OTC or its salt is about 30 mL-about 60 mL, and the concentration of OTC or its salt is about 0.625 mg / mL-about 1.125 mg / mL.

[0052] In some embodiments, the diameter of the trunk receiving the injection preparation is greater than about 6 cm, and the volume of the aqueous solution of OTC or its salt is about 60 mL-about 240 mL. In some embodiments, the diameter of the trunk receiving the injection preparation is greater than about 6 cm, and the volume of the aqueous solution of OTC or its salt is about 60 mL-about 120 mL. In certain embodiments, the diameter of the trunk receiving the injection preparation is greater than about 6 cm, the volume of the aqueous solution of OTC or its salt is about 60 mL-about 120 mL, and the concentration of OTC or its salt is about 0.625 mg / mL-about 1.125 mg / mL. In some embodiments, the diameter of the trunk receiving the injection preparation is greater than about 6 cm, and the volume of the aqueous solution of OTC or its salt is about 120 mL-about 240 mL. In certain embodiments, the diameter of the trunk receiving the injection preparation is greater than about 6 cm, the volume of the aqueous solution of OTC or its salt is about 120 mL-about 240 mL, and the concentration of OTC or its salt is about 0.625 mg / mL-about 1.125 mg / mL.

[0053] Solution

[0054] In some embodiments, the method comprises injecting a citrus plant with an injection formulation described herein.

[0055] In some embodiments, the method comprises measuring the diameter of a stem. In some embodiments, the method comprises measuring the perimeter of a stem, the diameter of a stem, or both. In some embodiments, the perimeter of a stem or the diameter of a stem is measured at about 10 cm above the graft.

[0056] In some embodiments, the injection of the injection formulation of any method described herein is performed by injecting the injection formulation into the rootstock of a crop. In some embodiments, the crop is a citrus plant. In some embodiments, the citrus plant is an orange tree.

[0057] In some embodiments, the injection of any of the methods described herein is performed by injecting the injection into the stem (e.g., young shoots) of a crop. In some embodiments, the crop is a citrus plant. In some embodiments, the citrus plant is an orange tree.

[0058] In some embodiments, the injection of the injection preparation or any method described herein is performed 4 times per year. In some embodiments, the injection of the injection preparation or any method described herein is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 times per year. In some embodiments, the injection of the injection preparation or any method described herein is performed more than 1 time, more than 2 times, more than 3 times, more than 4 times, more than 5 times, more than 6 times, more than 7 times, more than 8 times, more than 9 times or more than 10 times per year. In some embodiments, the injection of the injection preparation or any method described herein is performed less than 2 times, less than 3 times, less than 4 times, less than 5 times, less than 6 times, less than 7 times, less than 8 times, less than 9 times or less than 10 times per year. In some embodiments, the injection of the injection preparation or any method described herein is performed twice per year.

[0059] In some embodiments, the timing of injecting the injection formulation or any method described herein is determined by the growing season of the crop. For example, in some embodiments, the timing of injecting the injection formulation or any method described herein is determined by the harvest date or pre-harvest interval (PHI) of the crop. In some embodiments, the crop is a citrus crop. In certain embodiments, the citrus crop is an orange.

[0060] In some embodiments, the injection of the injection preparation or any method described herein is performed once a year. In some embodiments, the injection of the injection preparation or any method described herein is performed once a year, between about 0 days and 60 days after the final harvest of the crop. In some embodiments, the injection of the injection preparation or any method described herein is performed once a year, at least about 120 days before the crop is harvested (e.g., PHI). In some embodiments, the crop is a citrus crop. In certain embodiments, the citrus crop is an orange.

[0061] In some embodiments, the injection preparation or any method described herein is injected twice a year. In some embodiments, the injection preparation or any method described herein is injected twice a year, and the first injection is performed about 120 days before the crop is harvested (e.g., PHI). In some embodiments, the injection preparation or any method described herein is injected twice a year, and the first injection time is performed about 0 days-60 days after the final harvest of the crop. In some embodiments, the injection preparation or any method described herein is injected twice a year, and the second injection time is performed between about 60 days after the first injection time and about 120 days before the crop is harvested (e.g., PHI). In some embodiments, the injection preparation or any method described herein is injected twice a year, and the second injection time is performed at least about 90 degrees around the circumference of the trunk from the position of the first injection time. In some embodiments, the crop is a citrus crop. In certain embodiments, the citrus crop is an orange.

[0062] In some embodiments, the injection of the injection preparation or any method described herein is performed around March, around May, around June, around September, and / or around October. In some embodiments, the injection of the injection preparation or any method described herein is performed around January, around February, around March, around April, around May, around June, around July, around August, around September, around October, around November, around December, or any combination thereof.

[0063] In some embodiments, the injection of the injection formulation or any method described herein is performed on a citrus tree, such as an orange tree. In some embodiments, the orange tree is a late season orange variety (e.g., Valencia, OLL8). In some embodiments, the orange tree is a late season orange variety (e.g., Valencia, OLL8), and the injection of the injection formulation or any method described herein is performed around May, around October, or any combination thereof. In some embodiments, the orange tree is a short season orange variety (e.g., Hamlin). In some embodiments, the orange tree is a short season orange variety (e.g., Hamlin), and the injection of the injection formulation or any method described herein is performed around March, around April, around June, around August, or any combination thereof.

[0064] In some embodiments, injecting the injectable formulation or any of the methods described herein is performed after the final harvest of the crop from the previous growing season.

[0065] In some embodiments, injection close to harvest is avoided. In some embodiments, the injection of the injection preparation or any method described herein is performed at least about 16 weeks before harvest. In some embodiments, the injection of the injection preparation or any method described herein is performed at least about 12 weeks before harvest. In some embodiments, the injection of the injection preparation or any method described herein is performed about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks or about 20 weeks before harvest; or about 4 weeks to about 20 weeks before harvest.

[0066] In some embodiments, injecting the injectable formulation or any of the methods described herein is performed at the end of the previous growing season of the crop. In some embodiments, the crop is citrus. In some embodiments, the crop is orange trees. In some embodiments, the end of the previous growing season is after the final harvest of the previous growing season.

[0067] In some embodiments, the injection formulation or any method described herein is injected twice during the growing season. In some embodiments, the injection formulation or any method described herein is injected once after the final harvest of the previous growing season (e.g., injection formulation) and once at least about 120 days before the crop is harvested (e.g., second injection formulation). In some embodiments, the injection formulation and the second formulation are the same. In some embodiments, the injection formulation and the second formulation are different. In some embodiments, the crop is citrus. In some embodiments, the crop is an orange tree.

[0068] In some embodiments, the injection of the injection formulation or any method described herein is performed once after the end of the previous growing season (e.g., injection formulation) and once about 1 day to about 60 days before harvest (e.g., second injection formulation). In some embodiments, the injection of the injection formulation or any method described herein is performed (i) as soon as possible after the end of the previous growing season and (ii) about 1 day to about 60 days after (i) and at least about 120 days before harvest (e.g., PHI).

[0069] In some embodiments, the volume of the injection formulation injected into the citrus plant as described herein is about 30 mL to about 240 mL, about 30 mL to about 120 mL, about 50 mL to about 100 mL, about 30 mL to about 60 mL, about 60 mL to about 240 mL, about 60 mL to about 120 mL, or about 120 mL to about 240 mL; or about 30 mL, about 60 mL, about 120 mL or about 240 mL. In some embodiments, the total volume of the injection formulation injected into the tree during one growing season is about 30 mL to about 240 mL.

[0070] Citrus Plants & Diseases

[0071] In some embodiments, the citrus plant is a citrus tree or a citrus shrub. In some variations, the citrus tree is an orange tree, a lemon tree, a lime tree, a grapefruit tree, or a pomelo tree. In certain variations, the citrus plant is a lemon shrub or a lime shrub. In one variation, the citrus shrub is a dwarf citrus shrub. In other variations, the citrus tree is a mature tree.

[0072] In some variations, the citrus plants are afflicted with citrus greening disease caused by Liberibacter (e.g., L. asiaticus, L. africanus, L. americanus). In some variations, the disease is spread by Diaphorina citri and Trioza erytrea.

[0073] In some embodiments, the infected citrus plants exhibit at least one symptom caused by citrus greening disease. In some embodiments, the citrus plants to which the injection formulation is applied are infected. In some embodiments, the citrus plants to which the injection formulation is applied are not infected. In some embodiments, the methods described herein are only used for citrus plants with one or more symptoms caused by citrus greening disease. Such symptoms may include any one or more of the following: asymmetrical yellowing of veins and adjacent tissues; mottled patches on the entire leaf; premature defoliation; wilting of young branches; rot of absorbing roots and lateral roots; decreased vitality; poor growth, multiple off-season flowers; small, irregularly shaped fruits with thick and pale skin, green bottoms and bitter taste.

[0074] In some variations, to evaluate the efficacy of an injectable formulation for citrus plants, one or more of the following is evaluated: fruit Brix analysis, fruit yield, fruit drop, OTC residue levels in fruit, OTC concentration in citrus leaves, effect of treatment on Clas titer in leaves, and overall plant health.

[0075] In some embodiments, the present disclosure provides methods for enhancing or maintaining plant health in citrus plants and groves. In some such embodiments, the present disclosure provides methods for treating diseased plants and / or methods for controlling bacteria, fungi, viruses and / or other pathogens that cause citrus greening disease in citrus plants. In further such embodiments, the present disclosure provides methods for treating citrus plants whose xylem and / or phloem have been invaded by pathogenic bacteria, fungi, viruses and / or other pathogens, methods for controlling the pathogenic bacteria, fungi, viruses and / or other pathogens, and methods for preventing diseases by preventing sufficient colonization of plants by the pathogenic pathogens (such as bacteria, fungi and viruses).

[0076] In some embodiments, the systems, devices and methods herein are used to control citrus greening disease in citrus plants, including reducing the bacterial concentration (titer) in the vascular system. In some variations, the systems, devices and methods herein are used to control citrus greening disease in citrus plants, including reducing the bacterial concentration (titer) in the vascular system by enhancing the natural defense system of the plant. In certain embodiments, the systems, devices and methods herein can provide treatments that can suppress the disease to a level where citrus production is restored. In some variations, bacterial titer refers to the bacterial concentration in the vascular system of the infected plant. Bacterial titer can be measured using any suitable method and technique known in the art. For example, in one variation, bacterial titer is measured by quantitative PCR. In one variation, for example, the Clas titer is measured using any suitable technique known in the art.

[0077] In some variations, the treatment regimens provided herein can (i) reduce fruit drop; (ii) increase the Brix of the fruit; and / or (iii) increase fruit yield. In some embodiments, the treatment regimens provided herein can (i) reduce fruit drop by at least about 10%, (ii) increase the Brix by at least about 5%, and / or (iii) increase fruit yield by at least about 10%. In certain variations, the treatment regimens provided herein can (i) reduce fruit drop by at least about 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 30%, 40%, or 50%, or about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 25%, about 10% to about 25%, about 15% to about 25%, or about 17.5% to about 22.5%; (ii) increase Brix by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, or (iii) increase fruit yield by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, or 90%, or about 5% to about 90%, about 10% to about 80%, about 20% to about 70%, about 30% to about 70%, about 40% to about 70%, about 50% to about 70%, or about 55% to about 65%. In summary, in one variation, the treatment regimens provided herein can improve plant health recovery and produce healthier, tougher groves.

[0078] In some variations, the average OTC residues of fruits collected from plants using the injection formulation are less than 0.01ppm. In some variations, the average OTC residues of fruits collected from plants using the injection formulation are less than 0.0001ppm, less than about 0.001ppm, less than about 0.01ppm, less than about 0.015ppm, less than about 0.02ppm, less than about 0.025ppm, less than about 0.03ppm, less than about 0.04ppm, or less than about 0.05ppm, or there is no detectable level of OTC. In other variations, the average OTC residues are about 0.001ppm-about 0.01ppm.

[0079] In some variations, the average OTC residue of about 1 day to about 90 days after the last injection of the injection formulation is less than 0.01ppm for the fruits collected from the plants using the injection formulation. In some variations, the average OTC residue of about 1 day to about 250 days after the last injection of the injection formulation is less than 0.01ppm for the fruits collected from the plants using the injection formulation. In some variations, the average OTC residue of about 1 day to about 225 days after the last injection of the injection formulation is less than 0.01ppm for the fruits collected from the plants using the injection formulation. In some variations, the average OTC residue of about 1 day to about 125 days after the last injection of the injection formulation is less than 0.01ppm for the fruits collected from the plants using the injection formulation. In some variations, the average OTC residue of about 1 day to about 115 days after the last injection of the injection formulation is less than 0.01ppm for the fruits collected from the plants using the injection formulation. In some variations, the average OTC residue of the fruit collected from the plant to which the injection formulation was applied is less than 0.01 ppm from about 1 day to about 90 days after the last injection of the injection formulation. In some variations, the average OTC residue of the fruit collected from the plant to which the injection formulation was applied is less than 0.01 ppm from about 1 day to about 60 days after the last injection of the injection formulation.

[0080] In some variations, the average OTC residue of the fruit collected from the plant to which the injection formulation is applied is less than 0.1 ppm. In some variations, the average OTC residue of the fruit collected from the plant to which the injection formulation is applied is less than 0.1 ppm for about 1 day to about 35 days after the last injection of the injection formulation. In some variations, the average OTC residue of the fruit collected from the plant to which the injection formulation is applied is less than 0.1 ppm for about 1 day to about 10 days after the last injection of the injection formulation. In some variations, the average OTC residue of the fruit collected from the plant to which the injection formulation is applied is less than 0.1 ppm for about 1 day to about 7 days after the last injection of the injection formulation.

[0081] In some variations, the average OTC residue of about 0 days to about 3 days after injection of the injection formulation is about 0.05 ppm for fruits on plants to which an injection formulation is applied, which is reduced to less than about 0.01 ppm about 0 days to about 3 days after injection of the injection formulation. In some variations, the average OTC residue of about 0 days to about 3 days after injection of the injection formulation is about 0.05 ppm for fruits on plants to which an injection formulation is applied, which is reduced to less than about 0.01 ppm about 90 days after injection of the injection formulation. In some variations, the average OTC residue of about 0 days to about 3 days after injection of the injection formulation is about 0.5 ppm for fruits on plants to which an injection formulation is applied, which is reduced to less than about 0.01 ppm about 90 days to about 125 days after injection of the injection formulation.

[0082] In some variations, the leaves of plants to which an injection formulation comprising OTC or its salt is applied have an average OTC residue of less than 0.1 ppm at about 0 days to about 60 days after the injection formulation is injected. In some variations, the average OTC residue of leaves of plants to which the injection formulation is applied reaches a peak at about 1 day to about 3 days after the injection formulation is injected, and then drops to less than about 0.1 ppm at about 60 days after the injection formulation is injected. In some variations, the OTC residue of leaves on plants to which the injection formulation is applied is retained longer than plants treated with sprays of OTC or its salts, and the average OTC residue of the plants about 28 days after the spray treatment is less than about 0.1 ppm. In some variations, the average OTC residue of leaves on plants to which the injection formulation is applied is about twice as high as the average OTC residue of leaves on plants treated with sprays of OTC or its salts about 1 day to about 3 days after the injection formulation is injected.

[0083] In some variations, the average OTC residue in the roots of plants to which an injection formulation comprising OTC or a salt thereof is applied is less than about 0.1 ppm. In some variations, the average OTC residue in the roots of plants to which an injection formulation comprising OTC or a salt thereof is applied is less than about 0.1 ppm from about 0 days to about 21 days after injection of the formulation.

[0084] In some embodiments, the average OTC residue refers to the residue of OTC or its salt. In some embodiments, the average OTC residue refers to the residue of OTC or its salt and one or more metabolites of OTC or its salt. For example, the average OTC residue can include 4-epi-oxytetracycline, a metabolite of OTC.

[0085] In some variations, the average fruit drop of the plants administered the injection formulation is less than about 20. In some variations, the average fruit drop of the plants administered the injection formulation is less than about 25, less than about 20, less than about 15, or less than about 10; or about 10-about 25 or about 10-about 20.

[0086] In some variations, the average fruit yield of the plants to which the injection formulation is applied is at least about 55 pounds. In some variations, the average fruit yield of the plants to which the injection formulation is applied is at least about 35 pounds per plant, at least about 40 pounds per plant, at least about 45 pounds per plant, at least about 50 pounds per plant, at least about 55 pounds per plant, at least about 60 pounds per plant, at least about 65 pounds per plant, at least about 70 pounds per plant, at least about 75 pounds per plant, at least about 80 pounds per plant, or at least about 85 pounds per plant, or about 30-about 90 pounds per plant, or about 35-about 85 pounds per plant.

[0087] In some variations, the average Brix of the plants to which the injection formulation is applied is at least about 7.5. In some variations, the average Brix of the plants to which the injection formulation is applied is at least about 7.5, at least about 7.6, at least about 7.7, at least about 7.8, at least about 7.9, at least about 8, or at least about 8.5; or in the range of about 7 to about 9, or in the range of about 7.5 to about 8.5.

[0088] In other embodiments, controlling citrus greening disease in citrus plants using the systems, devices, and methods herein includes at least partially or completely restoring phloem function in infected citrus plants. In certain of the foregoing embodiments, this can restore the productivity of the plant and overall plant health, including the metabolic profile of the plant. In some variations, the metabolic profile of the plant can be used to measure plant health.

[0089] In other embodiments, controlling citrus greening disease in citrus plants using the systems, devices, and methods herein includes at least partial or complete restoration of production capacity. In some variations, the yield over the life cycle of the plant is increased compared to untreated control plants.

[0090] In certain embodiments, the method includes delivering a formulation comprising oxytetracycline and optionally one or more nutrients to a citrus plant. In certain embodiments, the method includes accurately delivering the formulation (also referred to as "precision injection") to a citrus plant. Accurate delivery refers to delivering the formulation only or substantially only to a target location in a citrus plant. For example, in some embodiments, the target location is the active vascular system of the plant. In certain embodiments, the method includes injecting the injection formulation into the active vascular system of the plant and not exceeding the active vascular system. In some variations, the active vascular system of the plant is xylem and / or phloem. In one variation, the active vascular system is active xylem (such as sap) and phloem. In further embodiments, accurate delivery includes delivering the formulation to the active vascular system of a citrus plant while minimizing damage to the plant relative to conventional forms of injection drilling systems. In other embodiments, accurate delivery involves using a system that can be configured to deliver the formulation to the active vascular system of a plant and not exceed the active vascular system of the plant.

[0091] In certain embodiments, the method comprises injecting an injectable formulation comprising oxytetracycline into a citrus plant, such as into the active vasculature of a plant, using a precision delivery device and system (e.g., the devices and systems mentioned herein). In some such embodiments, the method comprises precisely injecting an injectable formulation comprising oxytetracycline into a plant. In certain embodiments, the method comprises injecting an injectable formulation comprising oxytetracycline (e.g., a precision injection injectable formulation) into a plant, such as into the active vasculature of a plant susceptible to disease caused by citrus greening disease.

[0092] In some embodiments, the present disclosure also provides systems and devices for delivering injection preparations to the interior of plants. In some embodiments, the system includes an injection tool operably connected to a fluid delivery unit, wherein the injection tool is configured to accurately deliver the injection preparation to a target location in the plant. In some embodiments, the system is configured to accurately deliver the injection preparation to the active vascular system of a citrus plant. In some embodiments, the fluid delivery unit also includes a preparation. In other embodiments, the system includes an injection tool, a fluid delivery unit, and a preparation source in fluid communication with the fluid delivery unit.

[0093] Injection system

[0094] In some embodiments, the injection of the injection formulation is performed using an injection system, the injection system comprising an injection tool operably connected to a fluid delivery unit, wherein the fluid delivery unit is configured to deliver the injection formulation. In some embodiments, the injection of the injection formulation comprises piercing a trunk or stem of a citrus plant using the injection tool of the injection system. In some embodiments, the injection of the injection formulation comprises delivering at least a portion of the injection formulation from the fluid delivery unit through the injection tool to the active vasculature of the plant and not exceeding the active vasculature of the plant.

[0095] In some embodiments, the injection formulation is delivered to the citrus plant using an injection system. In some variations, the injection system includes: an injection tool operably connected to a fluid delivery unit. In certain variations, the injection tool includes: a base having at least one inlet; and a body including at least one dispensing reservoir, and at least one outlet. In some embodiments, the injection system includes: an injection tool, a fluid delivery unit, and a source of active ingredients (including, for example, nutrients) formulated as a liquid.

[0096] In some embodiments, the injection system described herein includes an injection tool connected to a prefilled and pressurized application container that contains an injection formulation containing OTC or its salt mixed in an aqueous solution for application. In certain variations, the injection system is configured to precisely deliver the aqueous injection formulation to the vascular system of the tree. In certain variations, when a liquid injection formulation containing OTC or its salt is used with the injection system described herein, the negative effects of Asian citrus huanglongbing fungus on tree health and fruit yield and quality are surprisingly reduced.

[0097] In some embodiments, the injectable formulation is distributed into the vasculature of the tree for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, or at least 7 days.

[0098] In some variations, the body is designed to pierce a plant, such as a stem or stalk of a plant. In some variations, the body is in the shape of a blade. In some variations, the body has a cutting edge at its tip, and the width of the cutting edge is narrower than the width of the area where the body is connected to the base.

[0099] In some variations, the body includes: at least one outlet for receiving the injection formulation from at least one inlet and at least one distribution reservoir for maintaining the injection formulation near the adjacent tissue of the plant. In some variations, the fluid delivery unit is configured to store and deliver the injection formulation. In some variations, the fluid delivery unit includes a pressurized container (e.g., a pressurized tank).

[0100] In some embodiments, the method includes: piercing the trunk or stem of a citrus plant using an injection tool of the injection system; and delivering at least a portion of the injection formulation from the fluid delivery unit to the vasculature of the citrus plant through the injection tool. In some variations, the injection formulation is delivered pneumatically or hydraulically.

[0101] In some embodiments, the injection formulation is precisely delivered. In some variations, when the injection tool is inserted into the trunk or stem of the plant, the injection formulation is delivered to the active vasculature of the plant and does not exceed the active vasculature. In one variation, when the injection tool is inserted into the trunk or stem of the plant, the injection formulation is delivered to the xylem or phloem or both of the plant and does not exceed the xylem or phloem or both.

[0102] In other embodiments, precisely delivering the injection formulation comprises inserting the injection tool into the living vasculature of the plant without exceeding the living vasculature. In certain variations, precisely delivering the injection formulation comprises inserting the body of the injection tool into the living vasculature of the plant without exceeding the living vasculature. In one variation, precisely delivering the injection formulation comprises inserting the injection tool such that the dispensing reservoir is positioned within the living vasculature of the plant without exceeding the living vasculature.

[0103] In some variations, the method delivers at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the injectable formulation into the viable vasculature of the plant. In one variation, the method delivers at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the injectable formulation into the xylem and / or phloem of the plant. In some variations of the foregoing, the method delivers the injectable formulation into the viable vasculature of the plant in an average maximum time of less than about 10 minutes or less than about 5 minutes.

[0104] In certain embodiments, the method comprises injecting the injection formulation into the vasculature through one or more sites on the stem or stem of the plant. In embodiments where the formulation is injected through multiple injection sites, multiple injection systems as described herein may be used. In some embodiments where the formulation is injected through multiple injection sites, the system comprises multiple injection tools operably connected to a single fluid delivery unit.

[0105] In some variations, the method further comprises removing at least a portion of the bark surrounding the injection site, such as prior to piercing the trunk.

[0106] The methods described herein generally provide one or more commercial advantages over methods currently known in the art for controlling citrus greening disease. For example, the advantages include one or more of the following: faster recovery to pre-infection yield, rapid response (e.g., cure), less formulation required, less formulation loss to the environment, less damage to plants, response in older plants, response to plants with significant disease symptoms.

[0107] In some embodiments, the injection system includes an injection tool, a fluid delivery unit, and an injection formulation source. In operation, the injection tool is operably connected to the fluid delivery unit so that the injection formulation flows from the source through the injection tool into the plant. In some embodiments, the injection formulation source is independent of the fluid delivery unit. In other embodiments, the injection formulation source is integrated with the fluid delivery unit. Certain embodiments of the injection system suitable for the methods described herein are described in further detail below.

[0108] Injection tools

[0109] In some embodiments, the injection tool includes a main body, at least a portion of which is designed to be accommodated in the trunk or stem of a plant. The main body has a channel system (e.g., one or more channels) through which the injection formulation can flow. In some variations, the liquid formulation enters the injection tool through one or more inlets and leaves the injection tool through one or more outlets, through which the injection formulation is delivered to the interior of the plant. In some embodiments, the size and shape of the housing portion of the main body are designed to reduce or minimize damage to the target plant when inserted into the plant, while maintaining the effective function of the injection tool, delivering the required dose of the injection formulation directly to the sapwood of the plant trunk rather than the core material within the required time period. In other embodiments, the size and shape of the housing portion of the main body are designed to reduce damage to the target plant when inserted into the plant, compared to conventional drilling injection systems. In some embodiments, the injection tool is configured to penetrate the young shoot or rootstock of the tree to a depth of about 4mm, 4.5mm, 5mm, 5.5mm, 6mm, 6.5mm, 7mm, 7.5mm, 8mm, 8.5mm, 9mm, 9.5mm, 10mm, 10.5mm, 11mm, 11.5mm, 12mm, 12.5mm, 13mm, 13.5mm, 14mm, 14.5mm or 15mm. In some embodiments, the penetration depth of the injection tool is selected based on the diameter of the young shoot. In some embodiments, an injection tool configured to penetrate the young shoot or rootstock of the tree to a depth of about 4mm, 4.5mm, 5mm, 5.5mm, 6mm, 6.5mm, 7mm or 7.5mm is selected to inject a tree with a young shoot diameter of less than 6cm. In some embodiments, an injection tool configured to penetrate the sprouts or rootstock of a tree to a depth of about 6.5 mm, 7 mm, 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, 11 mm, 11.5 mm, 12 mm, 12.5 mm, 13 mm, 13.5 mm, 14 mm, 14.5 mm or 15 mm is selected to inject trees having sprouts greater than 6 cm in diameter.

[0110] In some variations, the exemplary injection tool is Figures 1A-1C and 2A-2D. Figures 1A-1C , depicts an exemplary injection tool 6001 having a base 6010 and a body 6020. The body 6020 includes a cutting element. For example, in one variation, the body 6020 includes a cutting edge pointing distally away from the base 6010 along the front surface 6021. In some variations, the outlet 6027 and the dispensing reservoir 6028 are within the body 6020. Figure 3 As shown in the side view of FIG. 6C , the thickness of the body 6020 increases from the distal portion 6041 to the proximal portion 6043 and the base 6010 .

[0111] In some embodiments, the base 6010 optionally includes a ribbed external structure 6012, such as a connecting splint, to grip the base 6010 and securely connect the injection tool 6001 to the fluid delivery unit. In some variations, a step is provided at the transition between the base 6010 and the body 6020. The step forms an abutment surface 6013. The abutment surface 6013 extends relative to the body 6020 (e.g., away from the body 6020). During the insertion of the injection tool 6001, the abutment surface 6013 contacts the tree and prevents the injection tool 6001 from being further advanced into the tree. In some variations, a larger abutment surface 6013 is convenient for use with smaller and less sturdy trees with relatively soft shells or borders. The relatively larger abutment surface disperses the force generated by the insertion to the corresponding larger surface 6013, thereby minimizing damage to the tree. In some variations, the abutment surface 6013 also provides a closed surface for the injection tool 6001 to establish a stable connection with the tree.

[0112] like Figure 1B and 1C As shown, the base 6010 includes an inlet 6011 that receives the injection preparation of the fluid delivery unit. The injection preparation passes through the main channel 6025 and is released into the distribution reservoir 6028 through the outlet 6027. Figure 1A and 1B As shown, the outlet 6027 opens laterally into a corresponding dispensing reservoir 6028.

[0113] The injection formulation is delivered from the outlet 6027, for example transversely relative to the longitudinal body axis 6040 and the corresponding insertion direction 6030, into the distribution reservoir 6028. The distribution reservoir 6028 maintains the injection formulation in a position adjacent to the plant tissue. Figure 3 In the example shown in B, the outlet 6027 extends proximally to the base 6010 and laterally to the insertion direction 6030 of the injection tool 6001.

[0114] Due to the relatively low profile of the injection tool 6001, the injection tool 6001 is easily inserted and installed in relatively small or weak trees with softer plant material (e.g., tissue, etc.). For example, the injection tool 6001 is configured to tap or manually press the injection tool 6001 into the tree.

[0115] like Figure 1B As shown, the injection tool 6001 is inserted along an insertion direction 6030 corresponding to a longitudinal body axis 6040 of the injection tool 6001. When the injection tool 6001 is inserted into the tree, the body 6020 of the injection tool 6001 spreads the tree material. The spreading of the tree material can minimize damage to the tree material and promote enhanced absorption of the formulation in some examples.

[0116] like Figure 1B As further shown, the outlet 6027 extends along an outlet direction 6032 toward the dispensing reservoir 6028. The outlet direction 6032 is transverse to the insertion direction 6030 (and the longitudinal body axis 6040). For example, in some variations, the outlet direction 6032 is offset from the insertion direction 6030 (and the longitudinal body axis 6040) by an angle of 125 degrees or the like. The transverse direction of the outlet 6027 isolates the outlet 6027 from tree material that would otherwise be introduced into the outlet passageway by insertion. In addition, the dispensing reservoir 6028 facilitates positioning of the outlet 6027 within the body contour, such as by recessing the outlet 6027 from the exterior of the body contour.

[0117] See also Figures 2A-2D , depicts another exemplary injection tool 6101 having a base 6110 and a body 6120. The base 6110 and the body 6120 have similar features to the base 6010 and the body 6120 described above. For example, the body 6120 may include a cutting edge along the front surface of the body. The body further includes an outlet that distributes the injection formulation to a distribution reservoir 6128 within the body 6120.

[0118] However, with Figures 1A-1C Unlike the illustrated injection tool 6001, the injection tool 6101 has a base 6110 including two inlets 6111 that receives the injection formulation from the fluid delivery unit. The injection formulation passes through the main channel and is released into the dispensing reservoir 6128 through the outlet.

[0119] In some variations, the injection tool may have multiple inlets operably connected to the fluid delivery unit. In certain variations, the injection tool has two, three, or four inlets operably connected to the fluid delivery unit.

[0120] Reference again Figure 2A , 2B and 2D, the base 6010 optionally includes a ribbed external structure, such as a connecting clamp, to facilitate gripping the base 6010 and securely connecting the injection tool 6001 to the fluid delivery unit.

[0121] In some embodiments, the size of the injection tool is determined by the diameter of the stem (e.g., bud). For example, in some variations, the size of the injection tool is determined by the diameter of the stem (e.g., bud) about 10 cm above the graft of the tree receiving the injection formulation.

[0122] In some embodiments, the injection systems described herein do not require drilling holes or installing valves in the trunk or stem of a plant prior to injection of the injection formulation, and the injection systems include the exemplary injection tools shown in the accompanying figures.

[0123] Fluid transport unit

[0124] In some embodiments, the fluid delivery unit and the injection formulation source are integrated into the formulation cartridge, such as a pressurized container. In some variations, the formulation cartridge is a pressurized tank. In operation, the injection formulation flows from the fluid delivery unit into the plant by an injection tool. WO 2020 / 021041 and WO 2021 / 152093 (each of which is incorporated herein by reference) provide other embodiments and variations of injection systems and components thereof, including drawings therein.

[0125] In some embodiments, the injection system or its parts used in the methods described herein are as shown in the accompanying drawings. In some embodiments, the system is configured to apply an injection formulation comprising one or more active ingredients (including, for example, nutrients) to a plant or part thereof. In certain embodiments, such a system is mounted on a columnar portion of a plant, such as a stem or a stem of a plant.

[0126] In some embodiments, the method provided herein includes installing an injection tool in a trunk, stem, root or branch of a plant, operably connecting the injection tool to a fluid delivery unit, and activating the fluid delivery unit to start the flow of fluid from the fluid delivery unit through the injection tool and into the plant. In other embodiments, two or more injection tools are installed in one or more of the stem, trunk, root, branch, etc. of a plant to minimize damage to the plant (e.g., by minimizing the size of a single hole in a tree or spacing the tools apart along the plant). In some such embodiments, two or more injection tools are operably connected to the same fluid delivery unit. In other such embodiments, two or more injection tools are operably connected to an independent fluid delivery unit.

[0127] In some variations, the fluid delivery unit comprises a spring-loaded fluid delivery unit. In some of the foregoing variations, the spring-loaded fluid delivery unit is configured to operate at a pressure of 1.5-3 bar. In other variations, the fluid delivery unit comprises a fluid delivery unit comprising a pressurized container (e.g., a pressurized tank). Examples of suitable fluid delivery units include Figure 3 and 4A Variations shown.

[0128] Reference Figure 3 , shows an exemplary spring loaded fluid delivery unit 9900. The base 9912 will house two springs 9908 in the syringe 9910. A piston with a rubber seal separates the injection formulation from the spring chamber. Each syringe body 9910 has a tube 9904 attached, which is connected to a T-shaped connector 9902. The injection tip ( Figure 39902. The spring loaded fluid delivery unit 9900 can be filled through the connector 9902.

[0129] In other exemplary embodiments, the spring-loaded fluid delivery unit may have a base to hold one or more springs in one or more corresponding syringes. The design of the spring-loaded fluid delivery unit may vary based on the pressure, volume, time or other appropriate parameters of the delivered injection preparation. For example, in some variations, multiple springs (such as double springs) may be used in the fluid delivery unit to allow a larger volume of injection preparation to be injected. In other variations, a single spring with a larger syringe may be used, but the pressure range used by the injection preparation may be affected.

[0130] refer to Figure 4A , shows an exemplary chassis-type injection system, which includes a system housing for integrating various components of the injection system, including an injection tool. The injection system 9800 includes a chassis 9802, which has a delivery interface connected to the injection tool 9806. The delivery interface includes, but is not limited to, a delivery tube ( Figure 4A The delivery interface extends to the injection tool and connects the formulation fluid to the dispensing reservoir of the injection tool. The flange 9880 can engage the formulation barrel so as to activate the barrel and hold it in place. In some embodiments, the position of the flange is adjustable to accommodate tanks of different lengths and / or can be activated at a desired time. Figure 4A As further depicted in , in some embodiments, at least a portion of the chassis includes a flexible portion 9882, for example to mitigate damage to the tip during installation. Figure 4A In the exemplary injection system shown, the chassis 9802 also includes an anchor 9890 to further facilitate coupling with the tree. For example, a strap, a band, or the like can be passed through the anchor 9890 to hold the injection system 9800 in place along the tree.

[0131] refer to Figure 4B , describes a pressurized preparation barrel 9810. Exemplary barrel 9810 includes a preparation container 9854, which includes a preparation reservoir having a certain amount of injection preparation therein. A barrel cap 9856 closes the preparation container 9854. A barrel discharge port 9858 extends from the preparation barrel 9810. Optionally, in some variations, the barrel discharge port includes an opening feature that is configured to transition from a closed configuration to an open configuration. The opening feature includes, but is not limited to, a valve, a membrane, etc. that is opened before coupling with the chassis of the injection system.

[0132] In some embodiments, when the injection tool is inserted into the trunk or stem of the citrus plant, the injection tool can remain intact and in place during multiple re-injections. In some embodiments, the method further includes: replacing the fluid delivery unit with a second fluid delivery unit; and delivering at least a portion of the injection formulation from the second fluid delivery unit to the active vasculature of the citrus plant through the injection tool, without exceeding the active vasculature. In some variations, the injection tool remains in the trunk or stem of the citrus plant for at least one growing season, at least two growing seasons, or at least three growing seasons.

[0133] List of implementation plans

[0134] The first embodiment relates to a method for controlling citrus greening disease in a citrus plant, wherein the citrus plant further has an active vascular system that passes through the trunk or stem of the citrus plant to other parts, and the method comprises: injecting an injection preparation containing oxytetracycline (OTC) or a salt thereof into the active vascular system of the plant to control citrus greening disease of the citrus plant.

[0135] A second embodiment is directed to the method of the first embodiment, wherein the viable vasculature into which the injectable formulation is injected is viable vasculature in the trunk or stem of a citrus plant.

[0136] A third embodiment is directed to the method of embodiment 1 or 2, wherein the citrus plant is a citrus tree.

[0137] A fourth embodiment is directed to the method of any of embodiments 1-3, wherein after injection, the citrus plant has a reduction in fruit drop of at least about 5%, at least about 10%, or at least about 15%, or about 10% to about 25% compared to untreated citrus plants.

[0138] A fifth embodiment is directed to the method of any of embodiments 1-4, wherein after injection, the citrus plant produces fruit with an increase in Brix of at least about 1%, at least about 2%, at least about 3%, at least about 4% or at least about 5%, about 5%-about 10% compared to untreated citrus plants.

[0139] A sixth embodiment is directed to the method of any one of embodiments 1-5, wherein after injection, the fruit yield of the citrus plant is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, or about 40%-about 75% compared to untreated citrus plants.

[0140] A seventh embodiment relates to the method of any one of embodiments 1-6, wherein the injection of the injection preparation is performed using an injection system, the injection system comprising an injection tool operably connected to a fluid delivery unit, wherein the fluid delivery unit is configured to deliver the injection preparation.

[0141] An eighth embodiment relates to the method of embodiment 7, wherein injecting the injection formulation comprises: piercing the trunk or stem of the citrus plant using an injection tool of the injection system; and delivering at least a portion of the injection formulation from the fluid delivery unit through the injection tool to the active vascular system of the citrus plant without exceeding the active vascular system.

[0142] A ninth embodiment is directed to the method of any of the preceding embodiments, wherein the injectable formulation is distributed in the trunk or stem and other parts of the citrus plant.

[0143] A tenth embodiment is directed to the method of embodiment 9, wherein the other part of the citrus plant comprises a fruit.

[0144] An eleventh embodiment is directed to the method of embodiment 9 or 10, wherein the other part of the citrus plant comprises leaves.

[0145] A twelfth embodiment is directed to the method of any one of embodiments 7-11, wherein the fluid delivery unit is a spring-loaded fluid delivery unit.

[0146] A thirteenth embodiment is directed to the method of any one of embodiments 7-12, wherein the delivery unit comprises a pressurized formulation cartridge.

[0147] A fourteenth embodiment relates to the method of any one of embodiments 7-13, further comprising: replacing the fluid delivery unit with a second fluid delivery unit; and delivering at least a portion of the injection formulation from the second fluid delivery unit to the active vascular system of the citrus plant through an injection tool, without exceeding the active vascular system.

[0148] A fifteenth embodiment is directed to the method of any one of embodiments 7-14, wherein the injection tool remains in the trunk or stem of the citrus plant for at least one growing season.

[0149] A sixteenth embodiment is directed to the method of any one of embodiments 7-15, wherein the injection tool remains in the trunk or stem of the citrus plant after multiple re-injections.

[0150] A seventeenth embodiment is directed to the method of any one of embodiments 7-16, wherein the trunk of the citrus plant has bark, and the method further comprises: removing at least a portion of the bark prior to piercing the trunk.

[0151] An eighteenth embodiment is directed to the method of any one of the preceding embodiments, wherein the citrus plant is an orange tree.

[0152] A nineteenth embodiment is directed to the method of embodiment 18, wherein the average oxytetracycline (OTC) residue in the fruit of the orange trees to which the injectable formulation is applied is less than about 0.01 ppm.

[0153] A twentieth embodiment is directed to the method of embodiment 18 or 19, wherein said method results in an average fruit drop of less than about 20 in orange trees to which said injectable formulation is applied.

[0154] A twenty-first embodiment is directed to the method of any one of embodiments 18-20, wherein the method results in an average fruit yield of at least about 40 pounds per plant, or about 45 to about 90 pounds per plant, in orange trees to which the injectable formulation is applied.

[0155] A twenty-second embodiment is directed to the method of any one of embodiments 18-21, wherein said method results in an average Brix of at least about 7.5 in orange trees to which said injectable formulation is administered.

[0156] A twenty-third embodiment is directed to the method of any one of embodiments 18-22, wherein the method results in an average reduction of at least about 10% in fruit drop in orange trees to which the injectable formulation is applied, compared to orange trees to which the injectable formulation is not applied.

[0157] A twenty-fourth embodiment is directed to the method of any one of embodiments 18-23, wherein the method results in an average increase in fruit Brix of at least about 5% in orange trees to which the injectable formulation is applied, compared to orange trees to which the injectable formulation is not applied.

[0158] A twenty-fifth embodiment is directed to the method of any one of embodiments 18-24, wherein the method results in an average increase in fruit yield of at least about 10% in orange trees to which the injectable formulation is applied, compared to orange trees to which the injectable formulation is not applied.

[0159] The twenty-sixth embodiment is directed to a method for increasing the fruit harvest yield in a citrus plant, wherein the citrus plant has an active vascular system that runs through the trunk or stem of the citrus plant to other parts, the method comprising: injecting an injection formulation comprising oxytetracycline (OTC) or a salt thereof into the active vascular system of the citrus plant, wherein the injection formulation is injected into the citrus plant one or more times during the growing season, thereby increasing the fruit harvest yield of the citrus plant in one or more growing seasons of injection compared to the fruit harvest yield in untreated citrus plants.

[0160] A twenty-seventh embodiment concerns the method of embodiment 26, wherein after two growing seasons, the average fruit harvest yield is increased by more than 100% compared to the average fruit harvest yield of untreated citrus plants.

[0161] A twenty-eighth embodiment concerns the method of embodiment 26, wherein after one growing season, the average fruit harvest yield is increased by more than 30% compared to the average fruit harvest yield of untreated citrus plants.

[0162] A twenty-ninth embodiment is directed to the method of any one of embodiments 26-28, wherein the injectable formulation is injected once during the growing season.

[0163] A thirtieth embodiment is directed to the method of embodiment 29, wherein the injection formulation comprises 150 mg of OTC or a salt thereof in solution.

[0164] A thirty-first embodiment is directed to the method of any one of embodiments 26-28, wherein the injection formulation is injected twice during the growing season.

[0165] The thirty-second embodiment is directed to the method of any one of embodiment 31, wherein the injection formulation comprises 75 mg of OTC or a salt thereof in solution.

[0166] The thirty-third embodiment is directed to the method of any one of embodiments 26-28, wherein the injection formulation is injected three times during the growing season.

[0167] The thirty-fourth embodiment is directed to the method of embodiment 33, wherein the injection formulation comprises 37.5 mg of OTC or a salt thereof in a solution, administered as two injections; or the injection formulation comprises 75 mg of OTC or a salt thereof in a solution, administered as a single injection.

[0168] A thirty-fifth embodiment is directed to the method of any one of embodiments 26-34, wherein 100 mg-200 mg of OTC or a salt thereof is injected into the citrus plant during the growing season.

[0169] A thirty-sixth embodiment is directed to the method of embodiment 35, wherein about 150 mg of OTC or a salt thereof is injected into the citrus plant during the growing season.

[0170] The thirty-seventh embodiment is directed to the method of any one of embodiments 26-36, wherein the fruit harvest yield is determined based on harvest weight, fruit number and / or fruit drop.

[0171] A thirty-eighth embodiment concerns the method of embodiment 37, wherein after one or more growing seasons, the harvest weight is increased compared to the harvest weight of untreated citrus plants.

[0172] A thirty-ninth embodiment concerns the method of embodiment 38, wherein after one growing season, the harvest weight is increased by more than about 50% compared to the harvest weight of untreated citrus plants.

[0173] A fortieth embodiment concerns the method of embodiment 38 or 39, wherein after two growing seasons, the harvest weight is increased by more than about 100% compared to the harvest weight of untreated citrus plants.

[0174] A forty-first embodiment concerns the method of any one of embodiments 37-40, wherein after one or more growing seasons, the number of fruit is increased compared to the number of fruit of untreated citrus plants.

[0175] A forty-second embodiment concerns the method of any of embodiment 41, wherein after one growing season, the fruit number is increased by more than about 10% compared to the fruit number of untreated citrus plants.

[0176] A forty-third embodiment concerns the method of any of embodiments 41 or 42, wherein after two growing seasons, the fruit number is increased by more than about 100% compared to the fruit number of untreated citrus plants.

[0177] A forty-fourth embodiment is directed to the method of any one of embodiments 37-43, wherein after one or more growing seasons, the fruit drop is reduced compared to untreated citrus plants.

[0178] A forty-fifth embodiment is directed to the method of any one of embodiments 37-44, wherein after one growing season, the fruit harvest yield is increased by more than about 25% compared to the fruit harvest yield of untreated citrus plants.

[0179] A forty-sixth embodiment is directed to the method of any one of embodiments 26-41, wherein the Brix of harvested fruit of the citrus plant is increased compared to the Brix of harvested fruit of an untreated citrus plant.

[0180] A forty-seventh embodiment is directed to the method of any one of embodiments 26-42, wherein the Brix / acid ratio of harvested fruit of the citrus plant is increased compared to the Brix / acid ratio of harvested fruit of an untreated citrus plant.

[0181] The forty-eighth embodiment relates to a method for treating citrus greening disease and leaf mottling in a citrus plant having a shoot diameter ranging from about 1.3 to about 5.1 cm, wherein the citrus plant has an active vascular system that runs through the trunk or stem to other parts of the citrus plant, the method comprising: injecting an injectable formulation comprising oxytetracycline (OTC) or a salt thereof into the active vascular system of the citrus plant, wherein the injectable formulation is injected into the citrus plant once or more during the growing season to reduce citrus greening disease and reduce leaf mottling.

[0182] The forty-ninth embodiment concerns the method of embodiment 48, wherein the citrus plant is injected once with an injection formulation comprising 12.5 mg of oxytetracycline hydrochloride.

[0183] A fiftieth embodiment concerns the method of embodiment 48, wherein the citrus plant is injected once with an injection formulation comprising 37.5 mg of oxytetracycline hydrochloride.

[0184] A fifty-first embodiment concerns the method of embodiment 48, wherein the citrus plant is injected twice with an injection formulation comprising 18.75 mg of oxytetracycline hydrochloride.

[0185] A fifty-second embodiment concerns the method of embodiment 48, wherein the citrus plant is injected three times with an injection formulation comprising 18.75 mg of oxytetracycline hydrochloride.

[0186] The fifty-third embodiment concerns the method of embodiment 48, wherein two injections are administered with an injection formulation comprising 37.5 mg of oxytetracycline hydrochloride.

[0187] A fifty-fourth embodiment concerns the method of any one of embodiments 51-53, wherein the injections are spaced about 30 days apart.

[0188] A fifty-fifth embodiment concerns the method of any one of embodiments 51-53, wherein the injections are spaced about 45 days apart.

[0189] A fifty-sixth embodiment is directed to the method of any one of embodiments 51-53, wherein the injection interval is about 60 days.

[0190] A fifty-seventh embodiment is directed to the method of any one of embodiments 48-56, wherein the citrus plant is injected in the rhizome.

[0191] The fifty-eighth embodiment concerns the method of any one of embodiments 48-56, wherein the citrus plant is injected in the young shoots.

[0192] A fifty-ninth embodiment concerns the method of embodiment 57, wherein the rhizome is injected with an injection tool to a depth of but not more than 6.5 mm.

[0193] A sixtieth embodiment concerns the method of embodiment 58, wherein the sprouts are injected with an injection tool to a depth of but not more than 6.5 mm.

[0194] The sixty-first embodiment relates to a method for treating citrus greening disease and leaf mottling in citrus plants having a shoot diameter greater than 6 cm, wherein the citrus plant has an active vascular system that runs through the trunk or stem of the citrus plant to other parts, the method comprising: injecting an injectable formulation comprising oxytetracycline (OTC) or a salt thereof into the active vascular system of the citrus plant, wherein the injectable formulation is injected into the citrus plant once or more times during the growing season to reduce citrus greening disease.

[0195] The sixty-second embodiment concerns the method of embodiment 61, wherein the citrus plant is injected once with an injection formulation comprising 150 mg of oxytetracycline hydrochloride.

[0196] The sixty-third embodiment concerns the method of embodiment 61, wherein the citrus plant is injected twice with an injection formulation comprising 75 mg of oxytetracycline hydrochloride.

[0197] The sixty-fourth embodiment concerns the method of embodiment 61, wherein the citrus plant is injected three times with an injection formulation comprising 50 mg of oxytetracycline hydrochloride.

[0198] A sixty-fifth embodiment concerns the method of any one of embodiments 63 and 64, wherein the injections are spaced about 30 days apart.

[0199] A sixty-sixth embodiment is directed to the method of any one of embodiments 63 and 64, wherein the injections are spaced about 45 days apart.

[0200] The sixty-seventh embodiment is directed to the method of any one of embodiments 63 and 64, wherein the injections are spaced about 60 days apart.

[0201] A sixty-eighth embodiment is directed to the method of any one of embodiments 61-67, wherein the citrus plant is injected in the rhizome.

[0202] The sixty-ninth embodiment is directed to the method of any one of embodiments 61-67, wherein the citrus plant is injected in the young shoots.

[0203] A seventieth embodiment relates to the method of any one of embodiments 61-69, wherein the citrus plant is injected with 60, 70, 80, 90, 100, 110 or 120 mL of the injection formulation.

[0204] The seventy-first embodiment concerns the method of any one of embodiments 61-69, wherein the citrus plant is injected with 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mL of the injection formulation.

[0205] The seventy-second embodiment concerns the method of embodiment 57, wherein the rhizome is injected with an injection tool to a depth of but not more than 8.5-9 mm.

[0206] The seventy-third embodiment concerns the method of embodiment 58, wherein the sprouts are injected with an injection tool to a depth of but not more than 8.5-9 mm.

[0207] The seventy-fourth embodiment is directed to the method of any one of embodiments 61-73, wherein after injection, the citrus plant has a reduced fruit drop of at least about 5%, at least about 10%, or at least about 15%, or about 10% to about 25% compared to untreated citrus plants.

[0208] The seventy-fifth embodiment is directed to the method of any one of embodiments 61-73, wherein after injection, the Brix of the fruit produced by the citrus plant is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4% or at least about 5%, about 5%-about 10% compared to untreated citrus plants.

[0209] The seventy-sixth embodiment is directed to the method of any one of embodiments 61-73, wherein after injection, the fruit yield of the citrus plant is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, or about 40%-about 75% compared to untreated citrus plants.

[0210] The seventy-seventh embodiment concerns the method of any one of embodiments 61-76, wherein the citrus plant is injected 150-120 days before harvest.

[0211] The seventy-eighth embodiment relates to a method for treating citrus greening disease and leaf mottling in a citrus plant having a shoot diameter of about 2 cm to about 6 cm, wherein the citrus plant has an active vascular system that runs through the trunk or stem of the citrus plant to other parts, the method comprising: injecting an injectable formulation comprising oxytetracycline (OTC) or a salt thereof into the active vascular system of the citrus plant, wherein the injectable formulation is injected into the citrus plant once or more times during the growing season to reduce citrus greening disease.

[0212] The seventy-ninth embodiment concerns the method of embodiment 78, wherein the citrus plant is injected once with an injection formulation comprising 75 mg of oxytetracycline hydrochloride.

[0213] An eightieth embodiment concerns the method of embodiment 78, wherein the citrus plant is injected twice with an injection formulation comprising 37.5 mg of oxytetracycline hydrochloride.

[0214] The eighty first embodiment concerns the method of embodiment 78, wherein the citrus plant is injected three times with an injection formulation comprising 25 mg of oxytetracycline hydrochloride.

[0215] The eighty-second embodiment concerns the method of any one of embodiments 80 and 81, wherein the injections are spaced about 30 days apart.

[0216] The eighty-third embodiment concerns the method of any one of embodiments 80 and 81, wherein the injections are spaced about 45 days apart.

[0217] The eighty-fourth embodiment concerns the method of any one of embodiments 80 and 81, wherein the injections are spaced about 60 days apart.

[0218] The eighty-fifth embodiment is directed to the method of any one of embodiments 78-85, wherein the citrus plant is injected in the rhizome.

[0219] The eighty-sixth embodiment concerns the method of any one of embodiments 78-85, wherein the citrus plant is injected in the young shoots.

[0220] The eighty-seventh embodiment concerns the method of embodiment 85, wherein the rhizome is injected with an injection tool to a depth of but not more than 6.5 mm.

[0221] The eighty-eighth embodiment concerns the method of embodiment 86, wherein the sprouts are injected with an injection tool to a depth of but not more than 6.5 mm.

[0222] The eighty-ninth embodiment concerns the method of any one of embodiments 78-88, wherein the citrus plant is injected with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 120 mL of the injection formulation.

[0223] The ninetieth embodiment is directed to the method of any one of embodiments 78-89, wherein after injection, the citrus plant has leaf mottling reduced by at least about 5%, at least about 10%, or at least about 15%, or about 10% to about 25% compared to untreated citrus plants.

[0224] Example

[0225] The presently disclosed subject matter will be better understood by reference to the following examples, which are provided by way of illustration of the invention, not limitation.

[0226] Example 1: Field trial

[0227] This example demonstrates the effect of oxytetracycline (OTC) on the treatment of citrus greening disease in orange trees.

[0228] Field trial design.Field trials were initiated on March 30th with treatments of commercial orange trees with Hamlin cultivar shoots grafted onto X639 cultivar rootstocks. Treatments consisted of oxytetracycline (OTC) applied to the shoots of each tree using Invaio's proprietary injection system. The trial design was a randomized, fully closed, with six replicates of three treatments per replicate for each of four trees on three different treatment dates. A complete list of treatments is included in Table 1. OTC is a commercially available oxytetracycline product. All injections were made at a rate of 60 ml at the concentrations shown in Table 1. One OTC injection was made to each tree for the first two injections, and two OTC injections were made to each tree for the third application.

[0229] Table 1.

[0230]

[0231] Brix Analysis Fruit was collected from each treated tree on December 8th in duplicate and pooled by treatment and replicate to create separate fruit samples for Brix (BRIX) analysis. On average, trees injected with OTC had higher BRIX compared to untreated trees (Table 2). BRIX for untreated trees ranged from 7.1-7.7 across replicates, while BRIX for OTC-treated trees ranged from 7.6-8.4 across replicates.

[0232] Table 2.

[0233]

[0234]

[0235] Fruit yield . The fruit yield of each treated tree was determined as the total weight (pounds) of fruit picked (harvested) from each tree on December 21. The average of the replicates and the average of all six replicates were recorded as the fruit yield (Table 3). The average fruit yield per tree for each treatment was 48.7 pounds for the OTC treatment and 32.0 pounds for the untreated treatment. The average fruit yield of the untreated trees in the replicates ranged from 23.1 to 45.2 pounds per tree, and the average fruit yield of the OTC-treated trees in the replicates ranged from 37.4 to 81.0 pounds per tree.

[0236] Table 3.

[0237]

[0238] Fruit drop. Fruit drop for each treated tree was determined by counting the number of fruits on the ground beneath each tree on six counting dates (approximately 2 weeks apart) starting on October 13 and ending on December 21 before fruit harvest. The counted fruits were picked up from the ground after each counting day. The average fruit drop for each date and the average fruit drop for all six dates were recorded (Table 4). The average number of fruits that fell from the tree for each treatment was 17.8 for the OTC treatment and 22.0 for the untreated. The average fruit drop for the untreated trees ranged from 20.5-25.3 across replicates, while the average fruit drop for the OTC-treated trees ranged from 14.5-20.9 across replicates.

[0239] Table 4.

[0240]

[0241]

[0242] Residue analysis . Fruit was collected from OTC-treated trees on the December 7 replicate and collected by treatment and replicate to create a single fruit sample for OTC residue analysis. Similarly, fruit was collected from untreated trees in two replicates and collected by replicate to create a single fruit sample for OTC residue analysis. No OTC residues were detected in fruit harvested from untreated trees. OTC residues detected in fruit collected from trees treated with OTC were all below the established minimum residue limit of 0.01 ppm. On average, the residue detected in trees injected with OTC was 0.007 ppm, with a range of 0.002 ppm-0.009 ppm in replicates (Table 5).

[0243] Table 5.

[0244]

[0245] Example 2: Field Trial - Two Season, Single Location Study

[0246] This example demonstrates the use of ArborBiotic in combination with the exemplary injection system described herein. TM (systemic water-soluble injectable antibiotic for the control of certain bacterial diseases, MGF Scientific) is effective in improving fruit yield and juice quality in orange trees.

[0247] Field trial design. A field trial study was conducted over two growing seasons by applying treatments to commercial orange trees with Hamlin cultivar shoots grafted onto X639 cultivar rootstocks. The field trial was designed to evaluate the effects of using a commercially available water-soluble injectable formulation containing oxytetracycline hydrochloride (OTC-HCl) on yield components and juice quality of orange trees, applied using the exemplary injection system described herein, as a potential solution for overcoming the negative effects of HLB on fruit yield and juice quality. The composition of the injectable formulation is listed in Table 6. The injectable formulation used was ArborBiotic TM , and is currently approved for injection into the trunks of fruit-free trees, including citrus, against a range of diseases, excluding HLB (citrus greening disease).

[0248] Table 6. Composition of injection preparations

[0249]

[0250] a Equivalent to 36.7% oxytetracycline

[0251] The field trial was designed as a completely randomized block with six blocks (replications), each containing two treatments (12 plots). Of the 12 plots, six plots were treated with the injection formulation and six plots were treated with water (negative control). The plots were large enough to ensure a sufficient number of representative samples and sufficient amount of material for residue analysis. Each plot was represented by four trees and treatments were applied during two growing seasons. Each tree was clearly identified with a unique identification number. Due to the application method, no larger buffer zone was required between the water-treated plots and the OTC-treated plots. No other formulations containing OTC were applied to the trees during the trial.

[0252] deal with The number and timing of applications varied with the season, but the amount of OTC-HCl (150 mg) delivered to each tree remained the same during both seasons. A complete list of treatments for Seasons 1 and 2 is included in Table 7.

[0253] Table 7. Treatment

[0254]

[0255] During Season 1, treatments included a non-injected water control (untreated) and an OTC formulation injected into the young shoots of Hamlin orange trees. Season 1 treatments are described as Treatments 1-3 in Table 7. Each tree in the treatment plot received two applications of the OTC formulation 73 days apart. Applications included one or two injections. For Treatments 1 and 2, each application included two injections of 94.8 mg of the OTC formulation in 60 mL of distilled water, with a delivered dose of 37.5 mg of OTC-HCl per injection per tree. For Treatment 3, each application included one injection of 189.6 mg of the OTC formulation in 120 ml of distilled water, with a delivered dose of 75.1 mg of OTC-HCl per injection per tree. Each tree treated with the OTC formulation received a total of 379.2 mg of the OTC formulation during Season 1, equivalent to 150.1 mg of OTC-HCl.

[0256] During the second season, treatments included water injections into Hamlin orange trees that were not treated during the first season, and injections of the OTC formulation into Hamlin orange trees that received the OTC formulation treatment during the first season. The second season treatments were treatment numbers 4-7 in Table 7. During the second season, each tree in the treatment plot received two applications during the season, and the treatments were injected into the rhizomes of the trees. For treatment numbers 4 and 6, which included water as a negative control, each application included two injections of 60 ml of distilled water per tree, with a total volume of 120 mL per application, and the application interval was 3-5 months. For treatment numbers 5 and 6, each application included one injection of 189.6 mg of the OTC formulation in 120 ml of distilled water, with each injection delivering a dose of 75.1 mg of OTC-HCl per tree, and the application interval was 94 days. Each tree treated with the OTC formulation received a total of 379.2 mg of the OTC formulation during the second season, equivalent to 150.1 mg of OTC-HCl.

[0257] Application method The method of application of the treatment was by trunk injection using the exemplary injection system described herein. The application vehicle was bottled deionized water. Each container initially contained 60 ml of an aqueous solution of the OTC formulation. The solution was prepared on the day of application or the day before, and each application container was uniquely identified. The volume remaining in the container after 7 days of application indicated the amount of uptake by the tree. The injection system was removed from the tree 7-8 days after application.

[0258] analyze Treatment efficacy was determined based on the evaluation of different yield components, including harvest weight, number of fruits, percentage of fruit drop (total during the two months before harvest), and fruit size (weight per fruit), as well as different juice quality parameters, including Brix content, acid content, and Brix / acid ratio. Analysis of variance (ANOVA) was used to analyze the collected data.

[0259] result In the first and second seasons, the average harvested fruit weight of the OTC formulation-treated plots was about 58% ( Figure 5 ) and about 115% ( Figure 6 ), and in both years, the harvested fruit weight of the OTC treatment averaged about 82% more than the control (data not shown in the figure). The increase in yield was due to an increase in the number of fruits and a decrease in fruit drop. In the second season, the OTC-treated plots averaged about 120% more fruits than the control plots ( Figure 7 No fruit count data were collected in Season 1. In Season 2, the average fruit drop in OTC-treated plots was approximately 28% less than in control plots ( Figure 8 The analysis results are summarized in Table 8.

[0260] Table 8. Summary of results

[0261] <![CDATA[ArborBiotic TM Processing]]> Efficacy variables Treatment effect direction Season 1, Season 2 Harvest fruit weight Increase Season 2 Number of fruits Increase Season 2 Fruit drop percentage reduce Season 1, Season 2 Brix Increase Season 1, Season 2 acid reduce Season 1, Season 2 Brix / Acid Ratio Increase

[0262] in conclusion

[0263] The efficacy of OTC injections against HLB was determined by measuring yield components (harvest fruit weight, fruit number, fruit size, and % fruit drop) and juice quality (Brix content, acid content, Brix / acid ratio). These are the most economically significant and quantifiable consistent and accurate indicators of the effect of OTC formulations on orange trees infected with Clas and showing symptoms of citrus greening.

[0264] Data and statistical analysis of the field studies showed that the average harvested fruit weight (harvested fruit weight) of trees treated with 150 mg OTC-HCl had significantly higher yields than untreated controls in Season 1, and than water controls in Season 2. In fact, the average harvested weight of OTC-treated plots was 57.4% and 115.3% greater than the control in Season 1 and Season 2, respectively. The increase in yield improvement suggests that continued injection of the OTC formulation over time can have a cumulative effect on harvested fruit weight.

[0265] During Season 2, additional data on yield were collected, including fruit number (number of fruits on the tree at harvest), fruit size (weight of individual fruit at harvest), and total fruit drop percentage during the 2-month period prior to harvest until harvest. These results help understand which yield factors contribute to the increased harvest weight. Trees injected with the OTC formulation of 150 mg OTC-HCl had a significant increase in fruit number (120% increase in Season 2) and a significant decrease in fruit drop percentage (39% decrease in Season 2) relative to the water treatment (negative control). The OTC formulation did not affect fruit size because the ArborBiotic TM Both the treated fruit and the water treated fruit averaged 0.28 pounds per fruit.

[0266] The juice quality of the fruit harvested from the trees was evaluated, including the Brix content and acid content. The data from these evaluations showed that the Brix content of the fruit harvested from the trees treated with the OTC formulation in seasons 1 and 2 was 8.1% and 7.2% higher than that of the control trees, respectively, the acid content was 6.9% and 16.7% lower than that of the control trees, respectively, and the Brix / acid ratio was 14% and 32% higher than that of the control trees, respectively. Therefore, OTC injection not only increased the number of fruits harvested, but also improved the juice quality of the fruit.

[0267] Example 3: Field Trial - Environment, Concentration and Application Rate Study

[0268] This example demonstrates that an OTC formulation (ArborBiotic TM ) resulted in statistically significant improvements in orange fruit yield over a range of environments, concentrations, and application rates.

[0269] Field trial design . A field trial study was conducted by applying various treatments to commercial orange trees in different citrus growing regions during one growing season. The field trial was designed to develop the most efficient use pattern (e.g., concentration and injection rate) for applying the OTC formulation using an exemplary injection system as described herein to treat different varieties of orange trees located in different environments. The study included 8 locations (01-08) and contained both short-season (Hamlin) and long-season (Valencia, OLL8) varieties of orange trees, and the trial locations were representative of commercial citrus growing regions.

[0270] Treatment included administration of ArborBiotic TM (commercial oxytetracycline hydrochloride or products containing "OTC-HCl"), water as a negative control, and FireLine TM 17WP (a commercial product containing OTC-HCl, manufactured by AgroSource) was used as a positive control and commercial reference. ArborBiotic was injected using the exemplary injection system described herein. TM and water treatment agents, and foliar application (i.e. spray) of FireLine TM 17WP. ArborBiotic is reported in Table 9 TM and FireLine TM Composition of 17WP.

[0271] Table 9. ArborBiotic TM and FireLine TM 17WP Composition

[0272]

[0273] a Equivalent to 36.7% oxytetracycline

[0274] b Equivalent to 17.0% oxytetracycline

[0275] The field experiment was designed as a randomized complete block with 6 blocks (replications) of 6 treatments (36 plots) and replicates at each location. Each plot was represented by two trees. Of the 36 plots, 6 plots were treated with treatment 1 (T1), 6 plots were treated with treatment 2 (T2), 6 plots were treated with treatment 3 (T3), 6 plots were treated with treatment 4 (T4), and 6 plots were treated with FireLine TM 17WP spray treatment served as a positive control and commercial reference, and 6 plots were treated with water as negative controls. Each tree in the treated plots received one or two injections per application (depending on the treatment), with applications being made once or twice per year.

[0276] All treatments used are summarized in Table 10. Treatments were injected into the trunks of orange trees. Treatment applications were made once or twice during the growing season. For Treatment 1 (T1), each application consisted of one injection of 94.8 mg ArborBiotic™ in 60 mL distilled water, delivering a dose of 37.5 mg of OTC-HCl per injection per tree. For Treatment 2 (T2), each application consisted of two injections of 94.8 mg ArborBiotic™ in 60 mL distilled water (a total of 189.6 mg ArborBiotic™ and 120 mL distilled water), delivering a total dose of 75.1 mg of OTC-HCl per application per tree. For Treatment 3 (T3), each application consisted of one injection of 189.0 mg ArborBiotic™ in 60 mL distilled water, delivering a total dose of 74.8 mg of OTC-HCl per application per tree. Treatment 4 (T4) was administered as a single administration consisting of two injections of 189.0 mg of ArborBiotic™ in 60 mL of distilled water (a total of 378.0 mg ArborBiotic TM and 120 mL of distilled water), which delivered a total dose of 149.7 mg of OTC-HCl.

[0277] Table 10. Treatment

[0278]

[0279]

[0280] *FireLine values ​​estimated based on label application rates and estimated number of trees per acre

[0281] The timing of applications varied from site to site due to differences in varietal maturity at different sites; for trees receiving more than one application, the time between the first and second applications was 3-5 months. For example, the first application was made as soon as possible after the final harvest of the previous season, and the second application was made no less than 60 days after the first application and no later than 120 days before harvest (at one site, treatment occurred 119 days before premature harvest). Late season orange varieties were injected in May and October, and short season orange varieties were injected in March or April and June or August. FireLine TM Foliar applications of 17WP were consistent with injection applications of the other treatments and were applied using a backpack sprayer at the label rate of 24 oz / acre.

[0282] The plots were large enough to ensure adequate numbers of representative samples and sufficient material for residue analysis. The water-treated plot (negative control) had 2 trees, and the ArborBiotic TM (T1, T2, T3 and T4) and FireLine TM Treated plots had 2 trees (12 trees total per treatment). Each tree was clearly identified with a unique number (such as the experiment number, plot number, and tree number). TM At least 1 buffer tree was maintained between trees treated with 17WP spray and all other treatments. Each plot within a site contained the same orange cultivar (shoot and rootstock genotype), but cultivars varied between sites. TM No larger buffer zones were required between treated plots. No other formulations containing OTC-HCl were applied during the trial.

[0283] Application method The administration method was trunk injection using the exemplary injection system described herein. The administration vehicle was bottled deionized water. The administration solution was prepared on the day of administration or the day before administration. Each administration container was uniquely identified. Each container initially contained 60 ml of ArborBiotic TM Aqueous solution. The volume remaining in the container 7 days after application indicates the tree's uptake. Remove the injection system from the tree 7-8 days after application.

[0284] analyzeTreatment efficacy was determined based on the evaluation of different yield components, including harvest weight, fruit number, fruit drop percentage (total during the two months before harvest), and fruit size (weight per fruit), as well as different juice quality parameters, including Brix content, acid content, and Brix / acid ratio.

[0285] result Compared with the control, the ArborBiotic TM The yield increase observed after one season of treatment averaged about 28%-51%, and in some sites the yield increase was as high as about 130% ( Fig.11 The increase in yield was due to an increase in fruit number and a decrease in fruit drop, as described below. The average harvest weight (lbs / tree) for each treatment is shown in Fig.12 In different locations, it was observed that the ArborBiotic TM Changes in average yield growth of trees treated with (T2, T3, T4) Fig.13 and 14 ). Measure different locations using ArborBiotic TM Harvest weight of treated trees (lbs / tree) (T2, T3, T4) for water treatment and FireLine TM Compared with 17WP foliar spray treatment. Figure 15-21 . Fig. 22 Water use, FireLine TM The average number of fruits per tree in the experimental sites for treatments T1, T2, T3, and T4, and the percent change in increase compared to the water treatment. TM The average fruit drop percentages of each experimental site for treatments T2, T3, and T4 are shown in Fig.23 On average, these ArborBiotic TM The treatment reduced fruit drop by 18.6% compared to water treatment and by 18.6% compared to FireLine TM The 17WP spray group decreased by 18.4%.

[0286] in conclusion

[0287] Results from the field study showed that regardless of OTC-HCl concentration (0.625 mg / ml v. 1.25 mg / ml), injection volume (120 ml v. 240 ml), and the amount of OTC-HCl injected during the season (75 mg v. 150 mg), there was no significant difference in the effect of OTC-HCl on the soil compared to water injection treatment (negative control) and foliar application of FireLine TM Compared to all ArborBiotic TMThe average harvested fruit weight increased in all treatments (site ID 01, 02, 04, 05, and 07). TM The harvested fruit weight of the treatment was 19.6% higher than that of the water treatment (ArborBiotic TM treatments were 9.8%-32.3%), and the harvested fruit weight was higher than that of FireLine TM The group was 28.9% higher (ArborBiotic TM These results confirm the results of Example 2 and demonstrate that growers can flexibly use ArborBiotic TM OTC-HCl in the form of HCl was injected into orange trees to increase harvest fruit weight. These results also demonstrate that FireLine TM Foliar applications of OTC-HCl were ineffective as they provided no measurable improvement over the untreated negative control.

[0288] Additional data on yield were collected, including fruit number (number of fruits on the tree at harvest), fruit size (weight of individual fruit at harvest), and percentage of total fruit that dropped during the two months prior to harvest until harvest. Analysis of these data supported the conclusion that the increase in fruit number and the decrease in percentage of fruit dropped were the primary factors in the increase in harvest weight. Fruit number in the ArborBioticTM treated trees was 11.9%-31.2% higher than the water-injected group (average 19.3% in the ArborBioticTM treatment) and 10.9% higher than the FireLine™ treated trees. TM The ArborBiotic™ treatments had 13.8% to 33.5% higher fruit drop than the water treatments (average 21.4% in the ArborBiotic™ treatments). For fruit drop percentage, the ArborBiotic™ treatments had 15.7% less fruit drop than the water treatments (7.2% to 21.7% less in the ArborBiotic™ treatments) and 15.5% less than the FireLine™ group (7.0% to 21.5% less in the ArborBiotic™ treatments). Average fruit size (lbs / fruit) had little effect on harvest weight, averaging 0.32, 0.32, and 0.30 for the ArborBiotic™, water, and FireLine treatments, respectively.

[0289] Analysis of juice quality of fruit harvested from trees at each site ID 01, 02, 04, 05 and 07 showed that ArborBiotic™ injections had no significant effect on Brix content, acid content or Brix / acid ratio compared to water injections and FireLine™ foliar applications. Despite the lack of significance after one season of injections, the average Brix content and Brix / acid ratio of the ArborBiotic™ treatment that delivered 150 mg OTC-HCl each season (T2) were 6.1% and 6.2% higher, respectively, across sites on average, and 16.7% and 15.4% higher, respectively, at specific sites, compared to the water treatment.

[0290] OTC-Cl treatment with the injection system described herein in the form of ArborBiotic™ is superior to Fireline TM Foliar treatments, indicating that the method of application has a significant impact on the effectiveness of OTC-Cl treatments (and specifically ArborBiotic™) on orange trees to achieve such high efficacy on orange trees.

[0291] These results demonstrate that one or more injections using an exemplary injection system as described herein, over a range of environments, concentrations, and application rates, statistically significantly improved orange fruit yield relative to a water injection control and a commercial OTC product (FireLine™ 17WP) labeled for foliar application, and can be used to effectively improve fruit yield and juice quality in HLB-infected orange trees.

[0292] Example 4: Field Trial - Study on Residues of OTC and Its Metabolites in Orange Raw Agricultural Products and Processed Commodities

[0293] This example demonstrates the use of an exemplary injection system described herein to inject orange trees with a stem cell containing ArborBiotic TM After treatment with oxytetracycline hydrochloride (OTC-HCl) and its metabolites, residue levels in processed orange commodity (PC) and raw orange agricultural product (RAC) were compatible with the current tolerance level of OTC-HCl in citrus.

[0294] Field trial design . The study included eight test sites (01-08) containing orange trees of the Hamlin, Navel, or Valencia varieties. The test sites were at least 20 miles apart and in different municipalities. If the test sites were within 20 miles of each other, the sites also varied in variety and application date. The sites were selected based on representative growing areas of commercial citrus and were essentially free of weeds, diseases, and insect pests. The test sites were selected to avoid major sources of variability, including slope, drainage, or factors that could affect disease or crop development.

[0295] Each experimental site was set up identically and included 5 plots: 1 untreated control plot ("UTC"), and 4 treated plots (T1, T2, T3, T4). The plots were large enough to ensure that a sufficient number of representative samples and sufficient amount of material were provided for residue analysis. The control plot had at least 4 trees and the treated plot had a minimum of 8 trees. Each tree was clearly identified with a unique number (such as study number, treatment number, and tree number). At least 1 buffer tree was retained between treated and untreated trees. Due to the application method, a larger buffer zone was not required between the control and treated plots. No other formulations containing OTC were applied during the trial. The experimental plots were not treated with products containing the active ingredient OTC in the previous year. A historical record of maintenance pesticide applications for the previous 3 years was provided. Records obtained during the growing season included tillage, irrigation, and maintenance pesticide applications for the experimental plots.

[0296] At the time of application, record air temperature, soil temperature, wind speed and direction, relative humidity, cloud cover BBCH stage, crop height, and rainfall within 24 hours of application. During the trial, meteorological data including daily minimum / maximum temperature, monthly average daily minimum / maximum temperature, monthly overall average temperature, and total rainfall were collected at the trial site or, if not available, at the nearest weather station (within 25 miles). Data were collected from the start of the experiment (start of application) until the end of the test (last sample collection).

[0297] deal with Treatments were injected into the trunks of orange trees. The treatments used in the study are included in Table 11. Treatment applications were made once or twice during the growing season. For Treatment 1 (T1), each application consisted of an injection of 94.8 mg ArborBiotic in 60 mL of distilled water. TM , each injection delivered a dose of 37.5 mg of OTC-HCl per tree. For Treatment 2 (T2), two injections of 94.8 mg of ArborBiotic in 60 mL of distilled water were used per application. TM (Total 189.6 mg of ArborBiotic TM and 120 mL of distilled water), delivering a total dose of 75.1 mg of OTC-HCl per tree per application. For Treatment 3 (T3), each application consisted of injecting 189.0 mg of ArborBiotic TM , delivering a total dose of 74.8 mg of OTC-HCl per tree per application. Treatments T1, T2, and T3 were applied twice per season, 3 months apart. Treatment 4 (T4) was a single application consisting of two injections of 189.0 mg of ArborBiotic in 60 mL of distilled water. TM(378.0mg ArborBiotic TM and 120 mL of distilled water), which delivers a total dose of 149.7 mg of OTC-HCl. Long-season varieties (e.g., Valencia) receive injections in May and August, and short-season varieties (e.g., Hamlin, Navel) receive injections in March and June.

[0298] Table 11. Treatment

[0299]

[0300] Application method . The treatment was applied by trunk injection using the exemplary injection system described herein. The application vehicle was bottled deionized water. The application solution was prepared on the day of application. Each container was uniquely identified. The volume of application solution delivered to each tree was recorded. The injection system was removed from the tree on the 7th day. Trees with uptake <80% of nominal value in the cumulative application were excluded from residue sampling.

[0301] Tables 12-15 show the application details of treatments T1, T2, T3 and T4 for each of the eight sites used in the study. In addition to the nominal dose, the actual average dose absorbed by the trees is reported to record that the sampled trees had reached at least 80% of the cumulative nominal dose.

[0302] Table 12. Treatment table for T1 treatment plots

[0303]

[0304] Note: T1 administration includes two root injections, each with 94.8 mg ArborBiotic TM / tree (37.5 mg OTC-HCl / tree) in the form of 60 ml aqueous solution - 1 container / tree

[0305] a RTI = Retreatment Interval. Number of days between two applications.

[0306] b The application rates shown are the actual rates achieved, expressed as the average uptake achieved in the treated trees in the test plots at each application time point.

[0307] c The total rate mg / tree was calculated using only the trees remaining in the study after application 2. This value is not the sum of the average rates from each of the first and second applications.

[0308] Table 13. Treatment table for T2 treatment plots

[0309]

[0310]

[0311] Note: T2 administration includes two root injections, each with 189.6 mg ArborBiotic TM / tree (75.1mg OTC / tree), in the form of 120ml aqueous solution - 2 bottles / tree

[0312] a RTI = Retreatment Interval. Number of days between two applications.

[0313] b The application rates shown are the actual rates achieved, expressed as the average uptake achieved in the treated trees in the test plots at each application time point.

[0314] c The total rate mg / tree was calculated using only the trees remaining in the study after application 2. This value is not the sum of the average rates from each of the first and second applications.

[0315] Table 14. Treatment table for T3 treatment plots

[0316]

[0317] Note: T3 administration includes two root injections, each with 189.0 mg ArborBiotic TM / tree (74.8 mg OTC / tree), in the form of 60 ml aqueous solution - 1 can / tree.

[0318] a RTI = Retreatment Interval. Number of days between two applications.

[0319] b The application rates shown are the actual rates achieved, expressed as the average uptake achieved in the treated trees in the test plots at each application time point.

[0320] c The total rate mg / tree was calculated using only the trees remaining in the study after application 2. This value is not the sum of the average rates from each of the first and second applications.

[0321] Table 15. Treatment methods for T4 treatment plots

[0322]

[0323] Note: T4 administration includes 1 rhizome injection of 378.0 mg ArborBiotic TM / tree (149.7mg OTC / tree), in the form of 120ml aqueous solution - 2 bottles / tree

[0324] a RTI = Retreatment Interval. T4 plots were not applied and each tree was treated once.

[0325] b The application rates shown are the actual rates achieved, expressed as the average uptake achieved in the treated trees in the test plots at each application time point.

[0326] c The overall rate mg / tree was calculated using only the trees that remained in the study after the first application.

[0327] Sampling method . From the control plot (UTC), single composite samples were collected from sites 01, 02, 04, 05, 06 and 08; 24-fruit RAC samples were collected from sites 01, 02, 04, 05, 06 and 08; and a 200 kg bulk sample was collected from site 04. Replicate composite RAC samples of 24 fruits were collected from each of the treatment plots (T1, T2, T3, T4) at the sampling locations. During the sampling process, control and treated samples were always kept sufficiently spaced apart to avoid contamination. Samples were collected from all four quadrants, from inside to outside of the canopy, and from high to low.

[0328] Sampling time Citrus fruits were collected according to the schedule in Table 16 (samples were collected only from the test site).

[0329] Table 16. Sampling time summary

[0330] Plot ID <![CDATA[Sampling interval a > commodity <![CDATA[Minimum sample size b > UTC 90DALA Citrus, unripe fruit 24 fruits / 100g UTC ENCH Citrus, whole fruit 24 fruits / 2kg T1, T2, T3, T4 90DALA Citrus, unripe fruit 24 fruits / 100g T1, T2, T3, T4 90DALA Citrus, unripe fruit 24 fruits / 100g T1, T2, T3, T4 ENCH Citrus, whole fruit 24 fruits / 2kg T1, T2, T3, T4 ENCH Citrus, whole fruit 24 fruits / 2kg

[0331] a DALA = days after last administration; ENCH = earliest normal commercial harvest

[0332] b The sample size must contain at least 24 fruits and meet the weight requirements

[0333] Residue analysis Oxytetracycline (OTC) and 4-Epi-Oxytetracycline analytes were measured in the study samples according to the method described in Robaugh, E. “A Method for the Determination of Oxytetracycline and 4-Epi-Oxytetracycline in Apples, Pears, Peaches, and Nectarines by LC / MS / MS.” 2013.

[0334] result

[0335] Residue Reduction Studies . Figure 24-27 Data for residue reduction for the four treatments and sites ID 3 (T1-T4) and ID 7 (T1-T3) are presented graphically. For treatments T1, T2, and T3, initial total residue levels in immature fruit were consistently shown to be less than 0.20 ppm 3 days after application of 2×37.5 mg (T1) or 2×75 mg (T2 and T3) OTC-HCl per tree with repeated injections, and declined to levels of 0.01 ppm or less 60-90 days after the last injection. There were no measurable differences between the low ratio (T1) and the high ratio used in T2 and T3 as repeated applications. A single high dose in T4 produced a significantly higher initial concentration of 0.5 ppm in immature fruit at 3 days, followed by a faster rate of decline than observed in treatments T1-T3; however, the kinetics were insufficient to completely dissipate the residue to levels of 0.01 ppm or less at 90 DALA.

[0336] Residues on fruit 90 days after last application (DALA) and earliest normal commercial harvest Residue results (average of duplicate samples) were determined 90 days after the last treatment and thereafter until the earliest normal commercial harvest (ENCH). At all eight locations, the average residue levels in duplicate samples at 90 days after treatments T1, T2, and T3 were at or below the current tolerance limit of 0.01 ppm for OTC in citrus ( Fig.28 ). Treatment T4 had 3 results above 0.01 ppm at DALA 90 and 97. Residues above 0.01 ppm were observed at sites 03, 07, and 08. At ENCH, fruit residues for all treatments collected and processed to date were below the LOD of 0.003 ppm.

[0337] The residue data indicated that treatments T1, T2, and T3 were compatible with the current tolerance limit of 0.01 ppm if a 90-day PHI was considered. Treatment T4 required a slightly longer PHI to meet the tolerance target. Example 5: Field Trial - Study of Residues of OTC and Its Metabolites in Orange Raw Produce and Leaves

[0338] This example demonstrates that ArborBiotic TMAfter injection into the trunk, the residue levels of oxytetracycline hydrochloride (OTC-HCl) in the fruit and leaves of orange trees decreased significantly before the end of the crop cycle. The fruit and leaf residue data in this example were obtained from orange trees (Valencia, OLL8, or Hamlin) at 9 test sites (01-09) in the second season using the treatment methods described in Examples 2 and 3. The fruit residue data were obtained from test sites 01-09, and the leaf residue data were obtained from test sites 01, 02, 03, 05, 08, and 09. The fruit and leaf residue data in this example were obtained from orange trees (Valencia, OLL8, or Hamlin) at 9 test sites (01-09) in the second season using the treatment methods described in Examples 2 and 3. The fruit residue data were obtained from test sites 01-09, and the leaf residue data were obtained from test sites 01, 02, 03, 05, 08, and 09.

[0339] Test site 09 (ArborBiotic TM and water as negative control). Test sites 01-08 (ArborBiotic TM , water as negative control, and FireLine TM 17WP was used as a positive control and commercial reference). Fruit residue analysis Oxytetracycline hydrochloride (OTC-HCl) and 4-Epi-Oxytetracycline analytes were measured in orange fruit study samples according to the method described by Robaugh, E. 2013. “A Method for the Determination of Oxytetracycline and 4-Epi-Oxytetracycline in Apples, Pears, Peaches, and Nectarines by LC / MS / MS”. The study matrix analyzed included reported data for both unripe and ripe intact orange fruit.

[0340] Leaf residue analysis Oxytetracycline (OTC-HCl) was measured in orange leaf disc samples according to the method described in Nazari, B. 2022, “Determination of Oxytetracycline in Citrus Leaf Using LC-MS / MS”.

[0341] Fruit residues at 90-250 DALA and harvestFor each sampling date, a composite sample was collected and analyzed twice, yielding duplicate results. The two results were then averaged to yield a mean residue concentration representative of the treatment and sampling date. Residue results (mean of the duplicate results) at 90 days after the last application and subsequent collection dates until harvest are shown in Fig.29 It should be understood that the "average total residue" includes both OTC-HCl and 4-epi-oxytetracycline analytes. The current data set recommends a PHI of 120 days to meet the tolerance target.

[0342] ArborBiotic was injected with 4 different treatment schedules using the exemplary tree injection system described herein. TM The key observations are as follows. Rapid absorption and distribution of the active ingredient, OTC-HCl, into the canopy was observed, with the highest concentrations generally measured on days 1-3 after injection. Mean leaf concentrations of OTC-HCl were observed to be 0.875 ppm for treatment T1, 1.558 ppm for treatment T2, and 1.637 ppm for treatment T3. For treatment T4, data were only available from 2 sites and were more variable. Mean concentrations ranged from 0.957 ppm at site 03 to 4.524 ppm at site 08. Mean concentrations between T1 and T2-T3 showed 2x the expected dose response. Once the peak OTC-HCl concentration in the leaves was reached, it subsequently declined moderately rapidly with a mean half-life of 6.2 days (T2) to 10.5 days (T1), with no significant differences between the T1-T4 regimens. Mean concentrations were below the LOQ of 0.1 ppm for all treatments and sites on days 58-63. After injection application T1-T4, the residue level in leaves (average leaf residue level of 1.346 ppm) was approximately 1.044 ppm higher than that of FireLine 1-3 days. TM The average residue level in samples obtained from the 17WP spray treatment was twice as high, with an average leaf residue level of 0.751 ppm. TM Leaf residues declined rapidly, falling by more than 70%, from an average residue level of 0.751 ppm on days 1-3 to an average level of 0.204 ppm on days 6-8.

[0343] The leaf residue data demonstrate that the ArborBiotic TM Injection into orange trees ensures rapid absorption and distribution into the canopy. Simultaneous foliar application of FireLine TMPeak residual levels in leaves were approximately 2-fold higher than the levels achieved with 17WP. Residues of foliar-applied OTC-HCl will dissipate rapidly from treated leaves (>70% within 5 days). However, injection treatment with oxytetracycline HCl will maintain elevated levels of OTC-HCl in leaf tissue, but will drop to levels below the LOQ before subsequent applications. For ArborBiotic 17WP using an exemplary injection system as described herein TM The observed decay kinetics with an average half-life of 6.2-10.5 days indicated that leaf residues would decay quantitatively by the end of the crop cycle. TM Leaves dropped from trees do not become a source of low-level environmental contamination or pose a potential risk of resistance to non-target microorganisms.

[0344] Example 6: Field trial - Study on the residues of OTC-HCL and its metabolites in orange tree roots

[0345] This example demonstrates the use of the exemplary injection system described herein to deliver ArborBiotic TM Injection into the trunks of orange trees did not produce environmentally relevant HCl concentrations in the roots of treated orange trees.

[0346] Field trial design The field trial was conducted by treating commercial orange trees (Valencia variety) at two different test locations. Treatments included the administration of ArborBiotic TM applied to the young shoots of each tree, treated with water (used as a negative control), and with the commercial product FireLine TM 17WP treatment (used as positive control and commercial reference).

[0347] deal with Treatments were injected into the trunks of orange trees. A complete list of treatments used in the study is included in Table 17. For Treatment A (TA), each application consisted of two injections of 94.8 mg ArborBiotic TM (Total 189.6 mg of ArborBiotic TM and 120 mL of distilled water), delivering a total dose of 75.1 mg of OTC-HCl per tree per application. Treatment 4 (T4) was a single application consisting of two injections of 189.0 mg of ArborBiotic in 60 mL of distilled water. TM (Total 378.0 mg of ArborBiotic TMand 120 mL of distilled water), delivering a total dose of 149.7 mg of OTC-HCl per tree per application. Two applications of all treatments were applied during the growing season. The first application was made in June or July, and the second application was made between September and November.

[0348] Table 17. Treatment

[0349]

[0350]

[0351] Place . Two representative sites of commercial citrus growing areas were selected for this study. Each test site included 16 plots: 4 negative control plots (water treatment) and 4 treatment plots for each of the three treatments (TA, TB, FireLineTM 17WP spray). The plots were large enough to ensure sufficient representative samples and materials for residue analysis. Each treatment contained 4 replicate plots. All treatments had 1 tree per plot, for a total of 4 trees per treatment. Each tree was clearly identified with a unique number (such as experimental number, plot number, and tree number). FireLineTM 17WP spray was placed in adjacent blocks where the product was applied with a commercial sprayer. According to the application method. No larger buffer zone was required between the negative control (water treatment) and the treatment plots for treatment A and treatment B. No other formulations containing OTC-HCl were applied during the trial.

[0352] Application method . The method of application for water treatments, TA, and TB was trunk injection using the exemplary injection system described herein. The application vehicle was bottled deionized water. The application solution was prepared on the day of application. Each container was uniquely identified. The volume of application solution delivered to each tree was recorded. The equipment was removed from the tree on days 7-9. FireLineTM 17WP spray was applied using a commercial sprayer at a label rate of 24 ounces / acre (equivalent to 4.08 ounces of OTC-HCl per acre), and the solution was prepared on the day of application according to the label recommendations. Treatments using FireLineTM 17WP were applied consistently with the other treatments. The sampled trees received at least 80% of the cumulative nominal dose.

[0353] Root residue analysis and results

[0354] OTC residues in the study samples were measured according to the method described in Nazari, B. 2022, “Determination of Oxytetracycline in Orange Tree Root Using LC-MS / MS”. Orange tree root samples were analyzed for the study.

[0355] This example demonstrates that systemic OTC concentrations in the leaves of treated trees reach maximum concentrations within 2-3 days after injection and then decline, with average half-life values ​​of 6-10 days. Based on the data obtained during this study, ArborBiotic was repeatedly injected with the exemplary injection system described herein at a rate of up to 378 mg / tree (149.7 mg OTC-HCl / tree). TM , no environmentally relevant concentrations were produced in the roots of the treated orange trees. Since there were no OTC-HCl residues in the roots, it was ruled out that the soil environment around the three roots would be contaminated by trace amounts of OTC-HCl through root exudates.

[0356] Other data

[0357] Regarding the above embodiments, such as embodiments 2-6, Figure 30-32 Some additional data and results are provided in .

[0358] exist Fig.30 The OTC-HCl treatment showed an increase in yield of about 74% over the water treatment. This treatment delivered 150 mg of OTC-HCl per season.

[0359] exist Fig.31 The OTC-HCl treatment showed an increase in yield of about 53% over the untreated control. This treatment delivered 600 mg of OTC-HCl per season.

[0360] exist Fig.32 In the two-year trial, OTC-HCl treatment resulted in a reduction in disease and leaf mottling compared to the untreated control. Significant treatment differences were observed on new grafts in the two-year trial. In the two-year trial, trees were treated with three applications (injected using the exemplary injection system described herein), each application of 19 mg of OTC HCl, for a total of 56 mg of OTC HCl.

[0361] Example 7: Field Trial - Efficacy of OTC Treatment on Citrus Trees with Shoot Diameters in the Range of 1.3-5.1 cm

[0362] This study was conducted to test the efficacy of an injectable formulation containing oxytetracycline hydrochloride as the active ingredient against Huanglongbing (HLB) infection of young citrus trees with shoot diameters ranging from 0.5-2.0 inches (1.3-5.1 cm).

[0363] The study was conducted over two seasons at two field trial locations. A total of 12 orange trees were tested per treatment at each location. Treatments are summarized in Table 18 below. Treatments included root-shoot injections of ArborBiotic TM , foliar application of FireLine TM17WP (FireLine Spray), a registered OTC product manufactured by AgroSource (AgroSource, Inc, PO Box 3091, Tequesta, FL 33469), and rhizome injection water treatment (negative control). Injections were performed with a 6.5 mm injection tool connected to a 50 ml bag-on-valve canister containing the injection formulation of Season 1, and with a 6.5 mm injection tool connected to a 100 ml bag-on-valve canister containing the injection formulation of Season 2.

[0364] Table 18. Processing summary

[0365]

[0366] *FireLine values ​​are based on label application rates and an estimate of the number of trees per acre

[0367] Treatment efficacy was determined based on the assessment of disease symptom development. Disease symptoms were assessed by analyzing the amount of leaf spots that occurred on young leaves infected with HLB. Leaf mottle is an early visible symptom of HLB infection, which occurs when carbohydrates cannot be transported through damaged phloem tissue in the leaves. A 0-5 rating scale was used to estimate the percentage of fully hardened leaves showing mottle symptoms. In this scale, 0-grade scoring = no visible spots on any leaf on the tree, 1 = <20%, 2 = 21-40%, 3 = 40-60%, 4 = 60-80%, and 5 = >80% of leaves showed mottle symptoms. This scoring is best performed after the main leaves (flush) expand and harden, usually in early summer (June), late summer (August) and autumn leaves (flush) mature (November) (Gottwald et al.). ANOVA results showed that the independent variables of assessment date, treatment and treatment assessment date had a statistically significant effect on leaf mottle (P < 0.10).

[0368] On the four leaf mottling evaluation dates, the water injection control and foliar application FireLine trees had significantly (P<0.10) more HLB mottling symptoms than trees treated with 94.8 or 189.6 mg / ml ArborBiotic (37.5 or 75.0 mg OTC-HCL, respectively). The average leaf mottling values ​​for the water injection and FireLine spray were 2.4 and 2.4, respectively, and the mottling value for both ArborBiotic treatments was 1.6. See Table 19.

[0369] Table 19. Average responses by treatment on evaluation date

[0370]

[0371]

Claims

1. A method for controlling citrus greening disease in a citrus plant, wherein the citrus plant further has an active vascular system extending through the trunk or stem to other parts of the citrus plant, the method comprising: An injectable formulation comprising oxytetracycline (OTC) or its salts is injected into the active vasculature of the plant to control citrus greening disease in citrus plants.

2. The method of claim 1, wherein the active vasculature into which the injection formulation is injected is the active vasculature in the trunk or stem of the citrus plant.

3. The method of claim 1 or 2, wherein the citrus plant is a citrus tree.

4. The method of any one of claims 1-3, wherein after injection, the citrus plant has a reduction in fruit drop of at least about 5%, at least about 10%, or at least about 15%, or about 10% to about 25% compared to untreated citrus plants.

5. The method of any one of claims 1-4, wherein after injection, the citrus plant produces fruit with a Brix increase of at least about 1%, at least about 2%, at least about 3%, at least about 4%, or at least about 5%, about 5% to about 10%, compared to an untreated citrus plant.

6. The method of any one of claims 1-5, wherein after injection, the citrus plant fruit yield is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, or about 40% to about 75% compared to untreated citrus plants.

7. The method of any one of claims 1-6, wherein the injection of the injection preparation is performed using an injection system, the injection system comprising an injection tool operably connected to a fluid delivery unit, wherein the fluid delivery unit is configured to deliver the injection preparation.

8. The method of claim 7, wherein the injection of the injection preparation comprises: piercing the trunk or stem of the citrus plant using the injection tool of the injection system; as well as At least a portion of the injection formulation is delivered from the fluid delivery unit through the injection tool into the viable vasculature of the citrus plant without exceeding the viable vasculature.

9. The method of any one of the preceding claims, wherein the injectable formulation is distributed in the trunk or stem and other parts of the citrus plant.

10. The method of claim 9, wherein the other parts of the citrus plant include fruit.

11. The method of claim 9 or 10, wherein the other parts of the citrus plant include leaves.

12. The method of any one of claims 7-11, wherein the fluid delivery unit is a spring-loaded fluid delivery unit.

13. The method of any one of claims 7-12, wherein the delivery unit comprises a pressurized formulation cartridge.

14. The method of any one of claims 7-13, further comprising: replacing the fluid delivery unit with a second fluid delivery unit; as well as At least a portion of the injection formulation is delivered from the second fluid delivery unit through the injection tool into the viable vasculature of the citrus plant without exceeding the viable vasculature.

15. The method of any one of claims 7-14, wherein the injection tool remains in the trunk or stem of the citrus plant for at least one growing season.

16. The method of any one of claims 7-15, wherein the injection tool remains in the trunk or stem of the citrus plant after multiple re-injections.

17. The method of any one of claims 7-16, wherein the trunk of the citrus plant has bark, and the method further comprises: Remove at least a portion of the bark before piercing the trunk.

18. The method of any one of the preceding claims, wherein the citrus plant is an orange tree.

19. The method of claim 18, wherein the average oxytetracycline (OTC) residue in the fruit of the orange trees to which the injectable formulation is applied is less than about 0.01 ppm.

20. The method of claim 18 or 19, wherein the method results in an average fruit drop of less than about 20 in orange trees to which the injectable formulation is applied.

21. The method of any one of claims 18-20, wherein the method results in an average fruit yield of at least about 40 pounds per plant, or about 45 to about 90 pounds per plant, in orange trees to which the injectable formulation is applied.

22. The method of any one of claims 18-21, wherein the method results in an average Brix of at least about 7.5 in orange trees to which the injectable formulation is administered.

23. The method of any one of claims 18-22, wherein the method results in an average reduction of at least about 10% in fruit drop in orange trees to which the injectable formulation is applied, compared to orange trees to which the injectable formulation is not applied.

24. The method of any one of claims 18-23, wherein the method results in an average increase of at least about 5% in fruit Brix in orange trees to which the injectable formulation is applied, compared to orange trees to which the injectable formulation is not applied.

25. The method of any one of claims 18-24, wherein the method results in an average increase in fruit yield of at least about 10% in orange trees to which the injectable formulation is applied, compared to orange trees to which the injectable formulation is not applied.

26. A method of increasing fruit harvest yield in a citrus plant, wherein the citrus plant has an active vasculature extending through the trunk or stem to other parts of the citrus plant, the method comprising: An injectable formulation comprising oxytetracycline (OTC) or a salt thereof is injected into the active vasculature of the citrus plant, wherein the injectable formulation is injected into the citrus plant once or multiple times during the growing season, thereby increasing the fruit harvest yield of the citrus plant in one or more growing seasons of injection compared to the fruit harvest yield in untreated citrus plants.

27. The method of claim 26, wherein after two growing seasons, the average fruit harvest yield is increased by more than 100% compared to the average fruit harvest yield of untreated citrus plants.

28. The method of claim 26, wherein after one growing season, the average fruit harvest yield is increased by more than 30% compared to the average fruit harvest yield of untreated citrus plants.

29. The method of any one of claims 26-28, wherein the injectable formulation is injected once during the growing season.

30. The method of claim 29, wherein the injection formulation comprises 150 mg OTC or a salt thereof in solution.

31. The method of any one of claims 26-28, wherein the injectable formulation is injected twice during the growing season.

32. The method of claim 31, wherein the injection formulation comprises 75 mg OTC or a salt thereof in solution.

33. The method of any one of claims 26-28, wherein the injectable formulation is injected three times during the growing season.

34. The method of claim 33, wherein the injection preparation comprises 37.5 mg of OTC or a salt thereof in a solution, which is administered as two injections; or the injection preparation comprises 75 mg of OTC or a salt thereof in a solution, which is administered as a single injection.

35. The method of any one of claims 26-34, wherein 100 mg to 200 mg of OTC or a salt thereof is injected into the citrus plant during the growing season.

36. The method of claim 35, wherein about 150 mg of OTC or a salt thereof is injected into the citrus plant during the growing season.

37. The method of any one of claims 26-36, wherein the fruit harvest yield is determined based on harvest weight, fruit number and / or fruit drop.

38. The method of claim 37, wherein the harvest weight is increased compared to the harvest weight of untreated citrus plants after one or more growing seasons.

39. The method of claim 38, wherein after one growing season, the harvest weight is increased by more than about 50% compared to the harvest weight of untreated citrus plants.

40. The method of claim 38 or 39, wherein after two growing seasons, the harvest weight is increased by more than about 100% compared to the harvest weight of untreated citrus plants.

41. The method of any one of claims 37-40, wherein after one or more growing seasons, the number of fruit is increased compared to the number of fruit of untreated citrus plants.

42. The method of claim 41, wherein after one growing season, the number of fruit is increased by more than about 10% compared to the number of fruit of untreated citrus plants.

43. The method of any one of claims 41 or 42, wherein after two growing seasons, the number of fruit is increased by more than about 100% compared to the number of fruit of untreated citrus plants.

44. The method of any one of claims 37-43, wherein after one or more growing seasons, fruit drop is reduced compared to untreated citrus plants.

45. The method of any one of claims 37-44, wherein after one growing season, the fruit harvest yield is increased by more than about 25% compared to the fruit harvest yield of untreated citrus plants.

46. ​​The method of any one of claims 26-41, wherein the Brix of harvested fruit of the citrus plant is increased compared to the Brix of harvested fruit of an untreated citrus plant.

47. The method of any one of claims 26-42, wherein the Brix / acid ratio of harvested fruit of the citrus plant is increased compared to the Brix / acid ratio of harvested fruit of an untreated citrus plant.

48. A method of treating citrus greening disease and leaf mottling in a citrus plant having a shoot diameter ranging from about 1.3 to about 5.1 cm, wherein the citrus plant has an active vasculature extending through the trunk or stem to other parts of the citrus plant, the method comprising: An injectable formulation comprising oxytetracycline (OTC) or a salt thereof is injected into the active vasculature of the citrus plant, wherein the injectable formulation is injected into the citrus plant one or more times during the growing season to reduce citrus greening disease and reduce leaf mottling.

49. The method of claim 48, wherein the citrus plant is injected once with an injectable formulation comprising 12.5 mg oxytetracycline hydrochloride.

50. The method of claim 48, wherein the citrus plant is injected once with an injectable formulation comprising 37.5 mg oxytetracycline hydrochloride.

51. The method of claim 48, wherein the citrus plant is injected twice with an injectable formulation comprising 18.75 mg oxytetracycline hydrochloride.

52. The method of claim 48, wherein the citrus plant is injected three times with an injectable formulation comprising 18.75 mg oxytetracycline hydrochloride.

53. The method of claim 48, wherein the citrus plant is injected twice with an injectable formulation comprising 37.5 mg oxytetracycline hydrochloride.

54. The method of any one of claims 51-53, wherein the injections are spaced about 30 days apart. E 55. The method of any one of E 51-53, wherein the injections are spaced about 45 days apart. E 56. The method of any one of claims 51-53, wherein the injections are spaced about 60 days apart.

57. The method of any one of claims 48-56, wherein the citrus plant is injected in the rhizome.

58. The method of any one of claims 48-56, wherein the citrus plant is injected in young shoots.

59. The method of claim 57, wherein the rhizome is injected with an injection tool to a depth of up to but not exceeding 6.5 mm.

60. The method of claim 58, wherein the sprouts are injected with an injection tool to a depth of up to but not more than 6.5 mm.

61. A method of treating citrus greening disease and leaf mottling in a citrus plant having a shoot diameter greater than 6 cm, wherein the citrus plant has an active vascular system extending through the trunk or stem to other parts of the citrus plant, the method comprising: An injectable formulation comprising oxytetracycline (OTC) or a salt thereof is injected into the active vasculature of the citrus plant, wherein the injectable formulation is injected into the citrus plant one or more times during the growing season to reduce citrus greening disease.

62. The method of claim 61, wherein the citrus plant is injected once with an injectable formulation comprising 150 mg of oxytetracycline hydrochloride.

63. The method of claim 61, wherein the citrus plant is injected twice with an injectable formulation comprising 75 mg of oxytetracycline hydrochloride.

64. The method of claim 61, wherein the citrus plant is injected three times with an injection formulation comprising 50 mg of oxytetracycline hydrochloride.

65. The method of any one of claims 63 and 64, wherein the injections are spaced about 30 days apart.

66. The method of any one of claims 63 and 64, wherein the injections are spaced about 45 days apart.

67. The method of any one of claims 63 and 64, wherein the injections are spaced about 60 days apart.

68. The method of any one of claims 61-67, wherein the citrus plant is injected in the rhizome.

69. The method of any one of claims 61-67, wherein the citrus plant is injected in young shoots.

70. The method of any one of claims 61-69, wherein the citrus plant is injected with 60, 70, 80, 90, 100, 110, or 120 mL of the injection formulation.

71. The method of any one of claims 61-69, wherein the citrus plant is injected with 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 mL of the injection formulation.

72. The method of claim 57, wherein the rhizome is injected with an injection tool to a depth of but not more than 8.5-9 mm.

73. The method of claim 58, wherein the sprouts are injected with an injection tool to a depth of but not more than 8.5-9 mm.

74. The method of any one of claims 61-73, wherein after injection, fruit drop in the citrus plant is reduced by at least about 5%, at least about 10%, or at least about 15%, or about 10% to about 25% compared to untreated citrus plants.

75. The method of any one of claims 61-73, wherein after injection, the citrus plant produces fruit having a Brix increase of at least about 1%, at least about 2%, at least about 3%, at least about 4%, or at least about 5%, about 5% to about 10% compared to an untreated citrus plant.

76. The method of any one of claims 61-73, wherein after injection, the fruit yield of the citrus plant is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, or about 40% to about 75% compared to an untreated citrus plant.

77. The method of any one of claims 61-76, wherein said citrus plant is injected 150-120 days prior to harvest.

78. A method of treating citrus greening disease and leaf mottling in a citrus plant having a shoot diameter of about 2 cm to about 6 cm, wherein the citrus plant has an active vasculature extending through the trunk or stem to other parts of the citrus plant, the method comprising: An injectable formulation comprising oxytetracycline (OTC) or a salt thereof is injected into the active vasculature of the citrus plant, wherein the injectable formulation is injected into the citrus plant one or more times during the growing season to reduce citrus greening disease.

79. The method of claim 78, wherein the citrus plant is injected once with an injectable formulation comprising 75 mg oxytetracycline hydrochloride.

80. The method of claim 78, wherein the citrus plant is injected twice with an injectable formulation comprising 37.5 mg oxytetracycline hydrochloride.

81. The method of claim 78, wherein the citrus plant is injected three times with an injectable formulation comprising 25 mg oxytetracycline hydrochloride.

82. The method of any one of claims 80 and 81, wherein the injections are spaced about 30 days apart.

83. The method of any one of claims 80 and 81, wherein the injections are spaced about 45 days apart.

84. The method of any one of claims 80 and 81, wherein the injections are spaced about 60 days apart.

85. The method of any one of claims 78-84, wherein said citrus plant is injected in the rhizome.

86. The method of any one of claims 78-85, wherein the citrus plant is injected in young shoots.

87. The method of claim 85, wherein the rhizome is injected with an injection tool to a depth of up to but not exceeding 6.5 mm.

88. The method of claim 86, wherein the sprouts are injected with an injection tool to a depth of up to but not more than 6.5 mm.

89. The method of any one of claims 78-88, wherein the citrus plant is injected with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 mL of the injection formulation.

90. The method of any one of claims 78-89, wherein after injection, the citrus plant has leaf mottling reduced by at least about 5%, at least about 10%, or at least about 15%, or about 10% to about 25% compared to an untreated citrus plant.

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