Patent Application
20240099299
I hereby claim the benefit under 35 U.S.C., Section 120 of U.S. application Ser. No. 17/516,229 filed Nov. 1, 2021.
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Bacterial diseases of perennial plants have been a source of concern for agriculture for many years. Bacterial diseases spread rapidly, often by wind, rain, or vectoring by an insect. When conditions are favorable, these bacterial pathogens can grow quickly and cause irreversible damage to a crop. Of particular note are vascular tissue colonizing bacterial species. Due to a relatively slow growth rate, these pathogens may not be detected for several growing seasons and already will have begun to negatively impact tree and vine health. Because perennial plants require a number of years to achieve productivity, any colonization by a bacterial pathogen will have long-term implications on the economic viability of the crop. Though not exhaustive, Erwinia spp., Psuedomonas spp., Xanthamonas spp., Xylophilus spp., Xylella spp., Candidatus liberibacter spp., and Rickettsia spp. are representative difficult to control pathogens which require extensive use of foliar antibiotics and insecticides to improve plant productivity and to limit their spread.
Among the most difficult to mitigate and control pathogens are vascular-colonizing bacterial species: Candidatus liberibacter spp., a phloem-colonizing bacterial pathogen which is the causal agent of Citrus Greening Disease/HLB, and Xylella fastidiosa, a xylem-colonizing bacteria that is the causal agent of Pierce's Disease in grapes, Coffee Leaf Scorch Disease in coffee plants, Citrus Variegated Chlorosis in Citrus, and Olive Quick Decline Syndrome in Olives. Because these organisms reside in the vascular tissue of these perennial species, their control is difficult due to a lack of effective antimicrobials or delivery mechanisms. Common control practices include foliar applications of antimicrobials, which suffer from poor penetration into the foliage and poor mobilization through the vascular tissue, control of insect vectors, using limited resistant rootstocks or varieties, increased fertilizer applications as an attempt to grow through the diseases, or, in dramatic and costly cases, removal of infected plants and replanting. For insect vectored plants, initial bacterial titers upon infection are low, but can reach as high as 107 Colony Forming Units (CFUs) per gram fresh tissue. When multiplied out to account for the size of the roots, vines or tree limbs, and corresponding foliage, the number of bacterial colonies within the tree can seem overwhelming.
The disclosure relates to antimicrobial compositions and more particularly pertains to a new antimicrobial compositions and methods for treating plant diseases. The present invention discloses novel antimicrobial compositions and methods for their use in treating liberibacter infections in plants, and in particular infections of Candidatus liberibacter africanus, Candidatus liberibacter americanus, and Candidatus liberibacter asiaticus, or mutants thereof in citrus trees, where they cause what is commonly known as citrus greening disease. This invention also may be used to control other perennial vascular or tissue inhabiting bacterial pathogens including Xyllela spp. Erwinia spp., Psuedomonas spp., Xanthamonas spp., Xylophilus spp., and Rickettsia spp.
The prior art relates to antimicrobial compositions. Prior art antimicrobial compositions for treating citrus greening disease may comprise solutions of tetracycline antibiotics and bark penetrants at a pH of 5.5-6.5 and solutions of auxins and cytokinins at a pH of 5-7 applied by spraying on plants and soil, ampicillin formulated at a pH of 6.5 or liquids obtained from Paenibacillus polymyxa cultures at a pH of 6.0 applied by injection, and beneficial microorganisms and their fermentation products applied to leaves, fruit, seeds, and soil. The prior art also discloses methods for forming protective layers on plants, wherein the protective layers comprise antibiotics that may prevent infections. What is lacking in the prior art is an oxytetracycline (OTC) solution having a pH of 1.8 to 2.5, which is substantially stable for at least two hours at 100° F. and for at least 24 hours at °75 F, and which can be delivered to the vascular network of a citrus tree to treat citrus greening disease.
An embodiment of the disclosure meets the needs presented above by generally comprising oxytetracycline (OTC) and an acid substantially dissolved in water to generate an antimicrobial composition having a pH in a range of 1.8 to 2.5 and a concentration of the oxytetracycline sufficient to treat a bacterial infection in a plant. The OTC or the salt thereof has a purity sufficient to generate an OTC solution that is substantially stable for a time period required for its delivery to a plant. The OTC solution thus is configured for systemic delivery to a plant to treat a bacterial disease inflicting the plant.
Another embodiment of the disclosure includes a method for preparing an acidified OTC solution and its use in treating a bacterial infection in a plant. Yet embodiment of the disclosure includes a citrus tree treated according to the method.
There has thus been outlined, rather broadly, the more important features of the disclosure in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are additional features of the disclosure that will be described hereinafter and which will form the subject matter of the claims appended hereto.
The objects of the disclosure, along with the various features of novelty which characterize the disclosure, are pointed out with particularity in the claims annexed to and forming a part of this disclosure.
The disclosure will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes reference to the annexed drawings wherein:
With reference now to
The antimicrobial composition generally comprises oxytetracycline (OTC) and an acid substantially dissolved in water to generate an OTC solution having a pH in a range of 1.8 to 2.5 and a concentration of the oxytetracycline sufficient to treat a bacterial infection in a plant. The OTC or the salt thereof has a purity sufficient to generate an OTC solution that is substantially stable for a time period required for its delivery to a plant. For example, OTC having a purity greater than 95% can be used to prepare an OTC solution that is substantially stable for at least 2 hours at 100° F. and for at least 24 hours at °75 F. The present invention anticipates OTC having a purity of 92% being used to generate OTC solutions that are substantially stable. The present invention anticipates use of cosolvents, such as, but not limited to, alcohols, dimethyl sulfoxide, and the like. The OTC solution enables treatment of a citrus tree that has been infected by Candidatus liberibacter africanus, Candidatus liberibacter americanus, Candidatus liberibacter asiaticus, or the like. The OTC solution may have an OTC concentration of 500 to 25,000 parts per million. The OTC solution may have an OTC concentration of 500-12,000 parts per million.
The acid may comprise an inorganic acid, although the present invention also anticipates the acid comprising an organic acid, or a combination of an inorganic acid and an organic acid. The acid may comprise hydrochloric acid, phosphoric acid, nitric acid, acetic acid, citric acid, or the like.
The OTC solution is configured for systemic delivery to a plant to treat a bacterial disease inflicting the plant. The OTC solution may be configured for injection into the vascular network of a tree or vine.
The present invention anticipates a method (
Provided below are detailed examples of the antimicrobial compositions for treating plant diseases and methods for their use in treating bacterial infections in plants. These examples should not be viewed as limiting regarding compositions, methods of preparation and use, or plant species.
Tree Vigor Ratings: 1=Lowest Vigor; 5=Highest Vigor. LSD stands for least significant difference and C.V. stands for Coefficient of Variance. “a” denotes the corresponding value is significantly different from any other value that does not contain the letter “a”. Similarly, “b” denotes the corresponding value is significantly different from any other value that does not contain the letter “b”.
In seeking to assess the importance of manufacturing quality of OTC for stability and function of OTC solutions for use according to this disclosure, analytical testing first was performed on three lots each Pharmaceutical Grade OTC HCl (>95%, PG-OTC, Century Pharmaceuticals LTD, Gujarat, India, supplied TJ BioTech LLC) and non-sterile, non-Pharmaceutical Grade OTC HCl (putatively 95%, nS-nPG-OTC, undetermined manufacturer, supplied by AgroSource Inc). The results of these analytical tests are shown in Table 1. Values in bold are below those for the specification of PG-OTC, which is shown in column 2 of Table 1.
89.22
90.23
88.01
89.15
3.52
3.29
3.75
3.52
0.270
0.287
0.332
0.296
3.522
Next, OTC solutions were prepared by first reducing the pH of water to pH 2 using hydrochloric acid. 11.0 g of PG-OTC (>95%) and non-sterile, nS-nPG-OTC (putatively 95%) each were dissolved into 1.0 L of the pH 2 water to generate 11,000 ppm test solutions, which then were monitored visually at 10× magnification. Immediately upon preparation at 75° F., the PG-OTC solution was essentially homogeneous and the nS-nPG-OTC solution was moderately nonhomogeneous, as is shown in
The test article solutions (11,000 ppm) were prepared as above and were fluidly pressurized into latex injection devices (FLexInect™, TJ Biotech LLC, Buffalo, SD) and then were injected into rootstock of uniform, commercially grown, Valencia citrus trees. The injection devices were monitored to measure uptake rates of the solutions.
Table 2 presents the results of Trial 1, which used trees (n=10) having trunk diameters between 2.125″ and 3.00″. 50 mL of the test solutions were injected with the ambient temperature between 80° F. and 90° F., in which temperature range transpiration in the vascular system of the trees is most active.
Trial 1 conclusions: The PG-OTC solution had an injection time 6.9 minutes (55.1%) lower than nS-nPG-OTC solution. Uptake rates were statistically significant at LSD P=0.05.
Table 3 presents the results of Trial 2, which used trees (n=10) having trunk diameters between 3.00″ and 4.25″. 100 mL of the test solutions were injected with the ambient temperature between 80° F. and 90° F.
Trial 2 conclusions: The PG-OTC solution had an injection time 116.5 minutes (86.5%) lower than the nS-nPG-OTC solution. Uptake rates were statistically significant at LSD P=0.01.
Table 4 presents the results of Trial 3, which used trees (n=30) having trunk diameters between 3.00″ and 4.25″. Up to 100 mL of the test solutions were injected with the ambient temperature <70° F., at which temperatures transpiration in the vascular system is reduced. In this trial, the injections were stopped at 120 minutes regardless of if injections were complete.
Trial 3 conclusions: The PG-OTC solution had an injection time 36.2 minutes (30.9%) lower than the nS-nPG-OTC solution. Uptake rates were statistically significant at LSD P=0.01.
Overall Conclusions from Functional Testing
Our studies clearly demonstrate that PG-OTC solutions were readily absorbed by trees, whereas, after the first 50 mL of nS-nPG-OTC solution goes into a tree, there is a marked decrease in absorption rate. The decreased absorption rates could be due multiple factors including the tree's response to impurities or the interaction of crystals in the nS-nPG-OTC solution with the tree's Xylem. While residues are within tolerance (10 PPB) in the fruit when using PG-OTC solutions, the decreased absorption rates using nS-nPG-OTC solutions may lead to residue issues, which may potentially be harmful to consumers and/or to the trees. The decreased absorption rates also likely will induce bacterial resistance.
Research by others (Archer, L., & Albrecht, U. (2023). Evaluation of Trunk Injection Techniques for Systemic Delivery of Huanglongbing Therapies in Citrus. HortScience, 58(7), 768-778. Retrieved Dec. 2, 2023, from https://doi.org/10.21273/HORTSCI17172-23 has shown that sustained pressure and elevated chemical exposure time during treatment of trees by injection can cause an increase in tree wounding, irrespective of environmental conditions, such as temperature, water availability, or the like. Quicker injection processes would provide significant economic improvements by reducing labor costs, tree damage that results from wounding and chemical exposure, increasing injector reusability, and improving OTC distribution throughout the tree. The impact of tree injury can lead to an increased incidence of physical damage caused by factors such as wind, unwanted plant exposure to herbicides, and incidences of infections by other plant pathogen, all of which can reduce the short-term and long-term productivity of perennial crops.
Table 5 below provides the results of this study. For Treatment 1, a 5,500 ppm solution of PG-OTC was prepared using distilled water. This solution then was acidified to pH 2 using hydrochloric acid. Each tree (n=9) was injected with 50 mL of the OTC solution, delivering 0.275 grams of OTC·HCl. For Treatment 2, 2.5 g of PG-OTC was dissolved in 2 gallons of water. The resultant solution was sprayed onto the foliage of the trees at a rate of 1 quart (0.3125 g OTC·HCl) per tree (n=9). Vigor ratings were performed 60 days after application of treatments.
Trial conclusions: Trunk injection of PG-OTC solutions was more effective than application of unacidified PG-OTC solutions to foliage by spraying.
Table 6 below provides the results of this study. For Treatment 1, a 5,500 ppm solution of PG-OTC was prepared using distilled water. This solution was first acidified to pH 2 using hydrochloric acid. Each tree (n=9) was injected with 50 mL of the OTC solution, delivering 0.275 grams of OTC-HCl. For treatment 2, a 5,500 ppm solution of PG-OTC was prepared using distilled water and buffered to pH 7 using NaOH. Each tree (n=9) was injected with 50 mL of the OTC solution, delivering 0.275 grams of the OTC solution per tree. Treatment 3 was an untreated control. After injection, fruit counts were observed.
Trial conclusions: In this trial, injections of OTC at pH 2 using hydrochloric/muriatic acid reduced fruit drop by an average of 15 pieces of fruit (60% reduction) relative to the Untreated Control and 9 pieces of fruit (48.4% reduction) relative to the OTC pH 7 injection. This was statistically significant at LSD p=0.05.
Table 7 represents a working example on dosing a desired amount of OTC into a tree or vine. Factors that could alter the dosage include overall plant health, i.e., a decline in vascular biomass caused by the pathogen, hedging and pruning, and environmental factors, such as temperature, moisture, and stress.
Generally, the volume of treatment injected would be proportional to a diameter of a trunk of a tree. Trees having vascular networks damaged by disease would be treated at lower concentrations to prevent phytotoxicity. As the trees recover, the concentration of PG-OTC can be increased to mitigate risk of reinfection.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be readily apparent, in light of the teachings of this invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the following claims.
Therefore, the foregoing is considered as illustrative only of the principles of the disclosure. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the disclosure to the exact compositions and methods of use, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the disclosure. In this patent document, the word “comprising” is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. A reference to an element by the indefinite article “a” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be only one of the elements.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17516229 | Nov 2021 | US |
| Child | 18533651 | US |
