Progress 09/27/23 to 09/26/24
Outputs
Target Audience:Preliminary results were disseminated to the ECDRE PIs at the annual Directors' meeting in D.C. in November 2023, and to our project's advisory council, which met in April. Changes/Problems:Hurricane Milton caused severe damage to the trees in our ongoing field trial and caused major fruit drop, so we anticipaste thatPCR data will be the only useful readout from the trial, which is ongoing. What opportunities for training and professional development has the project provided?Three graduate students, one postdoctoral scholar, and two undergraduate researchers have been supported on this project; all have had the opportunity to develop research and communication skills as part of this ongoing effort. How have the results been disseminated to communities of interest?Because this research is still in an early stage, no major dissemination has taken place. Profs. Ristroph and Lowry have discussed the project's goals and preliminary data at academic seminars (~5) in the past year, and Prof. Rolshausen and graduate student Riley Jones have submitted a poster discussing preliminary results from this work to a UC Riverside internal poster session. What do you plan to do during the next reporting period to accomplish the goals?We will complete the field trial; compile and publish the results; disseminate our findings; and apply for followup studies as appropriate.
Impacts
What was accomplished under these goals?
We have successfully developed a suite of 16 organic nanocarrier formulations 70-350nm in diameter and encapsulating streptomycin at 100% encapsulation efficiency. These formulations are stabilized by one of two surface materials: HPMCAS (hydroxypropylmethylcellulosee acetate succinate) or soybean lecithin and demonstrate good stability over time in water onthe time scale of weeks. Some formulations also contain a hydrophobic co-core material that aids in nanocarrier formation and stability over time. Particles were prepared at Purdue and sent to collaborators at UC Riverside and Carnegie Mellon for evaluation. At UC Riverside,an in vitro optical density assay was used to evaluate nanoparticles (NP) efficacy against Liberibacter crescens, a culturable relative of Candidatus Liberibacter Asiaticus (C.Las) the bacterial cause of Huanglongbing disease. This in vitro assay enables to select for the most active NP formulations for field evaluation in Florida. All formulations tested exhibited similar antibacterial efficacy at streptomycin concentrations as low as 0.0015 mg/mL. AtCarnegie Mellon, NP foliar uptake and translocation were measured in citrus saplings by confocal microscopy and induced coupled plasma mass spectrometry. Up to approximately 10% of the applied NPs were found to translocate to the roots, which is a significant improvement over free streptomycin after foliar application. These results, combined with those from UC Riverside, enabled us to select a final formulation for evaluation in the field. A field trial was begun in April 2024. Sufficient amounts of NPs were prepared at Purdue, then driven to Lafe Alfred, FL for the test. Treatments were applied to 70 trees per group for 4 treatments: foliar sprays with (1) NPs, (2) unencapsulated streptomycin, or (3) water; and (4) the current industry standard trunk injection with oxytetracycline, which was contracted by a professional group. This field trial is ongoing. PCR data for CLas titer is being collected. Hurricane Milton caused severe damage to the trees and caused major fruit drop, so we anticipaste that the PCR data will be the only useful readout from the trial, which is continuing. A no-cost extension has been granted for this project through September 2025, at which point the final report will contained full details of all experiments and deliverables.
Publications
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Progress 09/27/22 to 09/26/23
Outputs
Target Audience:Target audience at this stage of the project was primarily faculty and graduate student researchers. The experimental results of this project are not yet at a stage where wider dissemination is appropriate. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two graduate students, one postdoctoral scholar, and two undergraduate researchers have been supported on this project; all have had the opportunity to develop research and communication skills as part of this ongoing effort. How have the results been disseminated to communities of interest?Because this research is still in an early stage, no major dissemination has taken place. Profs. Ristroph and Lowry have discussed the project's goals and preliminary data at academic seminars (~5) in the past year, and Prof. Rolshausen and graduate student Riley Jones have submitted a poster discussing preliminary results from this work to a UC Riverside internal poster session. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period we will conclude translocation studies; decide which of the 16 working streptomycin formulations should be evaluated at the field level based on the translocation results (since all formulations tested exhibit comparable antimicrobial activity); prepare enough material for the field trial, and begin the field trial. We anticipate requiring a no-cost extension on this project in order to conlcude one year of a field trial; if additional years are desired, we will apply for an extension as appropriate.
Impacts
What was accomplished under these goals?
PURDUE: We have successfully developed a suite of 16 organic nanocarrier formulations 70-350nm in diameter and encapsulating streptomycin at 100% encapsulation efficiency. These formulations are stabilized by one of two surface materials: HPMCAS (hydroxypropylmethylcellulosee acetate succinate) or soybean lecithin and demonstrate good stability over time in water on the time scale of weeks. Some formulations also contain a hydrophobic co-core material that aids in nanocarrier formation and stability over time. In parallel, we have developed a corresponding suit of nanocarriers with the same size and surface chemistry as the streptomycin formulations, but with a core consisting of (a) a metalloporphyrin that contains a rare earth metal for tracking NCs by ICP-MS and(b) a flourescent dye molecule that enables NC tracking by confocla fluorescent microscopy. We have supplied our collaborators at CMU and UCR with nanocarrier formulations for further evaluation (see below). CARNEGIE MELLON: To follow the association of NPs to the leaves and to determine the penetration pathways, we imaged citrus leaves upon application of NPs by confocal imaging. We compared the application of NPs with different shells, HPMCAS and Lecithin which were applied on the abaxial side of the leaf in the presence of Silwet 0.1%. The size and Zeta potential (ZP) of the NPs were determined by light scattering and while HPMCAS NPs' size is 219±11 nm and ZP is -15.7 mV, Lecithin NPs' size is 504±44 nm and ZP is -57 mV. Although its bigger size, Lecithin NPs demonstrated a better ability to flood the stomata apertures and to diffuse into the mesophyll air spaces. On the other hand, HPMCAS showed a higher tendency to distribute on the leaf surface. For both cases, we didn't find clear evidence of cell internalization. In addition, In order to follow NPs translocation to different organs of citrus trees, we developed a method for the efficient extraction and analysis of rare metals using ICP-MS. Initial results indicate that for HPMCAS NPs, most of the applied metal can be extracted (>90%) and about 8% of the applied NPs were translocated to the roots; however, these results should be further verified. We are improving the method by reducing the background concentrations which evolved from poly-atomic interferences. That is done by using Palladium as a metal tracer and by reducing the amount of organic materials that are being digested. UC RIVERSIDE: We used an in vitro optical density assay to evaluate nanoparticles (NP) efficacy against Liberibacter crescens, a culturable relative of Candidatus Liberibacter Asiaticus (C.Las) the bacterial cause of Huanglongbing disease. This in vitro assay enables to select for the most active NP formulations before field evaluations in Florida. Optical density assays, or OD assays, are a common microbiology technique. Optical density assays measure the change in absorbance levels of specific treatments in each well of a 96-well plate to determine the effect of these treatments on bacterial growth. An increase in absorbance correlates to an increase in bacterial cell growth, ideal for monitoring slow growing, fastidious bacteria like L. Crescens. Wells with little change in absorbance indicate the presence of a treatment that is inhibitory to bacterial growth and wells with a high change in absorbance indicate a treatment that allows for normal (or almost normal) growth. We have tested 10 streptomycin NP formulations. The formulations differ by the NP coating characteristics. We tested HPMCAS (hydroxy methyl cellulose acetate succinate), which is a synthetic cellulose derivative used in nanoparticles because it causes the particles to have a negative charge (due to the succinate groups) which helps with solubilization, increases stability, and reduces hydrophobic aggregation. We also tested lecithin, a naturallyoccurring component of cell membranes, it's very useful as a nanoparticle coating because it is extremely biocompatible. It is also negatively charged but unlike HPMCAS, it is a phospholipid instead of a polysaccharide. Some other NP formulations differed by the presence of different emulsifiers/surfactants including sodium oleate (OL), sodium dodecyl sulfate (SDS), sodium dodecyl benzene sulfonate (SDBS). Results showed that overall coating and surfactant have not impacted product efficacy with streptomycin concentrations as low as 0.0015 mg/mL. Final minimum inhibitory concentration (MIC) and half maximal effective concentration (EC50) data for these formulations will be included in the final report. These values will give us the smallest concentration necessary to induce an effect on bacterial growth as well as the concentration required to achieve 50 percent inhibition, respectively. These values will help determine the necessary concentrations to be applied in the field. UNIVERSITY OF FLORIDA: UF's portion of the research will start with field experiments in February 2024.
Publications
- Type:
Conference Papers and Presentations
Status:
Under Review
Year Published:
2023
Citation:
R. Jones and P. Rolshausen, poster submitted to UC Riverside Plants 3D NSF Research Traineeship Program poster session
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