Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 2020 - Engineering | 100% |
Foliar delivery of anti-HLB agents is a highly desirable but historically unsuccessful strategy for eliminating Candidatus Liberibacter asiaticus (CLas) in citrus trees. Most agrochemicals, when applied foliarly in the form of a bulk water dispersion, are poorly internalized into leaves and do not transit efficiently to the phloem and root, where CLas resides. The development of a cost-effective and scalable delivery system capable of translocating systemically an agrochemical from the exterior of a leaf to the phloem and root is therefore still needed.This work will develope and validate the efficacy of organic nanocarriers containing anti-CLas agents.
Nanocarriers (NCs) will be formulated by Flash NanoPrecipitation with hydrophobic ion pairing.NC size, surface chemistry, and stability over time will be quantified using dynamic light scattering and electrophoretic mobility measurement.Translocation of organic nanocarriersto phloem, roots, and other plant parts will be quantified usingby ICP-MS using red grapefruit as a model citrus plant.NCs will be foliarly applied to red grapefruit; plant sections will be digested with acid; and lanthanide mass in each section will be quantified using mass spectrometry.Three in vitro bioassays will be used to measure dose response to nanocarrier formulations. Product efficacy will be tested with each assay in three independent experiments with three technical replicates for each formulation.L. crescens will be grown on culture medium and NCs containing known concentrations of bioactive payload will be loaded at a center of the plate in a well. The zone of bacterial growth inhibition around the well will be recorded.Replicated field trials with mature HLB-positive citrus trees will test the efficacy of the anti-CLas NCs during a full season in year 2 to reduce HLB disease symptoms, CLas titer in the leaves, improve tree foliage density, canopy size, fruit set, size, quality and yield. Preharvest fruit drop will be assessed since HLB greatly increases losses.
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.
Training & Professional Development
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.
Dissemination Streams
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.
Next Reporting Steps
We will complete the field trial; compile and publish the results; disseminate our findings; and apply for followup studies as appropriate.
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
Training & Professional Development
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.
Dissemination Streams
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.
Next Reporting Steps
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. <br><br>
<br>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. <br><br><b>Publications</b><br>