Liberibacter crescens has been cultured under laboratory conditions and is considered a model system that can be used to develop a biocontrol for ‘Candidatus Liberibacter asiaticus’ (Las). The focus of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using L. crescens strain BT-1. We have developed an overlay assay for L. crescens and implemented the system to screen for phages, tailocins or antimicrobials active against the model organism. We have incrementally tested a large number of phage lysates, broad host-range tailocins, plant/psyllid extracts, water samples, and induced cultures, with no activity against L. crescens identified to date. While continuing the search for environmental phage(s) active against L. crescens, we have also initiated a recombinant approach to produce active hybrid tailocins. As proof of concept, we have deleted the tail fibers of a broad host range tailocin and complemented the deletion in trans to obtain active tailocins, in induced lysates, at concentrations similar to that of wild type. Our goal is to clone putative identified tail fibers genes from prophages in L. crescens strain BT-1 to obtain tailocins active against L. crescens BT-1. Identification of the proper protein junction will require an array of constructs. Once this system is optimized for L. crescens BT-1, the next step will be to complement with tail fibers from prophage identified in the Las genome. Previously we reported antimicrobial activity of several Actinomycetales against L. crescens. While we have been able to detect antimicrobial activity on solid medium, optimal conditions for production in liquid in quantities needed for structural analysis have not been achieved. Chromatographic analysis of organic extractions of Microbacterium strain TM 313 supernatants revealed three major components. Structural characterization of the active component(s) is an ongoing in collaboration with another group.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. Three compounds exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control, where the level of biofilm formation were reduced to 50% and 60% of control, respectively. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected Xac 306 infection of unwounded and wounded citrus fruits at sub-inhibitory concentrations. We have completed testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. In the NB medium, planktonic cells exhibited a MIC of 0.50 mM CuSO4 without biofilm inhibitor. In the presence biofilm inhibitors at sub-MIC concentrations , the MICs of CuSO4 against Xac 306 planktonic cells were decreased to 0.25 mM. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. The results have been published by Phytopathology in a manuscript entitled: Foliar application of biofilm formation-inhibiting compounds enhances control of citrus canker caused by Xanthomonas citri subsp. citri. One patent is filed based on the results. We also identified multiple new biofilm inhibitors. The effect of those biofilm inhibitors to control citrus canker is being investigated. We tested the survival of both biofilm deficient and QS mutants on fruit surface. Effects of biofilm formation inhibitors on Xac infection on detached immature citrus fruit were tested using spray inoculation. The inhibitors affected the infection of Xac on both unwounded and wounded citrus fruits. We are testing more potential biofilm inhibitors. We continue characterizing how quorum sensing and biofilm formation contribute to Xac infection of citrus fruit. Multiple virulence genes involved in quorum sensing and biofilm formation are being investigated. The involvement of ColR, RpfF, and three more genes involved in biofilm formation or quorum sensing is studied in details. Two manuscripts are under preparation. The field trial (two different sites) is ongoing to test the effect of the identified biofilm inhibitors to control citrus canker. One new compound is able to inhibit QS at a concentration of 100 .M based on observation of bacterial phenotype of aggregate formation. Plant test in greenhouse showed that the QS inhibitor (100 .M) treatment could reduce the formation of canker lesions and bacterial population on spray-inoculated grapefruit leaves simultaneously with the canker bacterium Xcc 306. Six applications were conducted by foliar spray every 3 weeks. The disease incidence survey has been conducted once in June.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). Currently, sprays with copper bactericides are the primary mean in controlling citrus canker, which causes adverse effects on the environment by contaminating ground water or accumulating in the soil and affecting tree health. The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene via its 17.5 tandem repeats, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. TALEN targeting the promoter of CsLOB1 is also being done using citrus protoplast.
The goal of the research is to control citrus HLB using small molecules which target essential proteins of Candidatus Liberibacter asiaticus (Las). In our previous study, structure-based virtual screening has been used successfully to identify five lead antimicrobial compounds against Las by targeting SecA. SecA is one essential component of the Sec machinery. Those compounds showed promising antimicrobial activity. However, further work is needed to apply the compounds. We will evaluate the important characteristics of our antimicrobial compounds including solvents and adjuvants, phytotoxicity, antimicrobial activities against multiple Rhizobia, antimicrobial activity against Las, application approaches, and control of HLB. Those information are critical to for the practical application of those antimicrobial compounds in controlling HLB. We also propose to further optimize the five lead compounds. In addition, we propose to develop antimicrobial compounds against lipid A of Las. The lipid A substructure of the lipopolysaccharides (LPS) of Sinorhizobium meliloti, which is closely related to Las, suppresses the plant defense response. Las contains the complete genetic pathway for synthesis of lipid A. We hypothesized that Las uses lipid A to suppress plant defense. Thus, targeting lipid A could activate plant defense response. Lipid A is also an ideal target and has been targeted for screening antimicrobial compounds for multiple pathogenic bacteria. We have identified multiple small molecular ‘or’ peptide inhibitors against LipidA using pharmacophore based methods and are finalizing the list of the compounds for the activity studies. For SecA inhibitors, we are optimizing the compounds in collaboration with IBM. Two compounds with slightly higher binding affinity than C16 were identified. We also identified multiple SecA inhibitors. We are testing their inhibitory effect against purified SecA right now. Currently, we are evaluating the best range of composition ratio among each component (%weight) of AIs, solvents and surfactants. The following characteristics are being evaluated: 1) emulsion stability and ease of emulsion; 2) stability of diluted concentrate; 3) freeze-thaw stability; and 4) phytotoxicity to citrus species. We have successfully identified one formulation suitable for all five compounds without phytotoxicity. Using the formulation, we have tested all five compounds against eight different bacterial species including Liberibacter crescens. Field test is being conducted. For the field trial, 120 trees of four years old were selected. HLB disease severity was suveyed. The Las titers were measured for each tree using qPCR. We have compared spray and trunk injection. Trunk injection seems to have better efficacy. We have synthesized one compound at large amount for field trial. The field trial is ongoing.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates was tested using citrus (grapefruit) seedlings in greenhouse. Greenhouse assays suggested that a consortium of three Bacillus and relative isolates (AY16, PT6 and PT26A) may delay the development of both HLB symptoms and pathogen population on citrus leaves after root inoculation. The potential of the consortium to recover the tree decline from HLB infection is being evaluated in greenhouse. The growth conditions of the three strains were optimized using a small fermenter. Three antifoam agents, A204, PPG200 or M-Oil did not affect the growth of the three bacterial strains. The initial neutral to alkaline pH values (7.0 ~ 8.0) favor growth of the three bacteria in LB, while acidic pH (5.0 ~ 6.0) suppress bacterial growth. The optimal cultural temperature was determined to be around 30C with average bacterial population of 109-1010 cfu/ml after 20-hour incubation, although the bacteria may grow slowly under room temperature (~ 23C). The shelf life of three different formulations of the bacterial culture is being evaluated under room temperature. In a six-month time course, the bacterial populations in LB broth, OPB broth and tape water are comparatively stable with initial and final both at ~ 108cfu/ml. Under room temperature, after a 20 month storage, the population reduced about 10E5 to 10E6 folds. Four field trials are being conducted including more beneficial bacteria. For one of the field trial, nine applications have been performed. We are evaluating the survival of the beneficial bacteria in the soil. The application method has been changed during application to improve the survival of microbes in the soil. We are developing new methods to further increase the survival of microbes in the soil and allow the microbes have easy access to the roots. We have completed 3 surveys of HLB disease severity and collected leaf samples for Las population analysis using PCR. We are also testing the colonization and survival of the tested microbes on citrus roots and in rhizosphere soil in greenhouse using conventional culture-based method and qPCR-based method. A population of 10E4 to 10E5 cfu/g root and 10E3 to 10E4 cfu/g of soil at one month was observed. We are evaluating plant immunity response by studying the expression of specific genes related to plant defense.
The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated and treatments already started. The follow up investigations are ongoing, including monitoring the HLB symptoms, disease incidence and Las titer in leaves. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We have applied different compounds at three separate field trials. We conducted disease severity survey in Sep. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. We are also testing the mechanism of those compounds showing positive effect on HLB control. Our results indicated that those compounds upregulated defense related genes, e.g., PR2, but not PP2 an callose synthase genes, in both field and greenhouse. Las titers were also lower in treated plants than non-treated controls with the four compounds. We have investigated the effect of those compounds on disease severity, yield, juice content and quality. We will repeat those treatments for one more year. Currently, the treatments are being conducted. SA hydroxylase is being expressed and purified. Multiple SA hydroxylase inhibitors have been identified using molecular modeling. The inhibitory effect of the SA hydroxylase inhibitors have been tested using the purified SA hydroxylase. Three compounds showed inhibitory effect against SA hydroxylase. The three SA hydroxylase inhibitors will be used to control HLB in greenhouse or field trials. For the putative sec dependent effectors, we have identified some putative targets using yeast two hybrid assays. We are currently confirming the protein-protein interactions using immunoprecipitation method.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We will mainly test the antagonistic effect of Bacillus, Paenibacillus, Streptomyces and Pseudomonas strains Agrobacterium tumefaciens, Sinorhizobium meliloti, and L. cresens; Control HLB using antibacterial-producing bacteria. For the field test, we will investigate how antibacterial-producing bacteria affect HLB disease severity, Las titres, and citrus yield, survival of the antibacterial-producing bacteria in the rhizosphere and expression of the antibacterial biosynthesis genes in vivo. We have isolated Streptomyces spp. Bacillus spp. Paenibacillus spp., and Pseudomonas spp. from Florida groves. Multiple isolates showed antimicrobial production activity. We tested 27 antibacterial compound producing bacteria. These strains had been recovered, purified and confirmed by 16S rDNA sequencing. The antagonistic activity against Agrobacterium, Sionrhizobium meliloti and Xanthomonas citri pv. citri was determined. 5 strains, belonging to Paenibacillus, Burkholderia, Paenibacillus, Streptomyces and Streptomyces showed good antagonistic activity. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi).
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (July 1 to September 30, 2014), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Plant Improvement Team o Site prep continues for new planting of rootstock and scion cultivars being evaluated for HLB tolerance/resistance o Preparation for NVDMC trees ‘ Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance o Continue evaluating two new Dow herbicide trials ‘ Applications of the ‘Boyd Program’, Keyplex and Ben Hill Griffin programs continued in the ‘commercial scale’ nutritional demonstration. ‘ Conducting fall fertilizer and pest management programs for the groves o Herbicide program on schedule o Psyllid management continued . Participated in coordinated area wide spray in early August o Mechanical weed/middle management evaluation continues o Mechanical vine removal ‘ Continue to maintain Dr. Bowman’s new USDA rootstock plantings ‘ Commercial Trials: o Eurofins evaluations on disease and insect management continue o Keyplex nutritional trial evaluations continue . Initiated soil applied nutritional program evaluating the Roots product o DuPont demonstration for row middles management with Matrix Herbicide continues o Syntech evaluations on insect management and disease management . ACP . Greasy spot and melanose o Florida Ag Solutions continue to make applications to herbicide and insecticide evaluations . Initiated new evaluation in Navel block ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas
The purpose of the additional funding for this project was to collect additional data from growers so that the efficacy of the enhanced nutrient programs could be assessed more fully after some promising results were obtained from the limited data collected last year. After extensive effort, some data were collected from three growers. However, none of the data sets was complete enough for analysis, and efforts to obtain the additional information failed. Consequently, we will not be able to complete the goals of this project. Because all involved (growers and researchers) were optimistic at the start of the study, some funds were spent so that graduate students would be prepared to conduct the analyses. However, when it became evident that data would not be forthcoming, expenditures stopped. We believe that the growers had full intentions of supplying the data. However, recent events, such as extensive premature fruit drop, have been devastating to the industry, and it is understandable that the growers no longer have the time or energy to help with this project.
The goal of this project is first to identify a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian citrus psyllid (ACP). The toxicity of the selected toxin will then be enhanced by addition of a peptide that binds to the gut of ACP. This modification of the toxin is expected to enhance both binding and toxicity against ACP. The proteolytic profile of 25 Bt isolates have been characterized at Iowa State University (ISU) by Teresa Fernandez-Luna so far. Toxin samples provided by Teddi Mitchell and Mike Blackburn, USDA ARS, MD were solubilized (sodium carbonate pH 10.5, 10 mM DTT, lysozyme 200 mg per ml) for 3 to 16 hours at 37’C, according to the solubility of each strain. Samples are then dialyzed for 22 hours against 50 mM Tris-Cl pH 8.5. To determine the proteolytic profile and protein stability, 10 ug of each sample was incubated with bovine trypsin at a final concentration of 10% at 37 ‘C for 1 or 2 hours. Finally samples were activated for 1 hour to obtain at least 2 mg per sample for ACP toxicity assays conducted by USDA ARS, FL. Trypsin was removed from samples using Benzamidine sepharose with a 30 min incubation at room temperature and removal of the sepharose by centrifugation. Eight groups of Bt strains were identified based on the number of protein bands (between 1 and 8) seen on SDS-PAGE following toxin activation. ACP bioassays were conducted in the lab of David Hall at USDA ARS, FL to test 13 Bt isolates for the presence of ACP-active toxins. Two of the 13 isolates showed toxicity against ACP, and an additional four isolates identified with potential activity against ACP. One of the toxic strains was selected for purification of individual toxins by ion exchange and gel filtration column purification at ISU. These purified toxins will then be tested individually against ACP. A series of experiments is underway at ISU to standardize procedures for screening of a phage display library for identification of an ACP gut-binding peptide. Following confirmation of binding of the selected peptide to the ACP gut, this peptide will be used for Bt toxin modification.
At the request of the CRB, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes the Morse lab’s recent citrus research under this coordinated project (all arthropod research except ACP which is a separate project, i.e. 5500-189). Our major efforts this last year have focused on helping the industry deal with Fuller rose beetle (FRB) in relation to citrus exports to Korea. For the last several years, Korea has put increasing pressure on the CA industry to reduce egg mass levels on export fruit and there is a concern that the situation will become quite severe in 2014-15. To better advise growers and pest control advisors what can be done, 10 industry presentations on Fuller rose beetle have been made over the last year (since 10-1-13) and a Korea pre-season export meeting will be held in Tulare 9-16-14. Grafton-Cardwell and Morse have also updated Fuller rose beetle pest management guidelines recently to reflect all recent information. In addition, statistical analyses were recently completed to relate egg mass infestation levels of fruit with the probability of passing phytosanitary inspection given the number of fruit that are inspected. These data have been presented to industry and will be discussed at upcoming conference calls. 2013 field FRB control trials were run at Lindcove by Grafton-Cardwell (15 treatments evaluated) and in Pauma Valley by Morse (10 treatments evaluated). None of the treatments results in 100% control but it appears that 2 applications are relatively effective — either 2 ground sprays of bifenthrin, 2 trunk sprays of bifenthrin, or 2 foliar sprays of combinations of carbaryl, cryolite, and/or thiamethoxam. Based on these studies, two new field trials were set up in 2014 at the same field sites as in 2013 (at Lindcove, the same trees were used so as to look at FRB reduction after 2 years of treatments; at Pauma, we selected new trees to obtain high resolution between treatment efficacy). Citrus thrips resistance to Delegate has been confirmed on citrus in the San Joaquin Valley and more recently, we have investigated several reports of high levels of citrus thrips resistance to both Delegate and Success on blueberries in Kern and Tulare Co. Our concern is that citrus thrips from blueberries might be selected with these products in late summer and fall and then fly into citrus with further selection in spring. MGK recently released an organic formulation of Veratran D for citrus thrips control. A lab trial showed that this new formulation is just as effective, if not more so, against citrus thrips as the old non-organic formulation produced by Dunhill (unopened bag from 2008). In addition, it was confirmed that acidification of the spray tank to pH 4.5 before adding the Veratran D improves performance. Growers now have two effective organic options for citrus thrips control – Entrust SC + oil and Veratran D. We are continuing to examine contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, we have processed 41 such loads with a diversity of commercial varieties examined. We plan to continue this work through Sept. 2015.
A heat treatment machine using steam as a source of supplemental heat was developed. The system is able to heat treat single citrus trees up to the height of six feet one tree at a time. The system consists of a hydraulically controlled enclosure which descends to cover the entire canopy. A steam generator, together with a water tank was mounted on the bed of a ‘goat’ truck. Steam from the steam generator is streamed into the enclosure. The system was used to test steaming scenarios using multiple combinations of temperature and heating duration. More than 800 trees were heat treated in five different groves. From the experiment, we determined that applying steam at temperatures above 60 C (140 F) are likely to cause damage to the trees. When testing treatments with a temperature of 65 C (149 F), we discovered that in most cases the trees did not recover. There were two scenarios which we tested using the steaming system and they both seemed to work equally well. The first one was steam treating the tree at 55 C (131 F) for 3 minutes and the second was steam treating the tree canopy up to either 60 C (140 F) or 30 seconds after reaching a temperature of 55 C (131 F), whichever came first. These two scenarios were selected after several trial runs of different temperature and heating duration combinations. From the trial runs, these two scenarios seem to provide sufficient heat without damaging trees or causing significant leaf lost which delays the recovery of the trees. The tested trees are still under observation and the effectiveness of the treatment will be studied over a longer period to evaluate the performance of heat treatment. At this time, three, six, and nine months post heat treatment samples have been collected from HLB-positive Valencia at three grove locations. Samples have been processed and analyzed for Las bacteria. In addition, fruit harvest for the 2013-2014 growing year has been determined for each tree in the study. Fruit set and first fruit abscission for the 2014-2015 year have also been counted. These counts will be compared to fruit counts of heat treated and control trees of the harvest in spring 2015. Initial results show that four-year-old Valencia trees responded better (increased canopy growth, decreased Las bacterial titer) than 15-year-old Valencia. Additionally, the treated HLB positive four-year-old Valencia located in a well-managed commercial grow (excellent fertilizer and insect management program) visually outperformed the other block of Valencia located in a grove with a weaker management program. In depth statistics of the three randomized block design experiments will be performed after one year samples are taken (August-September 2014).
The overall objective of this study was to deliver antibiotics into HLB infected citrus plants using nanoparticulate carrier systems such as emulsions, liposomes and polymer nanoparticles. During initial proof of concept experiments, dye-doped liposome and polymer nanoparticles encapsulating water-soluble dyes as surrogate for antibiotics have been prepared. In addition, double emulsions of water-in oil-in water have been developed with a stable inner aqueous phase for the delivery of the water-soluble antimicrobials to plants. Liposomes particles have been prepared using e.g 1,2-dioleoyl-3-trimethylammonium-propane , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol through the thin lipid film hydration, reverse phase evaporation and ethanol injection methods. Constituent lipids amounts were varied to encapsulate water-soluble dyes such as 9-aminoacridine, sulforhodamine B and fluorescein. In order to minimize the leaching of the dyes, cationic lipids were incorporated in the lipid composition. The particle size was restricted to less than 100nm by the use of appropriate filtration membrane and the choice of hydrating solvent. Cryo transmission electron microscopy of Liposomes prepared via thin film hydration (before extrusion) demonstrated particle size to be around 200nm. Dye-doped polymeric nanoparticles comprising of environmentally safe and biodegradable materials such as poly (lactic acid) (PLA) and encapsulating fluorescent dyes have been prepared using double emulsion method. Polymer particles were characterized for their particles size distribution, absorbance, and fluorescence properties. PLA particles mixed with adjuvants were sprayed on abaxial and adaxial sides of citrus leaves and analyzed for leaf uptake by IVIS spectrum imaging system. Whole leaf imaging allowed detection of the uptaken particles by leaves. Confocal experiments on select leaves showed the presence of the particles in the leaf surface. Future experiments will be planned to determine the particle and adjuvant conditions for increasing the uptake. In addition, double emulsion particles, i.e. water-in-oil-in-water or w/o/w were also developed. These emulsions contain an encapsulated aqueous phase suspended in bulk water were stabilized by varying the surfactants and relative ratio of the amounts of the internal and external aqueous phases. Emulsions containing oxytetracycline and dye in the internal aqueous phase were developed to be used for future experiments. Highlights: In these experimental trials three types of particles were developed for the delivery of water soluble compounds (antimicrobial agents) to citrus plants. ‘ Liposomes encapsulating water soluble dyes in the size range 100-200nm have been prepared and characterized. ‘ Polymer particles encapsulating dyes were prepared using safe and biodegradable polymers. The dye-doped polymer particles were applied to citrus plants and imaged after incubation. Whole leaf imaging and confocal imaging indicated the presence of the fluorescence particles near the surface. ‘ Double emulsions systems containing water soluble compounds in the internal phase were developed and analyzed using optical imagining. Emulsions exhibiting stability for about three weeks and encapsulating active ingredient were prepared for future studies.
Construction of the imaging system: An optical sensing system based on a modified DSLR camera is now largely constructed and is able to acquire images of isolated leaves and small citrus seedlings. At present, the device can take photos of fluorescent emissions at the following excitation and emission wave lengths: UV LED-364nm (emission filters: 500nm, 690nm and 730nm; Blue LED-470nm (emission filters: 500nm, 690nm and 730nm); Green LED-527nm (emission filters: 690nm and 730nm). In addition, the camera system can take images based on the optical rotation of polarized light by starch grains. Image analysis: Images of seedlings have been acquired and manipulated by focus stacking to produce a high resolution macro-image at visible wavelengths. The focus stacking approach will be applied to fluorescence and polarized images. In the initial stages, images are being taken of individual leaves from HLP-infected and non-infected sweet orange trees. Fluorescence images are combined as ratios to identify regions of leaves that show chlorophyll loss. The Optical rotation images are being used to map the location of starch accumulation within infected leaves. Psyllid-inoculation of citrus seedlings: Sweet orange seedlings are being germinated and are ready to be exposed to feeding by HLB-positive psyllids. However, these experiments have been temporarily delayed due to low population numbers in the infected psyllid colony. It is anticipated that psyllid feeding studies will be initiated within the next reporting period.
The project entitled ‘Further characterization of HLB resistant clones of selected citrus varieties’ (project no. 758) is in its second year. The aim of the project is to test plants that have exhibited tolerance/resistance to HLB in a previous field trial. After this confirmation, we want to conduct experiments to understand the basis of HLB tolerance. Research conducted this quarter (from April 2014 to June 2014) consisted of: a) As a part of the greenhouse evaluation trial, we have already challenged 66 selected plants by feeding them with Liberibacter positive Diaphorina citri in a no-choice situation. We are testing DNA extracts made from these plants at various time intervals to measure the Liberibacter titer in all the plants. We have also been collecting samples and extracting them to prepare RNA for future use in conducting RNAseq experiments designed to see differences between susceptible and resistant cultivars. b) During the spring of 2014, we conducted over 1000 pollinations using HLB susceptible and resistant cultivars belonging to the genera Citrus, Eremocitrus, Microicitrus and Poncirus. We have collected seeds from 2013 pollinations and we are testing them for the presence of genetic material from the pollen and seed parents. c) At this time we are in the process of psyllid challenge of another set of 66 plants in Fort Pierce. These plants were also selected based on the results of the previous field trial. We are on track as per our proposed milestones.
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