In this reporting period, nine different T-SOL variants made with agriculture-grade chemicals were included for plant uptake studies. Citrus seedlings (Cleopatra sp.) were used as model plants. The T-SOL variants involved three different Zn chelating agents and three different concentrations of a plant surface permeability enhancer (1, 0.5 and 1M with respect to metallic Zn). Citrus seedlings were treated for 24hours with 800ppm dose of different T-SOL variants. After 24hours of treatment whole seedlings were washed thoroughly with 5L of deionized water to clean the additional materials sticking to plant surface. Whole plant samples were then oven dried for 5days at 60 C. The treated plant samples were then ground to fine powder in a dry blender (Cuisinart, Model SG-10). 0.5gms of the dried samples were then digested in 20mLs of aqua regia solution to find out the zinc uptake by plants. Zinc uptake varied from 0-0.8mg/gram dry weight for different treatments compared to untreated control samples (0.09mg Zn/ gram dry weight). Maximum uptake of up to 0.8mg Zn/gram dry weight of plant sample was observed. It was observed that plant micronutrient based Zn chelating agent exhibited maximum uptake. In the coming reporting period, plant uptake studies will be conducted to determine the uptake and translocation of T-SOL inside different plant tissue which can help further to select the most effective T-SOL variant prepared with agri-grade chemicals that can be later used in green house and field trial studies.
The program research objectives are to develop an effective and sustainable bacteriophage (phage)-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. Our approach has been to develop a bank of virulent (lytic) phages and/or antibacterial particles called tailocins , which are derived from phages. We have identified seven tailocins with activity against Xac and developed a large bank of virulent phages representative of the Caudovirales (tailed phages). The tailocins are protein assemblages that function like phage tails and kill the target bacterial cell by adsorbing and puncturing the cell envelope. In two independent experiments, a cocktail composed of tailocins XT-1 and XT-4 showed efficacy in reducing canker symptoms, when applied as a foliar spray, post application of Xac. Current efforts are directed towards isolating additional tailocin producing strains that are active against Xac. As reported previously, we are also focused on completing the characterization of Xac phages. We have determined that the burst size for CCP504, a virulent KMV-like phage, is ~70 PFU/cell and that the burst size for CCP513, a siphophage, is ~80 PFU/ cell. Abortive lysogeny tests are ongoing to reconfirm virulent status of cocktail phages. Further characterization of the two non-type IV pilus dependent phages, previously reported, determined that both phages had limited host ranges and would not be candidates for development of phage cocktails.
April 2016 The objectives for this proposal are1) Conduct ground and aerial applications of fungicides to determine the efficacy and economics of fungicide treatments; 2) Determine if Luna Sensation has enough systemic activity to protect flowers from before they fully develop and open; 3) Determine if the period flowering of trees affected by huanglongbing can be narrowed to eliminate the offseason bloom that contributes to the PFD inoculum increase in groves. The project officially started March 1st, 2016 but site selection and plot layout was initiated prior to the start of the project because PFD was beginning to affect the blocks we were planning to use. Four trials were initiated. They were in the Ona, Polk City, and Fort Meade areas. Four weekly applications were made in March by air and ground in Ona. Two applications were made in Polk City as that only was predicted to be needed by the PFD-FAD prediction system prior to the flowering period finishing. Three applications were made in Fort Meade where the bloom was more attenuated than Polk City. It is too early to start taking disease data yet and so no economic analysis has begun. The field trials for the plant growth regulators are in the planning stage.
The objective of this research will 1) characterize Pr-D (FP3) and its role and disease suppression; 2) investigate the dynamics of the prophages/phages in Las bacteria by revealing the variations in gene expression and recombination; and 3) identify critical elements, such as heat and chemical stress that facilitates lytic activities of the prophages. In addition, we will demonstrate whether or not if the ‘cross protection’ using mild strains of Las bacteria will work for the HLB pathosystem along with quantitative detection protocols for prophage-based strain differentiation. We harvested various Las-infected citrus and periwinkle samples showing symptoms ranging from mild to severe, and used for isolation and enrichment of prophage/phage apart from plant and bacterial host materials by differential centrifugation, PEG precipitation, and CsCl density fractionation. Absolute and relative amounts of prophage/phage and Las bacteria existed in total DNA and in various fractions have been evaluated by using specific LJ900 primers targeting the repeat sequence in the LasAI gene located within a prophage region of the Las genome and 16S rDNA primers for detecting Las genomic DNA. We developed a protocol that could isolate and enrich lytic phages from fresh plant tissues. The Las phages were enriched in certain fractions of the purification process, particularly in PEG precipitated pellet and certain ClCs fractions, consistent with the biochemical and biophysical property of free phages. For further confirmation, these fractions were directly examined by electron microscopy, and we were able to find some particles consisting of head-tail fiber structure typical of small bacteriophages, with diameter of the heads varies from 50-70 nm and the length and the width of the tails varies from 140-190 nm and 10-20 nm respectively. We have establish a digital PCR (dPCR) system for accurate quantification of HLB and Las prophage/phage. After optimization, we are able to detect as low as 1-2 copy numbers of targeted DNA molecule/.L sample. To further improve the quantification accuracy of absolute and relative amounts of phages and the bacterium, we designed new sets of primers and probes targeting only single copy genes. Based on Las genome analysis, we targeted gene 05560 in the prophage region, .-operon gene in the Las genome, and a CitLGT gene (limonoid UDP-glucosyltransferase-like) in the citrus genome as an internal measurement reference. The specificity and sensitivity of the new primers is currently being tested and we are optimizing an dPCR-based assay for accurate measurement of lytic phage activities in Las-infected materials. Duan Lab to detect bacterial transcripts in mixed eukaryotic/ prokaryotic samples at set time points throughout a typical course of thermotherapy treatment. Overall, the analysis revealed that, depending upon the time at which the samples was taken, between 4% and 9% of the total predicted genes for Las appear to be differentially regulated during the thermotherapy process compared to a sample taken at time zero. These genes provide initial evidence of how the bacteria itself is modifying its transcriptional activity in response to the increase in temperature. Although a majority of the regulated genes found are defined as hypothetical, several do have a predicted function and their contributions to the effects of heat therapy are now under investigation. Their regulation has now been confirmed and transcripts are being quantified via real-time PCR. Additional plants have also been stressed with heat for verification purposes and to ensure the accuracy of the RNA-seq results. Identification of these genes is leading the way towards deciphering the molecular mechanisms behind thermotherapy in an effort to find alternative methods of achieving the same reduction in Las titer as seen with thermotherapy that will work in the roots as well as the foliage. Construction of a transcriptional reporter system is also currently in progress for the final verification of the genes identified as being involved in stress response to heat in plants subjected to thermotherapy. This system will also allow future experimentation to rapidly identify other catalysts that can produce the same reduction in bacterial numbers as thermotherapy. Purification of the ~10Kb FP3 region has been achieved from both periwinkle and citrus, though the amount purified from citrus appears to be less than that from periwinkle (as would be expected from the lower bacterial titer found in citrus). This should allow the region to be sequenced in its entirety and comparisons made to help characterize the role of FP3 found in citrus vs periwinkle.
Tissue cultivation Tissue culture plants started on 12-14-2015 were subjected to the same 4 durations of relative humidity (RH; 2,3,4,5) as previously. This group also included 3 reps of each cultivar hardened-off on heated benches (80 F). On 1 Feb. these plants were harvested for root and shoot growth. Citrus cultivars consisted of C-35, C-54 and sour orange. The next set of tissue culture plants for hardening were started on 9 March 2016. These will be harvested in late April. This set also includes 3 replications on 80 F heated benches. This set includes Kuharske, Swingle, C-35, sour orange and US 812. For the most part, survival rates have remained high on most cultivars, with perhaps more growth under the shorter hardening-off periods. Stem cuttings Kurharske cuttings stuck in the propagation benches under mist produced using the same protocols as in the past produced no roots or shoots when stuck on 1-4-16, nor when stuck on 1-12-16. This was independent whether there was bottom heat or not. In contrast, cutting similarly stuck on 2-5-16 once again began to produce some roots over the 6 week rooting period that ended on 3-28-16. Continuing in the sequence, cuttings of Kurharske stuck on 2-23-16 produced roots when harvested on 3-29-16. Cuttings of both X639 and C-54 were also stuck with this group of Kurharske. They also produced roots. These plants were harvested, but dry weights have not been measured. The last set of cuttings were stuck on 3-18-16. These consisted of Kurharske, X639, C-35 and US812. In late March, all available cuttings of the HLB resistant trees in Citra were taken and brought to the citrus propagation bay at MREC Apopka. These were treated with an auxin blend of 4000 ppm auxin and stuck in seedling trays.
This research project aims at developing Fixed-Quat as an alternative to Cu biocides. While Quat compounds are powerful antimicrobial agents they are not known to be used directly on plants because of potential toxicity to plant tissue. However, combining Quat with other inactive ingredients such as our silica gel delivery makes it safer (non-phytotoxic) thus producing Fixed-Quat. In the previous reporting period, a new nanoformulation, Fixed-Quat E nanogel was synthesized with a quat concentration of 13,500 ppm ( For Food Use Quat used). In this reporting period, Fixed-Quat A II and Fixed-Quat E were further optimized to a quat concentration of 20,000 ppm ( For Food Use Quat used). FTIR was used to confirm silica and Quat interaction of the concentrated formulation and peak shifts similar to previous batches were observed. The Fixed-Quat A-II and E nanogel s safety was tested by phytotoxicity studies carried out in a Panasonic Environmental Test Chamber (Model MLR- 352H) which allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate summer weather conditions (biocide application season). Studies conducted on Sour orange, a common citrus variety and Roma Tomato sp, an ornamental plant revealed no sign of plant injury when tested with Quat concentration as high as 1000 ppm. It is noted that EPA maximum concentration for Quat industrial use (as surfactant/flocculating agent) is 200 ppm, indicating a large therapeutic window. Antimicrobial studies of optimized Fixed-Quat E nanogel was conducted against several model bacterial species, Xanthomonas alfalfae subsp. citrumelonis (Citrus Canker Surrogate), Pseudomonas syringae pv syringae, a gram negative causative agent of bacterial speck in citrus and tomato sp and Clavibacter michiganensis subsp michiganensis, a gram positive causative agent of canker and systemic infections in tomato. Studies were conducted to determine the Minimum Inhibitory Concentration (MIC) and compared against Kocide 3000 and copper sulfate. MICs of Fixed-Quat A-II and E were found to be = 1.0 g/mL for X. alfalfae, = 1.0 g/mL for P. syringae and = 1.0 g/mL for C. michiganensis. MIC results indicate no loss in efficacy when combined with different silica sources, thus displaying strong potential for commercial usage. Optimized versions of Fixed-Quat A-II and Fixed-Quat E are currently undergoing Citrus Canker trials on Ruby Ray grapefruit in Vero Beach, Florida.
In this reporting period, nine different variants were included for the optimization of T-SOL with agriculture-grade chemicals. These variants involved three different Zn chelating agents and three different concentrations of a plant surface permeability enhancer (with respect to metallic Zn). Interaction of metal chelating agents with Zn was characterized by UV-Vis and FT-IR spectroscopy suggested binding of metal ions with the chelate functional groups (such as carboxyls, hydroxyls and amines) which resembles the data collected with lab-grade chemicals. Microplate Alamar blue assay was used to determine the minimal inhibitory concentration (MIC) of the different variants of the T-SOL. Antimicrobial properties of the newly made T-SOL samples (from agri-grade chemicals) were assessed by running a Minimal Inhibitory concentration (MIC) assay as delineated by Clinical Laboratory and Standards Institute. The MIC values of the three variants of T-SOL containing chelating agent- (1, 0.5 and 0.1) were 75 ppm against Escherichia coli, whereas the MIC values for the T-SOL variants containing chelating agents 2 and 3 was 150 ppm for E. coli. But, the MIC values for all the nine variants were 75 ppm when treated against Xanthomonas alfalfae, values similar to the reagent grade chemicals. Phytotoxicity study was conducted with citrus plants (Sp. Cleo) at a field spray (800ppm) rate with different T-SOL variants. No toxicity was observed for all the treated variants. TSOL materials were tested for their efficacy in protecting a citrus variety in the Vero Beach, Indian River County area of Florida. Six different materials were tested using 3 different capping agents and with /without a surface modifying agent. Materials were sprayed on 8 yr.-old ‘Ray Ruby’ grapefruit trees at an application rate of 0.5 lbs/ acre (750-800 ppm) every 21 days from April to September. Materials were compared against standard Cu and/or (Cu+Zn) commercial products. The untreated control displayed a total infection incidence of 60 % while T-SOL materials reduced the infection incidence to 17-24 %. T-SOL materials performed better to commercial products which displayed comparable protection. The two best performing T-SOL variants will be delivered this month for canker and HLB field trial. In the coming reporting period, plant uptake studies will be conducted to determine the uptake and translocation of TSOL inside different plant tissue which can help further to select the most effective T-SOL variant prepared with agri-grade chemicals that can be later used in green house and field trial studies.
This research project aims to develop an alternative to Cu biocides in the form of Quaternary ammonium compounds (Quat) as an antimicrobial agent. While Quat compounds are powerful antimicrobial agents they are not known to be used directly on plants because of potential toxicity to plant tissue. However, combining Quat with other inactive ingredients such as our silica gel delivery makes it safer by producing Fixed-Quat. In the previous reporting period, a new nanoformulation, Fixed-Quat E nanogel was synthesized with a quat concentration of 9,000 ppm (“For Food Use” Quat used). The formulation composition and interactions between the components (silica and Quat) was confirmed using Fourier Transform Infrared Spectroscopy (FTIR), with Si-O stretching and SiO-H stretching confirming the presence of silica. The Si-O stretching frequency changed from 1039 cm (-1) to 1031 cm (-1) whereas the SiO-H frequency changed from 3391 cm (-1) to 3388 cm (-1), suggesting interaction of positively charged Quat with the silica gel. The Quat N-H bending frequency changed from 1468 cm (-1) to 1431 cm (-1). This change also supports the interaction of Quat with silica gel. In this reporting period, Fixed-Quat E was optimized to a quat concentration of 13,500 ppm (“For Food Use” Quat used). FTIR was used to confirm silica and Quat interaction of the concentrated formulation and similar peak shifts were observed. The Fixed-Quat E nanogel’s safety was tested by phytotoxicity studies carried out in a Panasonic Environmental Test Chamber (Model MLR- 352H) which allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate summer weather conditions (biocide application season). Studies conducted on Cleopatra orange, a common citrus variety and Tomato sp, an ornamental plant revealed no sign of plant injury when tested with Quat concentration as high as 1000 ppm. It is noted that EPA maximum concentration for Quat industrial use (as surfactant/flocculating agent) is 200 ppm, indicating a large therapeutic window. Antimicrobial studies of optimized Fixed-Quat E nanogel was conducted against several model bacterial species, Xanthomonas alfalfae subsp. citrumelonis (Citrus Canker Surrogate), Pseudomonas syringae pv syringae, a gram negative causative agent of bacterial speck in citrus and tomato sp and Clavibacter michiganensis subsp michiganensis, a gram positive causative agent of canker and systemic infections in tomato. Studies were conducted to determine the Minimum Inhibitory Concentration (MIC) and compared against Kocide 3000 and copper sulfate. MICs of Fixed-Quat E were found to be = 1.0 ug/mL for X. alfalfae, = 1.0 ug/mL for P. syringae and = 1.0 ug/mL for C. michiganensis. Optimized versions of Fixed-Quat A-II and Fixed-Quat E have been prepared for 2016 Citrus Canker trials on Ruby Ray grapefruit.
OVERVIEW: The program is designed to provide partial technical salary support to all faculty at the Citrus Center to ensure continuity of the research. PROGRAM OPERATIONS Horticulture/ Breeding: The funds were used to partially support Sonia del Rio. During the reporting period, she worked on: (1) Testing Texas Red grapefruit selections for fruit quality through the season (2) Screening rootstock seedlings for Phytophthora resistance (some survive under intense infection pressure) (3) Embryo recovery of plants from aborted seed of greening-infected fruit for potential resistance Plant Pathology: The funds were used to support Perla Duberney. During this time she worked on the evaluation of the effect of soil conditioners in improving the effectiveness of Mefenoxam treatment for Phytophthora control. The results showed that (1) Phytophthora propagule counts were suppressed in treated plots after 4, 8 and 12 weeks post application; (2) the use of the soil conditioner had a detrimental effect on Mefenoxam effectiveness; (3) fruit size was increased in treated trees compared to untreated trees; (4) application of soil conditioners promote better absorption of micronutrients. Entomology and Plant Physiology will utilize their portions of the grant in the latter portion of the FY.
The central mission of this project is to combine existing fertilization, irrigation, scion and rootstock options in modern, high-density citrus groves that will be more productive sooner, and survive the onslaught of HLB more successfully than conventional Florida citrus groves. The resulting synergistic production system was called the Advanced Citrus Production System, and relied heavily on open hydroponics with liquid fertigation, and computer automation, as well as precocious, dwarfing rootstocks and high planting densities. The overall goals of this three-year project renewal are to continue the existing ACPS experiments at Auburndale (2.5 year old replant) and at the CREC (mature 20 year old ACPS retrofit) in order to obtain long-term data which is crucial for the successful recommendation and adoption of this technology. A demonstration ACPS experiment testing four different rootstocks was established in March 2011 on 5 acres of CREC’s Lake Placid grove, and will also be continued with this project. Finally, two new ACPS experiments will be established to fill gaps and to evolve the new ideas developing from our current research experiments. Specific objectives are: i) To install a ‘Valencia’ juice orange ACPS replant experiment at the CREC, testing two rootstocks (US897, Swingle), and two novel ultra-high planting densities with narrow grove equipment. ii) To install a grapefruit fresh fruit ACPS replant experiment in the Indian River region in order to adapt the technology for regional priorities, conditions and soil types. iii) To continue the existing ACPS experiments at Auburndale, CREC (Lake Alfred), and Lake Placid. The collective results from various ACPS experiments of this project were valuable in demonstrating to citrus growers the need for using higher planting densities and more intensive irrigation and balanced fertilization technologies in order to remain competitive in an HLB-endemic environment. Economic production in the young Hamlin experiment was achieved in the third year (220 boxes/acre), and a year later, reached full production at 622 boxes/acre), with the best combination of rootstock (C35), high density (363 trees/acre), and drip fertigation. Conventionally grown groves would typically reach full production only after 6-10 years. Unfortunately the HLB incidence in this grove grew rapidly, reaching 75% in the fifth year, and causing drastic yield decline of affected trees. The best yield obtained in the fifth year was only 436 boxes/acre, and continued to decline thereafter due to stunted tree canopies and root systems, low fruit set, small fruit size and excessive preharvest fruit drop. The recommended practice of HLB management by destroying HLB-affected trees was futile in this grove because it was surrounded by symptomatic dooryard citrus trees in neighboring properties, over which we had no control. Unfortunately the other trials in this study suffered similar early high HLB incidence rates, leading to unsustainable production scenarios. The new knowledge, and horticultural gains of tree growth and fruit yield made with the ACPS research in this project were remarkable, but were compromised by less successful psyllid control and HLB prevention practices. The outcomes and technologies of this research have been adopted by many Florida growers for replanting new groves, especially with higher densities, precocious rootstocks like C35, and using effective hydroponic fertigation through drip or microsprinkler irrigation systems. Ultimately when HLB-resistant rootstocks or varieties are developed and released, the new resistant groves can be established in the most efficient and rapid method possible by adopting ACPS techniques developed and tested in this project.
Progress made in the second quarter of this study has focused on grove selection and the initial sampling of leaf tissue and soil. Work has also continued regarding development of soil conditions for root hair proliferation Objective 1: Leaf nutrient thresholds All three study areas have selected groves and trees for this phase of the research. The Ridge focus area has selected 11 study grove blocks (round orange) with 5 trees per block. The Indian River area has selected 10 study grove blocks (both round orange and grapefruit). The South Florida area has also selected grove blocks for sampling. The initial measurements that have been taken include LAI, tree volume, PCR, leaf nutrition, leaf starch content, soil nutrition, organic matter content, and physical properties. Leaves for nutrition were measured for SPAD, leaf area, dry mass, and scanned using a flatbed scanner for color as well as leaf circumference and leaf area. Objective 2: Determine soil conditions that favor root hair and VAM proliferation i. As mentioned in Objective 1 above, initial soil sampling at the 0-6 inch and 6-12 inch depth has been completed and analysis is beginning. Percent OM, using Loss On Ignition has been completed for the ridge portion of the survey area and in the next quarter the initial samples from the remainder of the survey areas will be analyzed. Analysis includes, soil nutrient content, pH, CEC, and soil color. ii. The cuttings that were placed in aeroponics tanks are continuing to be monitored and maintained with replacement solutions bi-weekly. The solution chemistries are still being fine-tuned and analyzed. Growth of the cuttings has been slow but is progressing. Analysis of the root development will be the next step of this portion of the study.
Seed decontamination: 1. Develop guidelines to prevent seed contamination by canker. Part 1. Surface disinfection of seeds. Objective is to develop economical and practical cleaning methods for removing Xcc without loss of seed viability. Progress: Thirty freshly squeezed seeds of Kuharske were plated on LB plates. Concurrently rinsate of seeds rinsed in sterile water had serial dilutions plated on media plates viz. V8, PDA, LB and Dox. LB plates showed numerous distinct colonies, whereas the rest showed bacterial smears. Bacterial colonies on LB plates resembled X. citri and other unknown bacteria. Similar results occurred on LB plates plated with freshly squeezed intact seeds. Identity of these bacteria is unknown, but appear to be common environmental bacteria. Identification requires DNA sequencing, planned in the future. Seeds treated with different concentrations of chlorox and PPM appeared to display higher seed germination rates, to be investigated in the future. Part 2. Evaluating the 2006 CHRP decontamination procedure. Progress: Experiments are in progress along with experiments using sodium hypochlorite and PPM for decontamination. Part 3. Development of diagnostic methods for on-site canker detection. Progress: Experiments are ongoing. Cutting and tissue cultivation In October propagation beds with bottom heat were constructed. Sides and separators were installed on 3 benches, producing 12 independent beds. Beds were partially filled with perlite and heating cables installed with independent temperature controls. On 14 October, 4 schedules of acclimation of tissue cultured rootstocks were applied to C-35, C-54 sour orange, US-812, SW-13 and KC-13 root stocks. Any roots were removed and stems transplanted into peat:perlite in seedling trays. Plants were harvested on 11/16. Success was >92% for all rootstocks except sour orange (82%). The next set of tissue culture plants were planted on 12/14, with additional plants placed in 4 of the heated sections; with the same levels of light and heat exclusion. Changes in relative humidity and light levels were the same reported previously. The gap in the sequence was due to insufficient numbers of plantlets the first delivered, with delays in obtaining the remainder. Due to technical issues, humidity levels were insufficient the first week and many plantlets died. Plants in heated benches faired better due to higher humidity. These plants will be harvested in mid-February. Cutting propagation continued with single node cuttings of Kuharski initiated on 10/14, 11/3 and 12/1. Harvest of cuttings have continued to be every 6 weeks, with harvest occurring on 10/14, 11/3, 12/1. Auxin concentrations of 4000 and 7500 ppm have been consistent. Most woody plants require higher auxin concentrations during the winter months for good rooting. Rooted cuttings of the HLB resistant lines were transplanted in 5 tree pots the week before Christmas. Any cutting that had good root systems were transplant, independent of shoot growth. Less than 10 cutting failed to produce roots. Even plants with no additional shoot growth had abundant root growth at transplanting. These plants were placed in the production side of the greenhouse due to insufficient space in the propagation bay.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. Transgenic Arabidopsis plants expressing lasAI or lasAII showed a different degree of impaired growth. In particular, the LasAI contains domains responsible for abnormal growth of the root and/or meristem. Trangenic citrus plants expressing Las AI also display growth retardation. Meanwhile, to further study the function of LasAI in citrus, transgenic citrus were generated to express LasAI, LasAI N-terminal, LasAI C-terminal, LasAI repeat region, LasAII and GFP control, respectively. We have obtained transgenic citrus plants transformed with different domains of LasAI, Interestingly, transgenic plants show different degree of growth retardation, in particular the full length LasA1 and LasA1 C-terminal shows slower growth compared to the other constructs. Using RNAseq and RT-qPCR, we were able to identify the up- and down- regulations of some important genes involved in host-pathogen interactions and biosynthesis of secondary metabolites in these transgenic plants. Transient expression of LasAI and three different LasAI domains, LasAI-N-terminal, LasAI-repeat, LasAI-C-terminal allowed us to visualize the sub-cellular localizations of different domains. Because of high level expression of these effector proteins, we developed a novel in vitro screening system that evaluates small molecules against these Las effectors. The library consists of more than 30 million compounds obtained from the small molecule libraries of the TPIMS (Torrey Pines Institute for Molecular Studies). Interestingly, a few groups of compounds showed interference activity against the mitochondrial localization of LasAI. Meanwhile, to concert this screening, we developed another in vitro screening system in conjunction with the culture screening using Liberibacter cresence (Lcr). From these screening of 65 scaffold chemicals, we identified a number of chemical groups that disrupted the interaction between LasA1 and mitochondria and inhibit both Las and Lcr growth. We are narrowing down to individual compounds that inhibit the function of the Las AI effector or kill Las bacteria via other pathways, and measured the dosage effect of these potential candidate. The selected chemicals (individual or small groups) are in the evaluation process with graft-based assay. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein was able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants with high level expression of this transgene displayed HLB-like symptoms, yellow shoot and impaired growth. Further characterization of this effector revealed its unique sub-cellular localizations. We also analyzed the expression of LasA1 in Las-infected citrus plants. RT-PCR results indicate that LasA1 expression is correlated with severity of HLB symptoms. In particular, LasA1 was expressed more in the yellow leaves or the yellow spots than green spots of the symptomatic leaves with blotchy mottle. Since transgenic plants expressing Las A1 showed up-regulation of defense-related genes, these transgenic plants were propagated and graft-inoculated with Las bacteria. We detected LasA1 in Las infected citrus leaves using antibody-based tissue printing. The results indicate that LasA1 was abundant and diffusible in Las-infected vascular tissues. To further characterize LasA1, we identified several citrus proteins that interacted with LasA1 using Yeast Two-Hybrid screening system. It is worth noting that a couple of these proteins were also identified as up-regulated genes in our RNA-seq data as mentioned above.
The overall objective of our research project is to develop an effective and sustainable phage/phage component-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. We have previously reported on first and second round evaluations of phage cocktails and first round testing of tailocin cocktails in cooperation with Dr. Nian Wang (University of Florida-Lake Alfred). In two independent experiments, when the tailocin cocktail containing tailocins XT-1 and XT-4 was applied to foliage at a multiplicity of killing units (MOKU) of 16 at 6 h post- or pre-inoculation with 5 X 10^7 CFU/ml of Xac, there was a an average reduction of 49% and 53%, respectively, in lesion formation as compared to the plants inoculated only with Xac. It was of interest to determine a dose response curve to the tailocin treatment. Using the same condition as previously described (Jan. 2015 report), Hamlin sweet orange trees were inoculated with 1 10^8 CFU/ml of Xac. After 6 h, the tailocin was applied at a MOKU of 15, 10 or 5. Lesion numbers were assessed 21 days after inoculation with the pathogen for treated and untreated trees. In two independent experiments an average of 39%, 31% and 11% reduction in lesions were observed in leaves of trees treated with tailocin cocktails at a MOKU of 15, 10 and 5, respectively, as compared to non-treated trees. Using a delta-PilA mutant of Xac, we have isolated and purified two non-type IV pilus dependent phages that appear to be virulent. Base on their plaque morphology the phages appear to exhibit depolymerase activity. Ongoing studies will determine the morphology and lifestyle of the two phages. Bioinformatic analysis of Xac phages genomes and tailocin gene clusters is ongoing.
The focus of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using Liberibacter crescens strain BT-1. We have accomplished that goal and have moved forward to identify naturally occurring phages and to construct modified tailocins that are active against strain BT-1. Once Candidatus Liberibacter asiaticus (Las) is successfully cultured, the protocols developed for L. crescens can be translated to Las. We have demonstrated that the tail fibers of tailocins can be deleted and complemented. Since the R-type pyocin systems have been well studied, we have initiated studies using the R2 platform and designed fusions between N-terminal tail fiber region of the R2 and C-terminal portions of tail spike from BT-1 prophages. We have identified the BT-1 tail spike protein based on topology. We are continuing the search for naturally occurring phages active against L. crescens. Members of the Rhizobium, Agrobacterium and Liberibacter are in the family Rhizobiaceae. Because of their phylogenetic relationship, it is possible that members of the Rhizobiaceae could share receptors. Rhizobium spp., Agrobacterium spp. or L. crescens BT-1 were used as hosts for enrichments from environmental samples. Several new Agrobacterium and Rhizobium phages have been isolated and purified. Electron microscopy studies will confirm their morphology. It is our experience that growth conditions can affect the bacterial surface, and therefore in vitro testing for phage and tailocin sensitivity. The phages are currently being tested against BT-1 using several modifications of medium BM7.
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