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 hyvI 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. Based on the variations of Las prophages/phages, we recognized certain molecular mechanisms behind the symptom variations and their association with “mild strains” of Las bacteria and host tolerance/resistance.
This is a new project that is based on several successful outcomes from previously funded research. Las appears to be very well adapted to psyllid hosts, moving through multiple membrane barriers and multiplying in the hemolymph, all without causing obvious disease, and all without expression of any phage genes examined to date. By contrast, Las host range in plants appears to be tenuous, limited to phloem only. Our working hypothesis is that Las acquired key genes for plant adaptation by way of its phage and these phage genes are highly regulated; off in psyllids, and on in plants. We propose targeting specific regulators of key phage encoded virulence genes (such as the Las peroxidase) as well as key regulators of the (lethal) phage lytic cycle. Direct targeting of the Las peroxidase enzyme itself is also proposed. Objective 1 is control of HLB using the putative Las LexA repressor protein, potentially a key phage lytic cycle regulator, and to date we have expressed and purified both a truncated LexA (CLIBASIA_01645, missing C terminal domain) and full length CLIBASIA_01645 fused with a His-tag for purification and further analysis of the protein. We are now using these proteins in mobility shift assays (EMSAs) to verify specific binding to previously cloned SC1 and SC2 promoter fragments as well as its own promoter. Objective 2 is control of HLB using a repressor protein from psyllids as target. A psyllid-sourced repressor of unknown identity silences the Las phage holin gene, expression of which is lethal. We previously reported that the Las holin promoter was strongly active in Liberibacter crescens (Lcr), a culturable proxy for Las, but nearly silent in E. coli. Further, activity of the holin promoter in Lcr was suppressed by aqueous extracts from psyllids applied outside of the Liberibacter cells, indicating cell penetration. The suppressor activity was sensitive to heat and proteinase treatment, indicating a protein, and the molecular size is estimated to be10-50 kDa. Electrophoretic mobility shift assays (EMSAs) were used to demonstrate specific binding of a protein to the Las holin promoter, and small, overlapping holin promoter fragments were used as competitor DNAs to further delineate the target binding sites of the protein. The DNA-binding protein has now been purified by DNA affinity capture, and we provided multiple purifed samples for tandem liquid chromatography mass spectroscopy (LCMSMS) for the purpose of identifying the protein and the source of the gene encoding the protein (the gene could be a psyllid gene or any one of several bacterial endosymbionts living in psyllids). The initial results were inconclusive because of limited amounts of purified protein. This has now been rectified. We have also now determined that Las-free psyllids carry the repressor, and that similar extracts from Drosophila (fruit fly) do not. Objective 3 is control of HLB using the Las phage peroxidase and Las lytic cycle activator(s) as targets. We have acquired two Prestwick combinatorial libraries for use in screening chemicals that may bind to and denature CLIBASIA_01645 (focus of Objective 1) and also the Las phage peroxidase, which we demonstrated was a virulence determinant of Ca. Liberibacter asiaticus. We have also acquired and set up a microplate reader equipped for the spectrophotometry, fluorimetry and luminometry assays needed to screen the combinatorial libraries. A potential lytic cycle activator was identified from Liberibacter crescens extracts using the holin promoter as bait and demonstrated by(EMSAs). The DNA binding site within the holin promoter was delineated using competitive DNA fragments.
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 have propagated more Las-infected periwinkle and citrus plants that contain high titers of prophage/phage FP3, which will be used for isolation and characterization of prophage/phage FP3. Phages from infected periwinkle tissues have been enriched using a refined phage purification pipeline consisting of differential centrifugations and PEG precipitation. The partially purified phage preparation showed an enrichment of phage DNA by 20~50 folds and a trace amount of Las genomic DNA as determined by PCR. While phage-like particles were observed under electron microscope, the images were not conclusive due to the large amount of contaminating host materials present. Further purification will be carried with sucrose density gradient and/or CsCl equilibrium density gradient centrifugation. Different varieties of citrus plants inoculated with a mild strain have been evaluated in greenhouse. Intriguingly, different varieties showed different response to the “mild stains/isolates”. However, in a given variety, the mild strain status was maintained after three consecutive propagation. We are evaluating the factors that affect the symptoms and titers and determining if a mild strain can be maintained in major commercial citrus varieties. Over one hundred of pre-inoculated sweet orange and grapefruit plants have been subjected to field evaluation. The first group planted in the field show their consistency of mild symptoms as those in the greenhouse after 7 months. We have developed a digital PCR (dPCR) system for early detection of HLB and tracking of lysogenic and lytic activities of the Las prophage/phage. We show that as few as 1 to 2 copies of the targeted DNA molecules per microliter can be detected, with the prophage probe providing the best sensitivity. With this tool, we were able to detect the lytic activities of the Las bacteriophage in some sample. For example, 20 times more phage DNA than bacterial DNA was present in sample EL L2VC2 than other samples, indicating active induction and replication of the Las phage in the infected citrus. RNA-seq analysis for bacterial transcripts from Las-infected plants subjected to heat stress using the same parameters as those currently used in thermotherapy has been completed. This analysis utilized a novel technique developed by the 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. Reverse-transcription PCR is currently being used to confirm the results of the RNA-Seq studies. Additional plants subjected to a traditional course of thermotherapy are being used to verify that Las genes found to be regulated under heat stress are consistent amongst various trees. Alternative forms of heat stress will also be tested for a specific subset of the genes identified to determine their role in the bacterial/plant interactions.
T-SOL (TM) is a new class of Zn chelate based antimicrobial compounds developed against citrus pathogens particularly HLB bacteria. T-SOL is designed to be systemic with the potential to be effective against citrus canker and HLB. Tasks completed in this reporting period: 1) Six different formulations of T-SOL (varying the chelating components) were prepared (metallic zinc concentration 20,000 PPM in 16 liters) and sent to field trials on the months of April and August of 2015. 2) Preliminary greenhouse studies performed in X. citri subsp., citri infected citrus trees suggested that 2 out of 3 T-SOL formulations (3 variants of TSOL formulations used in this study) significantly reduced the canker leaf lesions to ~60% in comparison to untreated control (100%), demonstrating at least locally systemic activity. 3) In vitro plant uptake studies were conducted with four different concentrations of T-SOL UP (800, 500, 300 and 100ppm). T-SOL UP is used in this set of studies but the same studies will be replicated in the next reporting periods with all the other 5 formulations mentioned above. Tomato plants (Solanum lycopersicum, used as model system) treated through foliar spray application method with the above-mentioned concentration of T-SOL for 24hours. After 24hours of treatment plants were sampled for digestion and analysis of zinc uptake by different parts of plants (whole plant, leaf, stem, root, phloem and xylem) through atomic absorption spectroscopy (AAS). Our result suggested maximum uptake of zinc by whole plant, leaf, stem, root and xylem at 500ppm treatment. Systemic uptake of zinc by phloem was highest at 300ppm while lowest uptake was observed at 500ppm (leaf phytotoxicity was observed at 500 ppm). Overall, treatment with T-SOL at all the different concentrations (100, 300, 500 and 800 ppm) showed definitive uptake of zinc chelate ranging from 0.3 to 7.1 mg per gram dry weight by different parts of the plant when compared to untreated plants (through the stomatal pores regardless of treatment concentration of metallic zinc). 4) Fourier Transform Infra red spectroscopy (FTIR) studies confirmed the formation of zinc chelate. Most of the resemblances were found in the fingerprint zones of the spectra. In the coming reporting periods, optimization of the synthesis protocol of the other five variants of T-SOL will be performed and their antimicrobial and phytotoxicity studies will be conducted to narrow down the most effective T-SOL variant that can be later taken to green house and field trial studies.
Quaternary Ammonium Compounds (Quat) is a powerful antimicrobial agent. However, Quat cannot be directly applied to citrus plants as a film forming fungicide/bactericide as it exhibits severe phytotoxicity and poor rainfastness. This research uses a silica based Quat delivery system (Fixed-Quat). Fixed-Quat material is non-phytotoxic and it demonstrated good rainfastness. In the previous reporting period, a new Fixed-Quat A-II nanogel formulation was synthesized with EPA approved For Food Use active and inerts. Fixed-Quat A-II is an improved version of previously developed formulation. Fixed-Quat A-II nanogel demonstrated no plant safety issues while maintaining exceptional antimicrobial properties. In this reporting period, a new version of Fixed-Quat AP-II containing a secondary EPA approved active having additional antimicrobial modes of action was designed and developed. The purpose was to combat any potential antimicrobial resistance against Quat active materials. Quat concentration in Fixed-Quat AP-II nanogel formulation was at 13,500 ppm level, same as the previous version. It is expected that multiple mechanisms of killing will increase the likelihood of biocidal efficiency. Phytotoxicity studies were carried out on the optimized version of Fixed-Quat AP-II in a Panasonic Environmental Test Chamber (Model MLR- 352H). The chamber allowed for controlled day/night cycling temperatures, light intensity and humidity to simulate summer weather conditions (biocide application season). Studies conducted on Vinca sp, an ornamental plant revealed no sign of plant injury even as high as 500-1000 ppm Quat (EPA maximum concentration for Quat industrial use is 200 ppm). The composition and interactions between the components was confirmed using Fourier transform infrared spectroscopy (FTIR). The morphology of Fixed-Quat AP-II nanogel was examined using Scanning Electron Microscopy (SEM), displaying sub-micron to micron composites with irregular shapes in multiple layers. Antimicrobial studies of Fixed-Quat AP-II nanogel was conducted against several model plant pathogen surrogates, Xanthomonas alfalfae subsp. citrumelonis, Pseudomonas syringae pv syringae and Clavibacter michiganensis subsp michiganensis. Studies were conducted to determine the Minimum Inhibitory Concentration (MIC) and compared against Kocide 3000 and copper sulfate controls. MICs of Fixed-Quat A-II 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. Future studies will involve additional antimicrobial studies (CFU) and rainfastness evaluation.
We continue to screen antibiotics against Liberibacter crescens. We are using commercially available antibitoics for this purpose to identify potential antibiotics effective against Ca. Liberibacter asiaticus. We are also interested in those antibitoics that do NOT inhibit L. crescens because they can be used as a means to reduce contamination in the media we are using to attempt to perfect long-term cultiring of Ca. Liberibacter asiaticus. We recently identifed four such contaminants and tested antibiotics against those contaminants. We are now using an antibiotic in our media that inhibits these contaminants but not Ca. Liberibacter asiaticus. Our collaboration with NuFarm continues. We expect to have data this fall on the efficacy of Mycoshield treatments. Our role is primarily one of data analysis. We are keenly interested in this mostly because of the expected benefit to citrus growers but also because it fits well into a manuscript we are preparing.
July 2015 The objectives of this project are to optimize Guignardia citricarpa ascospore extraction procedures and qPCR with automated extraction system, determine if prototype passive ascospore traps will capture a sufficient number of Guignardia citricarpa ascospores to be an effective monitoring tool and monitor for G. citricarpa ascospores in six locations around state. Slides are still being collected but there are still very few spores in the groves so it has been difficult to detect them at such low concentration with qPCR. We have started to work out ways to better the qPCR assay in case a large number of spores is ever detected so we have the method for the future.
One of the primary goals of this work is to identify a small molecule treatment that can be used to activate the phage lytic cycle genes encoded by Las prophage, thus bringing about the death of Las bacteria carrying these prophage. The majority of Candidatus Liberibacter asiaticus (Las) strains carry bacteriophage similar to SC1 and SC2 of Las UF506. Lytic phage particles are formed and have been reported in both periwinkles and in citrus, but not in infected psyllids. We reported that relative mRNA expression levels of prophage late genes SC2-gp095 (“peroxidase”) and SC1-gp110 ( holin ) were much higher in periwinkle than in citrus. We now report that these genes were silent in psyllids. We also reported that both the prophage holin (SC1_gp110) and endolysin (SC1_gp035) were functional, and that strong expression of the holin gene alone in Las cells would be sufficient to kill the cells, whether or not phage particles were formed. This could be part of the explanation as to why Las has to date not been cultured (Fleites et al. 2014). The Las holin promoter was strongly active in Liberibacter crescens (Lcr), a culturable proxy for Las, but nearly silent in E. coli. Further, activity of the holin promoter in Lcr was suppressed by aqueous extracts from psyllids applied outside of the Liberibacter cells, indicating cell penetration. The suppressor activity was sensitive to heat and proteinase treatment, indicating direct repression by a protein, and size fractionation demonstrated the the size was 10-50 kDa. Electrophoretic mobility shift assays (EMSAs) were used to demonstrate specific binding of a protein to the Las holin promoter, and small, overlapping holin promoter fragments were used as competitor DNAs, to further delineate the target binding sites of the protein. The DNA-binding protein was purified by DNA affinity capture, and MALDI-TOF analyses are underway to identify the protein. We also reported that the peroxidase on SC2 is a reactive oxygen species (ROS) scavenger and confirmed that it has peroxidase activity. SC2-gp095 was cloned in a shuttle vector and transformed into L. crescens (Lcr). Transformed Lcr cells showed 20-25% enhanced resistance to hydrogen peroxide on agar plates, 47% higher enzymatic activity and enhanced growth in liquid cultures. A non-classical secretion potential was confirmed by enzymatic and Western blot analyses in Lcr. Transient expression of SC2_gp095 in planta resulted in strong transcriptional down-regulation of RbohB, the key gatekeeper of the H2O2 mediated defense signaling in plants, helping explain the surprisingly long incubation period (years) before HLB symptoms appear in Las-infected citrus. Las peroxidase is therefore a secreted, horizontally acquired effector that suppresses host symptom development, a tactic used by most biotrophic plant pathogens. In psyllids, stringent control of all lytic cycle genes, including SC1 excision and replication (early genes) as well as the lysis genes and the peroxidase (late genes), appears to be critical to prevent Las from being detected by the insect innate immune system. Upon examination of SC1 and SC2 promoter regions, we found strong matches to a predicted Las chromosomally encoded LexA-like repressor, The lytic cycles of many phages are under the control of the bacterial SOS (suicide program) response. In the stable “lysogenic” or “prophage” state, the phage is kept dormant by suppression of early gene activation, often by using a phage C1-like repressor. In Las, the C1-like repressor is chromosomally encoded and has a LexA domain. Externally applied chemicals, as well as heat shocks have been used in other bacterial systems to activate the SOS response and derepress various phage lytic cycle genes by disabling LexA. We have recently developed indirect evidence that Las LexA may repress SC1 early gene activity, making this a potential new molecular target for HLB control.
Testing continues with L. crescens assay through CRDF research contract. As of the end of March, there have been 804 compounds tested since the start of the project. The new additional compounds tested included tetracycline derivatives from two companies, Echelon and Paratek, antimicrobial peptides, bacterial fermentation products, GRAS oils, and a zinc product from a collaborative lab at UCF. Work was started again with samples that were collected from a collaboration experiment with Dr. Mirkov at Texas A&M. They set up a trial with tomato plants and the relative pathogen of citrus greening, Liberibacter solancearum. This trial was designed to test the effectiveness of oxytetracycline as a treatment sprayed to the infected plants as a foliar treatment. Other antimicrobials were tested at the same time, including Mycosheild, streptomycin, and emulsion of thyme oil. The intention is to determine the bacterial load at various time points of the experiment using qpcr technology. Work is underway to optimize the procedure from published work (Li et al 2009), as the initial trials following the paper s protocol with standard curves did not work. Work also continues with Dr. Sharma s group at UF PERC to use GC-MS to detect carvacrol after spraying to citrus plants. We have performed experiments on Duncan and Valencia citrus varieties with carvacrol, and multiple surfactants. So far no oil is being detected after foliar spray in the trunk of the plants. Trials have entailed spraying once a day in the morning for 3 days, and collecting and processing material on the 4th day. Care is taken to protect the trunk and soil and sprayed carefully tilted to prevent contamination with the oil to those areas. One trial done on Valencia tested the bark painting method, and samples were taken above and below treatment area, as well as in the leaves. When painting the trunk we used a q-tip dipped in the formulation, and thoroughly covered the treatment area 3x a day for 3 days. Plans for more trials are underway.
Since the last report, a defined media for Liberibacter crescens has been developed. Using the methods previously described, we utilized the literature to break down the undefined the components of the media as best as possible. Changing the ratio of the buffer and alpha-ketoglutarate also made a positive difference. The growth of L. crescens is not 100% comparable to BM7, however the rate of growth is identical up until 5 days, where the defined media cultures begin to die, and BM7 cultures continue to replicate for up to 10 days. Plans to do metabolomics on the two culture conditions is underway; perhaps the defined media is limiting in one or more components, or perhaps an toxic substance is building up. With this new break through, efforts to culture L. asiaticus have continued, integrating information from the other group members. Most prominently, we are using the defined medium as a base for the asiaticus medium, and supplementing with ingredients that the Killiney lab identified in citrus phloem sap. Such ingredients include a significant increase of sucrose and the addition of sugar alcohols such as xylitol and sorbitol, vitamins, and organic acids like ascorbic acid and citric acid. A we suspect a key ingredient in phloem sap to be gamma-aminobutyric acid, as well as cell wall components (lipids and fatty acids). Another vital change is using ferrous chloride instead of ferric chloride, which was discovered in the genome comparison of the Liberibacter species. Las does not have an Iron III transporter, but does contain an Iron II/manganese transporter, therefore we believe adding ferrous chloride will be essential for growth of Las. Although a defined media has been obtained, work to optimize the media formulation is still underway. Plans for optimization include metabolomics, proteomic and trasncriptomic analysis of the ancestral Liberibacter crescens strain BT-0, which we hypothesize to be more similar to Las since it has not been carried in culture at length since its isolation from babaco papaya, unlike the lab strain BT-1, in both the current defined medium as well as in BM7. Clues may be discovered about what nutrients may be missing in the asiaticus medium, or if Las is lacking an essential gene for exogenous culture.New methods for media inoculation and detection have also been explored. Various methods for obtaining cells from infected psyllid guts have been tested. Ideas on how to test for Las growth are also being tested. So far multiple methods are being used in each inoculation attempt, until we can confirm one method is better than any other. These methods include extracting single guts from psyllids and a)pooling multiple in one tube with an amount of the culture media, vortexing, and using the supernatant for inoculation b) using 2 guts per culture tube of 50ul and leaving c) and d) above with the addition of 5ppm cefixime to limit contamination chances. Analysis was done on contaminants that were obtained in one of the culture attempts for antibiotic susceptibility of antibiotics that were shown to not inhibit L. crescens. Cefixime was the only one that inhibited the contaminants of the few antibiotics we decided to test.Direct PCR was investigated to try to assess if Las is present in inoculated cultures. Unfortunately it did not work when taking 2 or 5ul of liquid culture to pcr tube. The use of 16s amplification was used to determine this. Suspicion of the complexity of the media being the inhibitor was then tested by adding known amounts of extracted bacterial DNA to the media and running direct PCR at various dilutions. Results showed amplification at a 1:10 dilution. This needs to be tested again with attempted cultures and 16s to ensure it produces results. If so, this will be an easy first assessment of whether Las is present in culture. More organized meetings would have been beneficial to the group, although there was a lot of great input from all members throughout the project. Work on identifying the components of the hemolymph of the psyllid is underway in the Killiney and Pelz-Stilinski labs. Media formulations still continue with the Davis lab. Hilf had success with isolating viable Las cells and we may still collaborate with the group to test the media with his cell isolation method. The task is to culture Las is very large and complex, however significant progress has been made in the endeavor.
Since the last report, 84 more compounds have been tested in L. crescens assay to search for a treatment for citrus greening, bringing the total tested to 888. Most of the testing was continuing with bacterial fermentation products from major companies. Multiple compounds have been tested from smaller start-up corporations that have been having mixed success. Optimization of qpcr methods has encountered many problems, such as contamination, standard curve dilutions, efficiency of reactions, and reproducibility of the results. It turns out the qpcr machine was producing a systematic error and needed to be repaired. After repair, optimization continued, however research also underwent for alternative methods. A few papers were found that utilized the SYBR green chemistry vs the TaqMan chemistry for Lso detection. We decided to test out the alternative methods in hopes of consistent success. Unfortunately those methods proved to be just as challenging. With one good standard curve, it was decided to return to the TaqMan chemistry and a few more optimization runs were performed. Results have been reliable and work on the samples is underway. Trials with PERC group also continue, this time with much higher concentrations of carvacrol. Results are interesting, but some results seem to indicate contamination with the negative control, however results are still being analyzed. There is one more trial being planned with this project.
Testing continues with L. crescens assay through CRDF research contract. As of the end of March, there have been 804 compounds tested since the start of the project. The new additional compounds tested included tetracycline derivatives from two companies, Echelon and Paratek, antimicrobial peptides, bacterial fermentation products, GRAS oils, and a zinc product from a collaborative lab at UCF. Work was started again with samples that were collected from a collaboration experiment with Dr. Mirkov at Texas A&M. They set up a trial with tomato plants and the relative pathogen of citrus greening, Liberibacter solancearum. This trial was designed to test the effectiveness of oxytetracycline as a treatment sprayed to the infected plants as a foliar treatment. Other antimicrobials were tested at the same time, including Mycosheild, streptomycin, and emulsion of thyme oil. The intention is to determine the bacterial load at various time points of the experiment using qpcr technology. Work is underway to optimize the procedure from published work (Li et al 2009), as the initial trials following the paper s protocol with standard curves did not work. Work also continues with Dr. Sharma s group at UF PERC to use GC-MS to detect carvacrol after spraying to citrus plants. We have performed experiments on Duncan and Valencia citrus varieties with carvacrol, and multiple surfactants. So far no oil is being detected after foliar spray in the trunk of the plants. Trials have entailed spraying once a day in the morning for 3 days, and collecting and processing material on the 4th day. Care is taken to protect the trunk and soil and sprayed carefully tilted to prevent contamination with the oil to those areas. One trial done on Valencia tested the bark painting method, and samples were taken above and below treatment area, as well as in the leaves. When painting the trunk we used a q-tip dipped in the formulation, and thoroughly covered the treatment area 3x a day for 3 days. Plans for more trials are underway.
Since the last report, a defined media for Liberibacter crescens has been developed. Using the methods previously described, we utilized the literature to break down the undefined the components of the media as best as possible. Changing the ratio of the buffer and alpha-ketoglutarate also made a positive difference. The growth of L. crescens is not 100% comparable to BM7, however the rate of growth is identical up until 5 days, where the defined media cultures begin to die, and BM7 cultures continue to replicate for up to 10 days. Plans to do metabolomics on the two culture conditions is underway; perhaps the defined media is limiting in one or more components, or perhaps an toxic substance is building up. With this new break through, efforts to culture L. asiaticus have continued, integrating information from the other group members. Most prominently, we are using the defined medium as a base for the asiaticus medium, and supplementing with ingredients that the Killiney lab identified in citrus phloem sap. Such ingredients include a significant increase of sucrose and the addition of sugar alcohols such as xylitol and sorbitol, vitamins, and organic acids like ascorbic acid and citric acid. A we suspect a key ingredient in phloem sap to be gamma-aminobutyric acid, as well as cell wall components (lipids and fatty acids). Another vital change is using ferrous chloride instead of ferric chloride, which was discovered in the genome comparison of the Liberibacter species. Las does not have an Iron III transporter, but does contain an Iron II/manganese transporter, therefore we believe adding ferrous chloride will be essential for growth of Las. Although a defined media has been obtained, work to optimize the media formulation is still underway. Plans for optimization include metabolomics, proteomic and trasncriptomic analysis of the ancestral Liberibacter crescens strain BT-0, which we hypothesize to be more similar to Las since it has not been carried in culture at length since its isolation from babaco papaya, unlike the lab strain BT-1, in both the current defined medium as well as in BM7. Clues may be discovered about what nutrients may be missing in the asiaticus medium, or if Las is lacking an essential gene for exogenous culture.New methods for media inoculation and detection have also been explored. Various methods for obtaining cells from infected psyllid guts have been tested. Ideas on how to test for Las growth are also being tested. So far multiple methods are being used in each inoculation attempt, until we can confirm one method is better than any other. These methods include extracting single guts from psyllids and a)pooling multiple in one tube with an amount of the culture media, vortexing, and using the supernatant for inoculation b) using 2 guts per culture tube of 50ul and leaving c) and d) above with the addition of 5ppm cefixime to limit contamination chances. Analysis was done on contaminants that were obtained in one of the culture attempts for antibiotic susceptibility of antibiotics that were shown to not inhibit L. crescens. Cefixime was the only one that inhibited the contaminants of the few antibiotics we decided to test.Direct PCR was investigated to try to assess if Las is present in inoculated cultures. Unfortunately it did not work when taking 2 or 5ul of liquid culture to pcr tube. The use of 16s amplification was used to determine this. Suspicion of the complexity of the media being the inhibitor was then tested by adding known amounts of extracted bacterial DNA to the media and running direct PCR at various dilutions. Results showed amplification at a 1:10 dilution. This needs to be tested again with attempted cultures and 16s to ensure it produces results. If so, this will be an easy first assessment of whether Las is present in culture. More organized meetings would have been beneficial to the group, although there was a lot of great input from all members throughout the project. Work on identifying the components of the hemolymph of the psyllid is underway in the Killiney and Pelz-Stilinski labs. Media formulations still continue with the Davis lab. Hilf had success with isolating viable Las cells and we may still collaborate with the group to test the media with his cell isolation method. The task is to culture Las is very large and complex, however significant progress has been made in the endeavor.
Since the last report, 84 more compounds have been tested in L. crescens assay to search for a treatment for citrus greening, bringing the total tested to 888. Most of the testing was continuing with bacterial fermentation products from major companies. Multiple compounds have been tested from smaller start-up corporations that have been having mixed success. Optimization of qpcr methods has encountered many problems, such as contamination, standard curve dilutions, efficiency of reactions, and reproducibility of the results. It turns out the qpcr machine was producing a systematic error and needed to be repaired. After repair, optimization continued, however research also underwent for alternative methods. A few papers were found that utilized the SYBR green chemistry vs the TaqMan chemistry for Lso detection. We decided to test out the alternative methods in hopes of consistent success. Unfortunately those methods proved to be just as challenging. With one good standard curve, it was decided to return to the TaqMan chemistry and a few more optimization runs were performed. Results have been reliable and work on the samples is underway. Trials with PERC group also continue, this time with much higher concentrations of carvacrol. Results are interesting, but some results seem to indicate contamination with the negative control, however results are still being analyzed. There is one more trial being planned with this project.
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 have propagated more Las-infected periwinkle and citrus plants that contain high titers of prophage/phage FP3, which will be used for isolation and characterization of prophage/phage FP3. Phages from infected periwinkle tissues have been enriched using a refined phage purification pipeline consisting of differential centrifugations and PEG precipitation. The partially purified phage preparation showed an enrichment of phage DNA by 20~50 folds and a trace amount of Las genomic DNA as determined by PCR. While phage-like particles were observed under electron microscope, the images were not conclusive due to the large amount of contaminating host materials present. Further purification will be carried with sucrose density gradient and/or CsCl equilibrium density gradient centrifugation to separate the Las bacteriophages from contaminating host materials. Different varieties of citrus plants inoculated with a mild strain have been evaluated in greenhouse. Intriguingly, different varieties showed different response to the “mild stains/isolates”. However, in a given variety, the mild strain status was maintained after three consecutive propagation. We are evaluating the factors that affect the symptoms and titers and determining if a mild strain can be maintained in major commercial citrus varieties. Over one hundred of pre-inoculated sweet orange and grapefruit plants have been subjected to field evaluation. The first group planted in the field show their consistency of mild symptoms as those in the greenhouse after 7 months. We have developed a digital PCR (dPCR) system for early detection of HLB and tracking of lysogenic and lytic activities of the Las prophage/phage. We show that as few as 1 to 2 copies of the targeted DNA molecules per microliter can be detected, with the prophage probe providing the best sensitivity. With this tool, we were able to detect the lytic activities of the Las bacteriophage in some sample. For example, 20 times more phage DNA than bacterial DNA was present in sample EL L2VC2 than other samples, indicating active induction and replication of the Las phage in the infected citrus trees. RNA-seq analysis for bacterial transcripts from Las-infected plants subjected to heat stress using the same parameters as those currently used in thermotherapy has been completed. This analysis utilized a novel technique developed by the 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. This will pave the way for possible alternative techniques and/or improved methodologies related to the elimination of Las via heat.
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