The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of December 2015, we have processed more than 37,200 grower samples, with 3,995 during 2015. This represents a 46% increase in the number of grower’s samples over the previous calendar year, which in itself had seen a 37% increase over 2013 numbers. These increases are likely due to the increased efforts to mitigate the HLB-associated tree stresses. Growers in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. Another evidence of increased grower usage of the lab is seen in the fact that 60% of the individuals who submitted samples during 2015 were new clients who had not previously submitted samples. Additionally, more than 42,700 samples have been received for research for the entire period of diagnostic service, supported by grant funding of individual researchers. This brings the grand total to nearly 80,000 plant samples processed. Grower samples are typically processed and reports returned within a two to four week time period. For this report, focusing on the quarter from October-December 2015, there were 1,015 growers samples processed and 492 research. Since the start of the current grant in July 2015, the lab has received 2,176 growers samples, which is even higher than the expected increases in sample volume. The HLB Diagnostic Lab continues to offer the service of detection of CLas in psyllids as funded in this grant. Current methods of sample processing have become streamlined and therefore seen no change in procedure.
Since the last report, we have developed several media for the culturing of Ca. L. asiaticus (CLas), being AM2 the most promising one. These new media build on the components of AM2. We have acquired several new ideas on culturing by using a multi-omics approach to L. crescens cultures. These methods tell us which components may be absent or missing in AM2. As a result, we have developed media AM3 through AM10. rThus, components based on the genome analysis of CLas (Duan et al, 2009), the comparative genomics of the culturable strain L. crescens with CLas (Fagen et al, 2014), and the biochemical composition of the citrus phloem (Hijaz and Killini, 2014) were added to evaluate growth of Ca. L. asiaticus. Similar to obligate bacteria, CLas appears to be able to scanvange energy in the form of ATP since it encodes for an ATP/ADP translocase able to uptake ATP from its surroundings (Duan et al, 2009). Therefore, we have added ATP to one of these media formations. CLas cannot synthesize NAD, but it is able to recycle it, so we added NAD to certain media as well (Fagen et al, 2014). Other components found in the biochemical composition of the citrus phloem were added to the media as well such as benzoic acid, threonic acid, maleic acid and quinic acid. Inoculations with psyllids guts have been done in these new media and possible growth is being evaluated using PCR and live/dead bacterial experiments. We were also evaluating other techniques to verify CLas growth such as peak-to-trough ratio (Korem et al, 2015) using digital PCR. Growing cells are active replicating with more active replicating forks, therefore, in this state the ratio near the origin of replication would be higher than near the terminus, providing a quatitative data for population growth (Korem et al, 2015). References Duan, Y., Zhou, L., Hall, D. G., Li, W., Doddapaneni, H., Lin, H., … & Dickerman, A. (2009). Complete genome sequence of citrus huanglongbing bacterium,’Candidatus Liberibacter asiaticus’ obtained through metagenomics. Molecular Plant-Microbe Interactions, 22(8), 1011-1020. Fagen, J. R., Leonard, M. T., McCullough, C. M., Edirisinghe, J. N., Henry, C. S., Davis, M. J., & Triplett, E. W. (2014). Comparative genomics of cultured and uncultured strains suggests genes essential for free-living growth of Liberibacter. PloS one, 9(1), e84469. Hijaz, F., & Killiny, N. (2014). Collection and chemical composition of phloem sap from Citrus sinensis L. Osbeck (Sweet Orange). Korem, T., Zeevi, D., Suez, J., Weinberger, A., Avnit-Sagi, T., Pompan-Lotan, M., … & Sirota-Madi, A. (2015). Growth dynamics of gut microbiota in health and disease inferred from single metagenomic samples. Science, 349(6252), 1101-1106.
We are following up on antibiotics that inhibit contaminants in our Ca. Liberibacter asiaticus media but not L. crescens. We continue the search for commercially available antibitoics that inhibit Ca. Liberibacter asiaticus. The first version of our pipeline paper in now complete. This paper describes a logical set of steps toward the identification of antibiotics effective against ctirus greening disease. The final pillar of data needed to submit the paper is data from NuFarm on their field trials. These data are now available and we expect to complete the paper by February for submission to a journal. Our role in the NuFarm trials has primarily been data analysis.
September 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. A method was worked out to quantify spores on the slides and was tested by our lab and Southern gardens employees. Few difficulties were encountered. Counting of slides continues but the numbers are very low.
This project is a continuation of the funding that has been granted to SGC to provide an HLB detection laboratory available to the Florida industry. For the first quarter (July 1-September 30, 2015), a total of 6,951 samples have been processed. Of those, 6,481 are plant samples and 470 are psyllid samples. Virtually all of the plant samples represent research or grower trial samples submitted by either private, corporate, CRDF, State or Federal personnel. At the request of some of the sample providers, DNA concentration and copy number estimates are now being provided in addition to the Ct values for the qPCR test. This is being done on a case by case basis and only by request.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, September 2015. Numbers specific to this report since July 1, 2015 are 1429 samples received from growers. This number represents an increase from previous 3 years. Typically, there are more samples processed December through June. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
A Ca. L. asiaticus medium, AM, was proposed based on the components of a defined medium for L. crescens plus the addition of some compounds found on the phloem biochemical composition of citrus. So far, we have developed 5 modifications to AM, being AM2 the most promising one. Different inoculations procedures have been done using either a mix of psyllids guts or spin guts supernatant into the different media. Traditional PCR results indicated the presence of L. asiaticus inoculum in the cultures after three weeks; however, not turbidity associated with growth has been seen to date. We are currently exploring different possibilities such as qPCR and sequencing to verify that L. asiaticus is actually growing in the media. With collaboration of Dr. Killiny s lab, we are currently analyzing other important components within the phloem sap of citrus to be added to AM2. We expect to propose new formulations based on this information.
A Ca. L. asiaticus medium, AM, was proposed based on the components of a defined medium for L. crescens plus the addition of some compounds found on the phloem biochemical composition of citrus. So far, we have developed 5 modifications to AM, being AM2 the most promising one. Different inoculations procedures have been done using either a mix of psyllids guts or spin guts supernatant into the different media. Traditional PCR results indicated the presence of L. asiaticus inoculum in the cultures after three weeks; however, not turbidity associated with growth has been seen to date. We are currently exploring different possibilities such as qPCR and sequencing to verify that L. asiaticus is actually growing in the media. With collaboration of Dr. Killiny s lab, we are currently analyzing other important components within the phloem sap of citrus to be added to AM2. We expect to propose new formulations based on this information.
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.
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