This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (Jan 2016 to March 2015), we continue to evaluate 1) the effect of Pen and SD on control of HLB disease by gravity bag infusion in the field; 2) the efficiency of effective chemical compounds (Pen, SDX, Pcy and Carv) against HLB disease by gravity bag infusion; 3) the effectiveness of a combination of chemotherapy, thermotherapy and nutrient therapy against HLB in the field trials; 4) the efficacy of the new adjuvants to improve the uptake of antimicrobials. The chemical compounds (Pen and EBI-602) and additional nutrients were applied to the heat-treated citrus for three times by foliage spray, using our optimized nano-delivery system. The preliminary results showed that Pen was the more effective to control Las bacterium than EBI-602. The disease severity index (SDI) decreased by 6% after applied with Pen. The integrated practices (antimicrobial treatment coupled with heat treatment and nutrition fertilization) could decrease the fruit drop by 10~20 %, increase the fruit and juice weight by 3~13 %, and decrease the ratio of brix to acid by 0.2~5.0 %. The preliminary results from the other five antimicrobials (SD, Pen, SDX, Pcy and Carv) applied by gravity bag infusion showed that there were not different in the Las bacterial titers among the treated antimicrobials. Compared to the untreated plants, all antimicrobials reduced the Las bacterial titers, especially PEN. Both SD and Pen reduced the DSI through two years application. In last quarter, we tried to evaluate two new adjuvants (Bio and MF200) for improving the effectiveness of Pen by foliar spray. The preliminary results indicated that Pens formulated in both Bio and MF200 decreased the Las bacterial titers a lots. Ten antimicrobials were prepared in two different concentrations of the nano formulations (0.1 % and 1.0 %) in the greenhouse test. The Ct values kept over 36.o in the PEN-treatment. In next quarter, we will keep our application. Pcy and Carv will be changed application from trunk-injection to foliar spray. One papers has been published in the Crop Protection.
Citrus blight continues to be a major economic problem in citrus groves in Florida. Thousands of trees each year succumb to citrus blight, with estimated losses at over $60 million per year. The disease can occur on all common citrus cultivars, and Carrizo citrange are especially susceptible. Early symptoms are zinc deficiency in the leaves which may disappear, zinc accumulation in the phloem and eventually high zinc levels in the xylem. Blockage of xylem tissues with amorphous plugs follows with reduced water uptake. The causal agent of citrus blight is unknown. However, symptoms and all of the characteristics associated with citrus blight can be reproduced by root graft inoculations. Therefore in a project previously funded by CRDF we used NGS RNA sequencing protocols to look for novel viruses in roots of sweet orange with blight, but not present in roots of healthy trees, or trees affected by HLB. We identified several related endogenous pararetroviruses related to Petunia Vein Clearing Virus (PVCV) using a collection of 10 RNA libraries prepared from 10 different root samples collected from healthy trees or those with blight or HLB. In the quarter just ending we have focused our efforts on the remaining objectives: generating complete genome sequences for any and all active blight associated pararetroviruses and developing a active virus specific assay comprehensive enough to detect all blight associated pararetroviruses. Progress towards the generation of a complete genome sequence for the blight associated pararetroviruses was stymied by the discovery of a variable region in the genome. As of the last quarter we were reporting 90% complete genome sequence. This quarter we are pleased to announce complete genome sequence. It should be noted that the complete sequence generated represents a single isolate from an individual tree, and pararetroviruses are known to exhibit considerable genetic diversity. Therefore the complete genome sequence of a single isolate was used to further generate primers to sequence the pararetroviruses from additional infected trees, which is necessary to develop a diagnostic assay capable of detecting all active pararetroviruses. Regardless, we moved ahead with developing assay for the detection of active pararetroviruses, and began screening multiple assays on the 50 tree sample set that was collected in the first year of the project. Of the eight assays developed, two show siginficant promise for being both comprehensively inclusive and highly senstive for detecting the active blight associated pararetrovirus.
We built passive spore traps and deployed six in an Immokalee area grove with a history of citrus black spot and three in the Lake Alfred, CREC groves that do not have a history of black spot. The traps were installed at the heights of 0.5, 1.0, and 1.5 m. The goal was to determine if passive traps could capture enough Guignardia spp. ascospores to serve as a detection method to monitor for the spread of G. citricarpa prior to disease expression. The disease is often not detected for years after the fungus is introduced to the grove. In our study, we were not able to detect many Guignardia spp. ascospores with the passive traps. When compared to the Burkard style spore trap that has a flow of air across the tape used to trap the ascospores of 10 L/min, there was at least a 10 fold reduction in ascospore counts. We were able to establish that there is no difficulty in extracting DNA from G. citricarpa conidia on the silicone coated slides so if a sufficient number of ascospores were detected, then qPCR could be used to identify the species and approximately how many without the tedium of counting spores. While we were not able to capture enough ascospores to declare the trap design a success, there is some promise and perhaps with a different trap design this could be a useful tool in ascospore monitoring.
During the 769 project, we attempted to bring a group of people together to culture Ca. Liberibacter asiaticus (CLas). Mike Davis gave us his latest recipe for culturing CLas. Dean Gabriel did experiments that showed that the bacteriophage was not expressed in Mike Davis’s cultures. Mark Hilf had some ideas but these did not contribute to the progress made to date. Nabil Killiny was very helpful. He completed and published a metabolome of citrus phloem. He is now working on a similar metabolome of the psyllid gut. We used the citrus information to add several components to the medium we are now using. A defined medium for L. crescens was developed. This has served as the foundation for optimizing a medium for CLas. Meanwhile, several approaches suggested refinements to the medium. Comparative genomics of various Liberibacter showed that CLas probably requires a high osmotic environment where L. crescens does not. As a result, we added various sugars to the medium at the levels Dr. Killiny observed in citrus phloem. A transcriptomic analysis of L. crescens showed a high expressed of an ascorbate transported in the medium suggesting that ascorbate was limiting in the medium. A small RNA analysis of L. crescens suggested that the potassium levels we were using was too high. We have since reduced potassium levels. Proteome analysis of L. crescens showed the expression of 50 proteins in L. crescens whose gene homologs are lacking in CLas. Of these, 30 are of known function, 20 are of unknown function. Determining the functions of these 20 proteins may be important in solving the riddle of CLas culturing. The 30 proteins of known function mainly code for amino acid synthesis suggesting that certain amino acids be added to the medium. In addition, proteomics taught us that nicotinate and zinc are needed in any CLas medium. A new round of multi-omics analysis of L. crescens in a defined (that provides slow growth) and undefined medium (that provides faster growth) is in progress and should tell us more about factors limiting the growth of Liberibacter strains in culture. An essential gene set was determined in L. crescens under BM-7 medium growth conditions. Mutagenesis of L. crescens identified 314 genes that are essential for growth in culture, of those, 238 have homologs in CLas. The 76 essential genes of L. crescens that are needed for growth of L. crescens in BM-7 medium likely code for functions required for growth of CLas on that same medium. These results suggested that folate, fatty acids, thiamine, and pantothenate are required for any CLas growth medium. As a result of all of these studies, we now have a medium called AM13 that allows very slow growth of CLas in culture. We now need to confirm the purity of this culture and optimize it for faster growth. We also need to sequence the genome from CLas in culture since it is likely to be a more accurate genome sequence than was obtained from psyllid metagenomes in the past. This is in progress and we found a novel way to do this without amplifying the DNA. The growth observed in AM13 is very slow and the cultures never become turbid. Thus, improvements to the medium are still needed to get the cell numbers necessary for many future experiments.
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. In this last quarter, we expressed and purified LexA (CLIBASIA_01645) fused with a His-tag for purification and further analysis of the protein. Mobility shift assays (EMSAs) have now been used to confirm that this repressor protein both binds to its own promoter, as is typical of LexA repressors, and also binds specifically to the primary SC1 lytic prophage early gene promoter. This binding was quite specific to a particular small fragment in the SC1 early gene promoter region and we are now characterizing the region. This finding makes this LexA repressor a promising target for chemical inhibitors, since inhibition of SC1 is likely critical to Las survival in psyllids. Objective 2 is control of HLB using a repressor protein of unknown identity from psyllids as target. This repressor has been was published by us as silencing the Las phage holin gene, expression of which is lethal. We reported earlier that Las-free psyllids carry the repressor, and that similar extracts from Drosophila (fruit fly) do not. The repressor protein from the psyllid extract was immuno-captured using holin promoter DNA immobilized on magnetic DynaBeads. Sufficient protein was captured to enable visualization by Commassie Blue sent for liquid chromatography tandem mass spectrometry (LCMSMS) analyses to attempt to identify the protein. Twenty-five candidate peptide fingerprints were identified, of which two were of bacterial origin. A Wolbachia protein that seems to be unique to Wolbachia found in psyllids was identified that is within the characterized size range of 10- 50 kD (a small protein) and that is also potentially a DNA-binding protein. This protein candidate is not found in Wolbachia sequenced from Drosophila or other insects and is being cloned into an expression vector for use in activity assays with the holin promoter reporter construct used to identify the psyllid repressor activity. Objective 3 is control of HLB using the Las phage peroxidase and Las lytic cycle activator(s) as targets. We are now standardizing the inhibitory dose of hydrogen peroxide against Liberibacter crescens wild type strain BT-1 (lacking peroxidase) and BT-1 transformed with Las peroxidase from prophage SC2. These will be used for high throughput peroxidase inhibition screening assays using two Prestwick combinatorial chemical libraries in a 96-well plate format using a spectrophotometric plate reader. Once an ID50 (50% growth inhibition) value is established, inhibition of SC2 peroxidase by candidate combinatorial library chemicals may be possible. Alternatively, crude cell free protein extracts of wild type and peroxidase containing BT-1 strains will be assayed using an in vitro peroxide/ peroxidase assay kit in a 96-well format screen.
In this reporting period, nine different variants were included for the optimization of T-SOL. 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 which suggested binding of metal ions with the chelate functional groups (such as carboxyls, hydroxyls and amines). Microplate Alamar blue assay was used to determine the minimal inhibitory concentration (MIC) of the different variants of the T-SOL. The antibacterial efficacy of T-SOL variants with the chelating agents (1, 0.5 and 0.1M with respect to the metallic Zn) were screened against Escherichia coli and Xanthomonas alfalfae. The MIC of the T-SOL variants (all three chelate concentrations) was found to be in the range 80 ppm 160 ppm (metallic zinc) for both E. coli and X. alfalfa. Interestingly, the MIC of the T-SOL prepared with agriculture-grade Zinc source was found to be 40 ppm (metallic zinc) for all the nine T-SOL variants when screened against both X. alfalfa and P. syringae suggesting that the antibacterial efficacy was not affected by the source of zinc. Preliminary trunk injection study has been initiated with T-SOL which contains a plant tissue permeable compound (inert) using a 2 Gallon size citrus plant. No noticeable phytotoxicity was observed after a week of injection of about 4.0 mL T-SOL (1000 ppm metallic Zn) formulation. In the coming reporting period, optimization of the synthesis protocol of T-SOL with agriculture-grade chemicals will be performed and their antimicrobial efficacy and phytotoxicity studies (foliar spray and trunk injection) will be conducted to narrow down the most effective T-SOL variant that can be later taken to green house and field trial studies.
This research project aims at developing an alternative to Cu biocides. Fixed-Quat is a non-phytotoxic highly potent antimicrobial formulation which are being evaluated in this project. In the previous reporting period, Fixed-Quat A-II and AP-II nanogels were studied for their antimicrobial and plant safety properties. These formulations demonstrated strong potential for further development on which study is being continued. In this reporting period, a new environmentally-friendly, minimally processed silica core material (EPA approved for food use; cost effective) was used to create a new version of Fixed-Quat, referred to as Fixed-Quat-E. Fixed-Quat E nanogel was synthesized with a quat concentration of 9,000 ppm (the quat active is cleared for EPA for food use ). The composition and interactions between the active and inert components were confirmed using Fourier Transform Infra-Red (FTIR) spectroscopy, with Si-O stretching, SiO-H stretching and Si-H bending confirming the presence of silica. FTIR confirmed the presence of quat with N-H stretching and bending. The morphology of Fixed-Quat E materiall was examined using Scanning Electron Microscopy (SEM) which revealed sub-micron to micron composites with irregular shapes in multiple layers. X-ray Diffraction (XRD) was used to study the crystallinity of the new silica core material and results indicated that the material is primarily amorphous in nature. Phytotoxicity study of the Fixed-Quat E material was 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 Tomato sp, an ornamental plant revealed no sign of plant injury even as high as 1000 ppm Quat. It is noted that EPA maximum allowed concentration for industrial use of Quat is 200 ppm), indicating a large therapeutic window. Antimicrobial studies of Fixed-Quat E material was conducted against 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 = 2.0 g/mL for X. alfalfae, = 2.0 g/mL for P. syringae and = 2.0 g/mL for C. michiganensis. Future studies will involve rainfastness evaluation and preparation of formulations for 2016 citrus canker field trials.
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.
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