A heat treatment machine using steam as a source of supplemental heat was developed. The system is able to heat treat single citrus trees up to the height of six feet one tree at a time. The system consists of a hydraulically controlled enclosure which descends to cover the entire canopy. A steam generator, together with a water tank was mounted on the bed of a ‘goat’ truck. Steam from the steam generator is streamed into the enclosure. The system was used to test steaming scenarios using multiple combinations of temperature and heating duration. More than 800 trees were heat treated in five different groves. From the experiment, we determined that applying steam at temperatures above 60 C (140 F) are likely to cause damage to the trees. When testing treatments with a temperature of 65 C (149 F), we discovered that in most cases the trees did not recover. There were two scenarios which we tested using the steaming system and they both seemed to work equally well. The first one was steam treating the tree at 55 C (131 F) for 3 minutes and the second was steam treating the tree canopy up to either 60 C (140 F) or 30 seconds after reaching a temperature of 55 C (131 F), whichever came first. These two scenarios were selected after several trial runs of different temperature and heating duration combinations. From the trial runs, these two scenarios seem to provide sufficient heat without damaging trees or causing significant leaf lost which delays the recovery of the trees. The tested trees are still under observation and the effectiveness of the treatment will be studied over a longer period to evaluate the performance of heat treatment. At this time, three, six, and nine months post heat treatment samples have been collected from HLB-positive Valencia at three grove locations. Samples have been processed and analyzed for Las bacteria. In addition, fruit harvest for the 2013-2014 growing year has been determined for each tree in the study. Fruit set and first fruit abscission for the 2014-2015 year have also been counted. These counts will be compared to fruit counts of heat treated and control trees of the harvest in spring 2015. Initial results show that four-year-old Valencia trees responded better (increased canopy growth, decreased Las bacterial titer) than 15-year-old Valencia. Additionally, the treated HLB positive four-year-old Valencia located in a well-managed commercial grow (excellent fertilizer and insect management program) visually outperformed the other block of Valencia located in a grove with a weaker management program. In depth statistics of the three randomized block design experiments will be performed after one year samples are taken (August-September 2014).
The overall objective of this study was to deliver antibiotics into HLB infected citrus plants using nanoparticulate carrier systems such as emulsions, liposomes and polymer nanoparticles. During initial proof of concept experiments, dye-doped liposome and polymer nanoparticles encapsulating water-soluble dyes as surrogate for antibiotics have been prepared. In addition, double emulsions of water-in oil-in water have been developed with a stable inner aqueous phase for the delivery of the water-soluble antimicrobials to plants. Liposomes particles have been prepared using e.g 1,2-dioleoyl-3-trimethylammonium-propane , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol through the thin lipid film hydration, reverse phase evaporation and ethanol injection methods. Constituent lipids amounts were varied to encapsulate water-soluble dyes such as 9-aminoacridine, sulforhodamine B and fluorescein. In order to minimize the leaching of the dyes, cationic lipids were incorporated in the lipid composition. The particle size was restricted to less than 100nm by the use of appropriate filtration membrane and the choice of hydrating solvent. Cryo transmission electron microscopy of Liposomes prepared via thin film hydration (before extrusion) demonstrated particle size to be around 200nm. Dye-doped polymeric nanoparticles comprising of environmentally safe and biodegradable materials such as poly (lactic acid) (PLA) and encapsulating fluorescent dyes have been prepared using double emulsion method. Polymer particles were characterized for their particles size distribution, absorbance, and fluorescence properties. PLA particles mixed with adjuvants were sprayed on abaxial and adaxial sides of citrus leaves and analyzed for leaf uptake by IVIS spectrum imaging system. Whole leaf imaging allowed detection of the uptaken particles by leaves. Confocal experiments on select leaves showed the presence of the particles in the leaf surface. Future experiments will be planned to determine the particle and adjuvant conditions for increasing the uptake. In addition, double emulsion particles, i.e. water-in-oil-in-water or w/o/w were also developed. These emulsions contain an encapsulated aqueous phase suspended in bulk water were stabilized by varying the surfactants and relative ratio of the amounts of the internal and external aqueous phases. Emulsions containing oxytetracycline and dye in the internal aqueous phase were developed to be used for future experiments. Highlights: In these experimental trials three types of particles were developed for the delivery of water soluble compounds (antimicrobial agents) to citrus plants. ‘ Liposomes encapsulating water soluble dyes in the size range 100-200nm have been prepared and characterized. ‘ Polymer particles encapsulating dyes were prepared using safe and biodegradable polymers. The dye-doped polymer particles were applied to citrus plants and imaged after incubation. Whole leaf imaging and confocal imaging indicated the presence of the fluorescence particles near the surface. ‘ Double emulsions systems containing water soluble compounds in the internal phase were developed and analyzed using optical imagining. Emulsions exhibiting stability for about three weeks and encapsulating active ingredient were prepared for future studies.
Construction of the imaging system: An optical sensing system based on a modified DSLR camera is now largely constructed and is able to acquire images of isolated leaves and small citrus seedlings. At present, the device can take photos of fluorescent emissions at the following excitation and emission wave lengths: UV LED-364nm (emission filters: 500nm, 690nm and 730nm; Blue LED-470nm (emission filters: 500nm, 690nm and 730nm); Green LED-527nm (emission filters: 690nm and 730nm). In addition, the camera system can take images based on the optical rotation of polarized light by starch grains. Image analysis: Images of seedlings have been acquired and manipulated by focus stacking to produce a high resolution macro-image at visible wavelengths. The focus stacking approach will be applied to fluorescence and polarized images. In the initial stages, images are being taken of individual leaves from HLP-infected and non-infected sweet orange trees. Fluorescence images are combined as ratios to identify regions of leaves that show chlorophyll loss. The Optical rotation images are being used to map the location of starch accumulation within infected leaves. Psyllid-inoculation of citrus seedlings: Sweet orange seedlings are being germinated and are ready to be exposed to feeding by HLB-positive psyllids. However, these experiments have been temporarily delayed due to low population numbers in the infected psyllid colony. It is anticipated that psyllid feeding studies will be initiated within the next reporting period.
The project entitled ‘Further characterization of HLB resistant clones of selected citrus varieties’ (project no. 758) is in its second year. The aim of the project is to test plants that have exhibited tolerance/resistance to HLB in a previous field trial. After this confirmation, we want to conduct experiments to understand the basis of HLB tolerance. Research conducted this quarter (from April 2014 to June 2014) consisted of: a) As a part of the greenhouse evaluation trial, we have already challenged 66 selected plants by feeding them with Liberibacter positive Diaphorina citri in a no-choice situation. We are testing DNA extracts made from these plants at various time intervals to measure the Liberibacter titer in all the plants. We have also been collecting samples and extracting them to prepare RNA for future use in conducting RNAseq experiments designed to see differences between susceptible and resistant cultivars. b) During the spring of 2014, we conducted over 1000 pollinations using HLB susceptible and resistant cultivars belonging to the genera Citrus, Eremocitrus, Microicitrus and Poncirus. We have collected seeds from 2013 pollinations and we are testing them for the presence of genetic material from the pollen and seed parents. c) At this time we are in the process of psyllid challenge of another set of 66 plants in Fort Pierce. These plants were also selected based on the results of the previous field trial. We are on track as per our proposed milestones.
The purpose of this project is to determine methods to effectively eliminate Candidatus Liberibacter asiaticus (Las), the bacterium associated with huanglongbing (HLB) in Florida, from citrus. Emphasis was placed on cryotherapy with conventional shoot tip grafting being used for comparison purposes. Application of crytherapy to recover plants free of HLB from budwood approached 95 percent efficiency. The project also included determining the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. Infected material was sent to Ft. Collins, CO under permit for cryotherapy treatment. Recovered plants were allowed to grow for 12-14 weeks following therapy before testing for the presence of HLB or other pathogens as appropriate. Personnel were trained in the cryotherapy technique at Riverside, CA and at Ft. Pierce, FL. With pre-treatment and cryotherapy, results indicated the procedure was very effective at eliminating Citrus tatterleaf virus and citrus viroids. These pathogens are the most difficult to eliminate by thermaltherapy and/or by shoot tip grafting. Forty USDA Pummelo hybrids were passed through the standard cryotherapy treatment. The treated buds were recovered, and trees grown from this treatment have been maintained in a greenhouse and monitored for Las. To date none of these trees have tested positive for Las, even though the material which supplied the buds for the cryotherapy treatment did test positive. Approximately 75 advance USDA scion selections that existed in the field and were infected with HLB have been rescued by collecting budwood from the least symptomatic branches on the trees and subsequently grafting onto rootstocks in the greenhouse. As the trees grew in the greenhouse, they were monitored for Las at intervals and trees testing Las positive were destroyed. The trees that test Las negative will be maintained and testing will continue. In Riverside, a system has been developed using Jiffy pots for the seedling used as indicator plants, allowing for a grow out of about 75-90 days after the seed was sown until the young plants can be inoculated and used as indicators. The inoculum bud survival is very high, greater than 98 percent. The entire indexing procedure can be done with the plants in the Jiffy pots. A trial was performed to compare the results obtained by using the mini-plant biological indexing compared with the traditional standard indexing protocol, using indicator plants 10-14 months old, for 15 accessions. The results from the mini-plant index revealed the presence of Citrus vein enation virus in one accession which had been missed using the traditional standard indexing protocol. The advantage of using the mini-plants for biological indexing is that 35 indicator plants may be contained in the space normally occupied by three traditional indicator plants; quicker turnover of the small plants for indexing means more accessions may be tested per year; biological indexing for pathogens expressing only under cool temperatures may be indexed for year-round as the smaller plants may be placed close to the cool pads in the greenhouse, and because they occupy less space, the whole trial can be done utilizing the cooler temperatures. This project was to have been funded beginning April 2012 and to continue for two years, ending April 30, 2014. The agreement was not executed until September 28, 2012, and funding allocated on December 18, 2012 but due to inefficiencies in the various systems, it was not possible to submit an invoice for funds for the research performed the first year. A request for a no-cost extension was denied at the end of the second year, so the results reported are the result of of one year’s funding for what was to be a two year project.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. In the enhancement project, we are expanding the field test to include two more field trials in two more locations, larger scale of field test, test more beneficial microbes, and test different approaches to enhance the survival of the beneficial microbes in the soil. The field trial has been done for 9 nine months for one experiment site and 5 months for a second experiment site. For one site, nine applications were conducted. We have surveyed the disease incidence twice for this site. For another site, the first application was conducted in February, 2014. The experiment is ongoing with monthly treatment. We conducted the background survey regarding HLB disease incidence, severity, Las titers, Phytophthora, nematodes, root (root density and health status), and microbial diversity right now. We will be collecting most meaningful data including disease incidence, Las titers, gene expression of defense genes, survival of beneficial bacteria around 12 months after first application. For a better understanding of the Microbe Program, all the tested products were analyzed for the microbes in the products. The survival of the tested microbes are being investigated.
The overall objective of this research project is to develop an effective and sustainable phage/phage component-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. We currently have 39 Xac high titer phage lysates that are assembled into 13 groups based on host range and type IV pilus dependency for host infection. First round efficacy protection and therapeutic evaluation of a phage cocktail was conducted in cooperation with Dr. Nian Wang (University of Florida-Lake Alfred). A cocktail composed of four characterized phages from four different groups was used. Hamlin sweet orange plants grown in a citrus canker quarantine greenhouse were trimmed and fertilized to induce new growth. Plants were spray treated with sterile tap water (STW), Xac or Xac/phage cocktail mix. Xac inoculated plants were treated with the cocktail at a multiplicity of infection (MOI) of 20 or 0.5, 6 h-pre- or 6 h-post-Xac inoculation (5 X 10^6 or 5 X 10^8 CFU/ml, respectively). Control plants were inoculated with STW or Xac. Symptom development was monitored and the Xac population was quantified for a 21-day period. In in two independent experiments, when phage was applied, pre, post or at the time of the inoculation of Xac, reduced canker lesions numbers were observed, as compared to the Xac only inoculated plants. The most effective canker lesion control was observed when the phage cocktail was applied simultaneously with Xac at an MOI of 20, with the same treatment at an MOI of 0.5 resulted in reduction too. Treatment of plants 6 h pre- or post-Xac inoculation at an MOI of 20 or 0.5 also resulted in a reduction in lesions. The bacterial populations recovered from the leaves correlated with the visual observations over the 21-day post-inoculation period. In a separate study, phage movement experiments were conducted using Hamlin plants grown in the TAMU greenhouse facility. Plants were trimmed and fertilized to induce new growth. Phage CCP504 [8 X 10^8 plaque forming units (PFU)] was injected into each of two sites/stems, inoculating multiple stems with branching to determine phage movement and/or persistence over a three week period. Samples were obtained at time of inoculation and at 1, 2, and 3 weeks post inoculation, all in duplicate. Time point samples were sub-divided into 2-inch segments below and above the point of inoculation (POI) and homogenized in buffer. The samples were processed and the PFU/g of plant tissue determined both by direct plating and qRT-PCR. Initial studies indicated that phages survived three weeks in citrus plants without a host and moved from the POI into leaves. We have also identified two new tailocins, XT-4 and XT-5, the former exhibiting an active broad host range (killing13/13 Xac isolates-similar to XT-1) and the later a narrow host range (4/13). However, the host range of XT-5 does not overlap with that of the previously reported XT-2 or XT-3 tailocins. In our continued expansion of the phage pool, we have isolated 11 new potentially unique phages that are currently being characterized.
The focus of this project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using Liberibacter crescens strain BT-1 as the host. It is hypothesized that once ‘Candidatus’ Liberibacter asiaticus (Las) is successfully cultured, the protocols developed for L. crescens will be adaptable to Las. We developed an overlay assay for L. crescens that to date has been used to test 329 individual phage lysates from diverse hosts, as well as 12 broad host-range tailocins. Utilizing the same system, 131 plant extracts, 33 citrus psyllid extracts, and 55 water samples have been assayed for the presence of phage/tailocins active against L. crescens BT-1. No phage or tailocins active against L. crescens BT-1 have been detected to date. Additionally, liquid cultures of 32 diverse bacterial isolates were mitomycin C and/or UV-induced and tested for phage, tailocin, and/or bacteriocin activity against L. crescens BT-1, with no activity detected. Previously we reported antimicrobial activity against L. crescens by a Microbacterium isolate. We expanded our search to include a wide range of bacteria, including alpha, beta, and gamma proteobacteria, as well as actinobacteria; with most microorganisms exhibiting no antibacterial activity when tested against L. crescens using the overlay assay. However, several Actinomycetales produced large zones of growth inhibition against L. crescens. The diameters of the zones produced are indicative of small molecular weight compounds. To identify the compound(s) responsible for the observed inhibitory activity, strains producing inhibitory zones were grown using a range of conditions. Minimal or no production was achieved for most strains using standard liquid production techniques. Production conditions for the Actinomycetales antimicrobials are currently being optimized. Fractionation of the previously reported AC-313 inhibitory compound from Microbacterium (strain TM-313) indicates that the active compound is a mixture of three compounds. Large-scale production using strain TM-313 (10 liters) has been initiated to obtain quantities of the AC-313 compound(s) required for structural characterization. We are continuing the search for environmental phage(s) active against L. crescens, while initiating a recombinant approach. Sequences encoding for putative phage tail fibers have been identified in the L. crescens BT-1 genome. Experiments will be initiated to use sequence of identified phage tail fibers in the construction of a tailocin with activity against L. crescens BT-1.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. Three compounds exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control, where the level of biofilm formation were reduced to 50% and 60% of control, respectively. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected Xac 306 infection of unwounded and wounded citrus fruits at sub-inhibitory concentrations. We have completed testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. In the NB medium, planktonic cells exhibited a MIC of 0.50 mM CuSO4 without biofilm inhibitor. In the presence biofilm inhibitors at sub-MIC concentrations , the MICs of CuSO4 against Xac 306 planktonic cells were decreased to 0.25 mM. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. The results have been published by Phytopathology in a manuscript entitled: Foliar application of biofilm formation-inhibiting compounds enhances control of citrus canker caused by Xanthomonas citri subsp. citri. One patent is filed based on the results. We also identified multiple new biofilm inhibitors. The effect of those biofilm inhibitors to control citrus canker is being investigated. We tested the survival of both biofilm deficient and QS mutants on fruit surface. Effects of biofilm formation inhibitors on Xac infection on detached immature citrus fruit were tested using spray inoculation. The inhibitors affected the infection of Xac on both unwounded and wounded citrus fruits. We are testing more potential biofilm inhibitors. We continue characterizing how quorum sensing and biofilm formation contribute to Xac infection of citrus fruit. Multiple virulence genes involved in quorum sensing and biofilm formation are being investigated. The involvement of ColR, RpfF, and three more genes involved in biofilm formation or quorum sensing is studied in details. Two manuscripts are under preparation. The field trial (two different sites) is ongoing to test the effect of the identified biofilm inhibitors to control citrus canker. One new compound is able to inhibit QS at a concentration of 100 .M based on observation of bacterial phenotype of aggregate formation. Plant test in greenhouse showed that the QS inhibitor (100 .M) treatment could reduce the formation of canker lesions and bacterial population on spray-inoculated grapefruit leaves simultaneously with the canker bacterium Xcc 306.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). Currently, sprays with copper bactericides are the primary mean in controlling citrus canker, which causes adverse effects on the environment by contaminating ground water or accumulating in the soil and affecting tree health. The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene via its 17.5 tandem repeats, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Next, we will use both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach.
The goal is to develop short term approaches to control citrus Huanglongbing (HLB) using antibacterial-producing bacteria. Recent studies indicate that HLB severely damaged citrus roots. The destructive effect of HLB on roots partly results from Candidatus Liberibacter asiaticus (Las) infection of the roots. In this study, we will conduct the following objectives: Screen bacteria that can produce antimicrobial compounds against Liberibacter crescens and related Rhizobiaceae bacteria; purify and characterize antimicrobial compounds produced by the screened bacteria; illustrate the regulation of antimicrobial production by producing bacteria at different environmental conditions and with different inducers; investigate the growth of the producing bacteria in different conditions; and control HLB by delivering antimicrobial compounds using the producing bacteria in the soil. We have isolated 84 strains of Streptomyces spp., Bacillus spp., Paenibacillus spp., and Pseudomonas spp. producing antibacterials from Florida citrus groves. We have also acquired 28 different species of Streptomyces spp., Bacillus spp., Paenibacillus spp., and Pseudomonas spp. strain producing different antibacterials. The antagonistic activity against Agrobacterium and Sionrhizobium meliloti was determined. The primary screen was done, and 5 strains showed good activity. The second round of antagonism (quantitative) is being performed to quantify the activity. These strains are being inoculated to citrus roots and the colonization is being determined by inoculation and recover method. These strains will also be labeled with GFP and the tissues (roots, branches and leaves) will be observed under florescent microscope to determine the colonization and movement of these strains. The effects of inoculation to the HLB infected plants are being determined.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates was tested using citrus (grapefruit) seedlings in greenhouse. Greenhouse assays suggested that a consortium of three Bacillus and relative isolates (AY16, PT6 and PT26A) may delay the development of both HLB symptoms and pathogen population on citrus leaves after root inoculation. The potential of the consortium to recover the tree decline from HLB infection is being evaluated in greenhouse. The growth conditions of the three strains were optimized using a small fermenter. Three antifoam agents, A204, PPG200 or M-Oil did not affect the growth of the three bacterial strains. The initial neutral to alkaline pH values (7.0 ~ 8.0) favor growth of the three bacteria in LB, while acidic pH (5.0 ~ 6.0) suppress bacterial growth. The optimal cultural temperature was determined to be around 30C with average bacterial population of 109-1010 cfu/ml after 20-hour incubation, although the bacteria may grow slowly under room temperature (~ 23C). The shelf life of three different formulations of the bacterial culture is being evaluated under room temperature. In a six-month time course, the bacterial populations in LB broth, OPB broth and tape water are comparatively stable with initial and final both at ~ 108cfu/ml. Four field trials are being conducted including more beneficial bacteria. For one of the field trial, nine applications have been performed. We are evaluating the survival of the beneficial bacteria in the soil. The application method has been changed during application to improve the survival of microbes in the soil. We are developing new methods to further increase the survival of microbes in the soil and allow the microbes have easy access to the roots. We are also testing the colonization and survival of the tested microbes on citrus roots and in rhizosphere soil in greenhouse using conventional culture-based method and qPCR-based method. We are evaluating plant immunity response by studying the expression of specific genes related to plant defense.
Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated and treatments already started. The follow up investigations are ongoing, including monitoring the HLB symptoms, disease incidence and Las titer in leaves. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We have applied different compounds at three separate field trials. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. We are also testing the mechanism of those compounds showing positive effect on HLB control. Our results indicated that those compounds upregulated defense related genes, e.g., PR2, but not PP2 an callose synthase genes, in both field and greenhouse. Las titers were also lower in treated plants than non-treated controls with the four compounds. We have investigated the effect of those compounds on disease severity, yield, juice content and quality. We will repeat those treatments for one more year. Currently, the treatments are being conducted. SA hydroxylase is being expressed and purified. New SA hydroxylase inhibitors are being identified using molecular modeling.
The goal of the research is to control citrus HLB using small molecules which target essential proteins of Candidatus Liberibacter asiaticus (Las). In our previous study, structure-based virtual screening has been used successfully to identify five lead antimicrobial compounds against Las by targeting SecA. SecA is one essential component of the Sec machinery. Those compounds showed promising antimicrobial activity. However, further work is needed to apply the compounds. We will evaluate the important characteristics of our antimicrobial compounds including solvents and adjuvants, phytotoxicity, antimicrobial activities against multiple Rhizobia, antimicrobial activity against Las, application approaches, and control of HLB. Those information are critical to for the practical application of those antimicrobial compounds in controlling HLB. We also propose to further optimize the five lead compounds. In addition, we propose to develop antimicrobial compounds against lipid A of Las. The lipid A substructure of the lipopolysaccharides (LPS) of Sinorhizobium meliloti, which is closely related to Las, suppresses the plant defense response. Las contains the complete genetic pathway for synthesis of lipid A. We hypothesized that Las uses lipid A to suppress plant defense. Thus, targeting lipid A could activate plant defense response. Lipid A is also an ideal target and has been targeted for screening antimicrobial compounds for multiple pathogenic bacteria. We are identifying the small molecular ‘or’ peptide inhibitors against LipidA using pharmacophore based methods and finalizing the list of the compounds for the activity studies. For SecA inhibitors, we are optimizing the compounds in collaboration with IBM. Two compounds with slightly higher binding affinity than C16 were identified. Currently, we are evaluating the best range of composition ratio among each component (%weight) of AIs, solvents and surfactants. The following characteristics are being evaluated: 1) emulsion stability and ease of emulsion; 2) stability of diluted concentrate; 3) freeze-thaw stability; and 4) phytotoxicity to citrus species. We have successfully identified one formulation suitable for all five compounds without phytotoxicity. Using the formulation, we have tested all five compounds against eight different bacterial species including Liberibacter crescens. Field test is being conducted. We have compared spray and trunk injection. Trunk injection seems to have better efficacy. We are synthesizing two compounds at large amount for field trial.
The routine leaf tissue analyses for this project are behind schedule because we were prevented from submitting samples to our regular analytical lab due to restrictions on the movement of plant materials and soil. These rules are being enforced by DPI as part of the citrus quarantine for Florida. Once the necessary permitting is in place, we will resume leaf nutrient analysis and reporting for this project.
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