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 have identified multiple small molecular ‘or’ peptide inhibitors against LipidA using pharmacophore based methods and are finalizing the list of the compounds for the activity studies. Several lipid A inhibitors have been ordered and are being tested against Las and its relatives. For SecA inhibitors, we are optimizing the compounds in collaboration with IBM. Two compounds with slightly higher binding affinity than C16 were identified. We also identified multiple SecA inhibitors. We are testing their inhibitory effect against purified SecA right now. 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. For the field trial, 120 trees of four years old were selected. HLB disease severity was surveyed. The Las titers were measured for each tree using qPCR. We have compared spray and trunk injection. Trunk injection seems to have better efficacy. We will repeat one more time to compare the efficacy of different application methods. We have synthesized two compounds at large amount for field trial. The field trial is ongoing on small scale. We are currently analyzing Las population at different times after treatment. The yield data will be compared.
The goal of this project is first to identify a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian citrus psyllid (ACP). The toxicity of the selected toxin will then be enhanced by addition of a peptide that binds to the gut of ACP. This modification of the toxin is expected to enhance both binding and toxicity against ACP. The proteolytic profiles of toxins from 42 Bt isolates have been characterized so far at Iowa State University. Methods used for toxin solubilization, and characterization of the proteolytic profile and stability were as described in the previous report. The results indicate the presence of several different toxin groups based on the protein profile seen on SDS-PAGE following trypsin-treatment of each isolate. In bioassays conducted at USDA ARS Florida, of the twenty-three Bt isolates tested, two isolates show promise with high ACP mortality rates relative to control treatments. One strain was selected as the most promising strain for further purification of individual toxins by ion exchange and gel filtration column purification. A phage disulfide-constrained heptapeptide library (Ph.D.-C7C; New England Biolabs) was screened for ACP gut binding peptides. Adult ACP were fed on sucrose diet containing the phage library by membrane feeding and the guts dissected. Phage that bound to the ACP gut membrane were eluted from the dissected gut in pH 2.2 buffer. Eluted phage were amplified and the screen repeated three times for enrichment of phage displaying peptides that bind to the ACP gut membrane. To analyze the selected clones, overnight cultures of E. coli were diluted 1:100, infected with single phage clones, grown for 5 hr with shaking at 37’C, and the culture harvested. Single phage ssDNA was purified and used to determine peptide-encoding sequences. Five sequences were obtained after the three rounds of selection; two of them were enriched in the second and third rounds of selection. These two peptide sequences provide the most promising candidates for use in modification of a selected Bt toxin with basal activity against ACP.
Update on the Lake Placid ACPS / Open Hydroponics (OHS) / Controlled Release Fertilizer (CRF) trial: The objective of this experiment was to evaluate an advanced production method for replanting in HLB-endemic groves. A 4.7-acre ACPS demonstration experiment was planted with ‘Vernia’ orange trees at 8×18′ spacing (303 trees/acre) in April 2011. Main treatments for research and demonstration are three rootstocks (Rough Lemon, C35, and X639) in factorial combination with three fertilizer / irrigation methods (Microsprinkler OHS, Microsprinkler CRF, Drip OHS) A field day was hosted in the Lake Placid experiment on March 7, 2012, and a useful handout with photos and more information is available at http://www.crec.ifas.ufl.edu/extension/extension_meetings/PDF/Field%20day%20handout.pdf’ An update of the trial with photos and data was also published in the August 2014 edition of “Citrus Industry” The 180 acres of grove surrounding the new research block was already close to 100% HLB-positive in 2011. An aggressive psyllid control program of neonicotinoid soil drenches and foliar pesticide sprays kept the HLB infection rates in the new trees low for the first year, but by age 3 years, the 4.7-acre block was 50% HLB-symptomatic. So far the affected trees have continued to grow vigorously, assisted by foliar and soil-applied nutritional treatments. Tree height at 3.5 years age averaged 9.7 to 12.4 feet, depending on the rootstock (Table 1). ‘Vernia’ orange on rough lemon rootstock grew significantly taller (by 2.6 feet) than on either X639 or C35 rootstocks but the three different fertilizer programs performed equally well in this experiment. Table 1: Average tree height (in feet) of ‘Vernia’ orange trees at 3.5 years of age on three rootstocks and with three ACPS fertilizer programs. Average heights in columns identified by different letters (a,b) are significantly different. Fertilizer treatment NS Roostock*** MS-OHS MS-CRF Drip-OHS Rough Lemon 12.387a 12.379a 12.392a C35 9.971b 9.779c 10.325b X639 9.692c 9.700c 9.913b We plan to measure the 2014/15 fruit yield in the plots in order to determine the economic production of this ACPS block, and the best combination of rootstock and fertilizer / irrigation treatment.
Report for the nano-particle “enhancement” project dealing with antimicrobials: The last set of nanoparticles consisting of a double liposome configuration has been synthesized by Sharma’s laboratory and delivered to the testing laboratory in Lake Alfred. The double liposome constructed for our purposes consists of a larger liposome of approximately 200 nm encapsulating smaller liposomes of 10 nm containing Alexa-488. The matrix is an aqueous buffer at pH 5.5. Following the established protocol, trees have been treated with the double liposome solution and are now in the growing period before final analysis. This is the last nanoparticle treatment in the series of 4 types of liposomes used for this study. There are no new results to report for the nutrient uptake experiments during this period.
HLB is characterized by impaired phloem performance, hormonal imbalance, poor root function and architecture, limited nutrient supply resulting in immature fruit drop and ill tree health/death. Our goal is to restore the proper functioning of the phloem tissue and consequently restore tree health by using the newest plant hormone ‘Strigolactones’ (SL). As opposed to other growth regulators, SL are carotenoids derived hormones demonstrated to induce cambial activity, fruit growth and development and symbiotic arbuscular mycorrhizal (AM) fungi association in many crops. SL applications have resulted in substantial increases in vascular tissue including phloem. Besides SL regulates root and shoot architecture by increased formation of primary roots, lateral roots, and elongation of root hairs. Application of SL either foliar and/or drench to citrus trees in nano- to micro- mole quantities, should to induce new phloem, roots in and regulate shoot architecture of HLB trees resulting in restore tree health. month intervals. To carry out this project, a Postdoctoral associate was hired. The individual is already established in the lab and is working full time. Greenhouse experiments: For the greenhouse trial,s 50 Valencia on Carrizo trees were purchased. Trees were planted on 3.5 gal pots and placed in a greenhouse. An automatic irrigation system was established and trees were allowed to acclimate for 30 days. Treatments have been applied to both healthy and HLB trees and growth patterns being recorded. A second spray is scheduled for January. Field experiments: Field grown HLB-affected Valencia trees have been selected in a block at CREC. Trees have been flagged, area under the tree has been cleared, fruit per tree counted and experimental fruit flagged. Petiole measurements have been taken and fruit drop is under observation. A preliminary concentration curve experiment for strigolactone was conducted with mid-season fruit to verify the optimal concentration to be used. HLB trees have been sprayed as scheduled. “Healthy” appearing Valencia trees have been identified at a grove in Wiersdale, FL. These trees will serve as control for the field experiments.
Psyllid-inoculation of citrus seedlings: Sweet orange seedlings have been growing at the CREC (psyllid growth room of Dr. William Dawson) for 7 weeks. During that period the seedlings were exposed to feeding by HLB-positive psyllids. These plants are scheduled for harvesting after week 8. Preparations are underway to catalogue every leaf on each plant using three emission filters (500nm, 690nm and 730nm) using three excitation wavelengths (UV 364nm, Blue 470nm, Green 527nm) in addition to polarization images at 594nm). Results from image acquisition and PCR results will be included in the next progress report.
Sequence analysis and comparison of pathogen genomes can provide valuable insights into epidemiological spread, sources of phenotypic variation, and the genetic bases for pathogen survival and interaction with host plant and insect vector. Although experimental intractability has slowed generation of sequence data for Ca. Liberibacter, the number of sequenced strains continues to grow, with those of Japanese strain Ishi-1 (Katoh et al, 2014) and the California isolate HHCA (Zheng et al, 2014), released in the last two months. These bring the total number of sequenced Ca. Liberibacter strains and species to seven. The CG-HLB Genome Resource Website has previously hosted a GBrowse based genome viewer for viewing sequence data and analyses of Liberibacter genomes. Though an ‘industry standard’ with many valuable capabilities, the user interface for GBrowse is not optimal. To supplement and eventually replace the GBrowse viewer, a JBrowse viewer has been installed on the CG-HLB website and currently hosts seven Ca. Liberibacter genome sequences. Gene and CDS features are shown by default with additional analytical tracks available for selection by the user. As with GBrowse, tracks in JBrowse are hyperlinked to other databases, maximizing the network of information available for different genome features. For example, the JBrowse view of reference strain CLas psy62 includes tracks for metabolic pathways, subcellular locations, structural predictions generated by the Grishin lab, expression data, repeat regions, and genome comparison information. However, JBrowse has the added advantage of a fast and seamless user interface that allows for smooth panning, zooming, and navigation. JBrowse also accommodates easy viewing of the locations of single nucleotide polymorphisms (SNPs). Sources of variation among CLas strains sequenced thus far occur largely at the level of SNPs. For instance, Las gxpsy was shown to have 1766 SNPs relative to Las strain psy62 and Las A4 to have 1410 SNPs relative to Las psy62. Mapping of SNP locations can be used as markers for tracking strain movement as well as providing insights into genetic sources of phenotypic variation. Additional information about the use of the JBrowse viewer is available on the CG-HLB Genome Resources website at http://www.citrusgreening.org/HLB-JBrowse.html. The previously installed Gbrowse viewer will continue to be available for the foreseeable future.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. Although we previously reported that LasAI and LasAII target host mitochondria, we had to co-inoculate the gene silencing suppressor P19 to have a detectable expression of LasAI and LasAII (PLoS ONE8:e68921, 2013). After optimization for a variety of parameters that are critical for efficient gene expression in plants, high expression level of LasAI/LasAII were detected without co-inoculation of P19. Transgenic Arabidopsis plants expressing lasAI or lasAII showed a different degree of impaired growth. In particular, the LasAI contains domains responsible for abnormal growth of the root and/or meristem. Transient expression of LasA1 and three different LasAI domains, LasAI-N-terminal, LasAI-repeat, LasAI-C-terminal allowed us to visualize the sub-cellular localizations of different domains. Furthermore, Western blot tests revealed potential post translational processing of LasAI protein. Meanwhile, in order to study the function of LasAI in citrus, transgenic citrus are being generated to express different domains of LasAI. Because of high level expression of these effector proteins, we are able to recognize an unique phenotype in inoculated tobacco leaves that can be used for rapid screening of small molecules. Perfection of this screening system and screening small molecules are underway. The crystal structure and immunolocalization of LasAI protein should be reported in the following progress report. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein was able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants with high level expression of this gene displayed HLB-like symptoms, yellow shoot and impaired growth. Further characterization of this effector and identification of mechanism involved in symptom development are underway.
The project entitled ‘Further characterization of HLB resistant clones of selected citrus varieties’ (project no. 758) is currently in its second year. The overall aim of the project is to study tolerance/resistance to Citrus huanglongbing. We have field data that indicates different levels of disease tolerance or resistance in certain citrus relative genera. In this project, we are further testing the resistance by conducting controlled experiments in green house conditions. In the current period (July 1- Oct 1, 2014), we have challenged about 70 plants with Liberibacter positive and negative psyllids in a no-choice situation. A representative part of the psyllids used for the experiment were checked for the presence of the bacterium and also the titer of Liberibacter was estimated. After a two week exposure of the plants to the Liberibacter containing (or, Liberibacter negative control psyllids), the individual psyllids were collected and checked again for the Liberibacter titer. The plants are then maintained in the greenhouse and observed for symptoms. We have also collected plant tissue at different times and conducted DNA extractions. In the past, we have used the plant DNA extractions for real-time PCR assay in order to determine the Liberibacter titer in the different treatments. At present, we have the capability of detecting very minute amounts of Liberibacter by conducting a droplet digital PCR assay. More sensitive than real time PCR, this method can give an absolute quantification of the pathogenic bacteria. We have standardized the assay to be used for the plant samples. We are in the process of testing plant samples using this methodology. This will enable us to determine the tolerant and resistant cultivars in our experiments with accuracy and will complement the other methods that we are using to test resistance or tolerance.
The citrus pathogen Xanthomonas axonopodis pv. citri (Xac) can cause extensive damage to twigs, leaves, and fruit of susceptible varieties. The overall objective of this research program is to develop a bio-control system for citrus canker that utilizes virulent (lytic) phages and/or phage components (tailocins). We have previously reported on first round evaluation of a phage cocktail in cooperation with Dr. Nian Wang (University of Florida-Lake Alfred). In two independent experiments, when phage was applied, pre, post or at the time of the inoculation of Xac, reduced canker lesion numbers were observed, as compared to the Xac only inoculated plants. Second round testing of a phage cocktail is currently being conducted. Xac inoculated plants, with appropriate controls, will be treated with the phage cocktail at a multiplicity of infection of 20. Symptom development and Xac population levels will be monitored and quantified for a 21-day period. In addition, a cocktail consisting of two high titer broad host range tailocins XT-1 (killing 13/13 Xac isolates) and XT-4 (killing 12/13 Xac isolates) will be evaluated. We will test the hypothesis that one-hit one-kill kinetics of tailocins can act to reduce Xac populations and disease symptom development. The five identified tailocins, with activity against Xac, are being further characterized. Conditions for optimal production of the tailocins and identification of genes encoding for the tailocins are currently underway. We are continuing to expand the pool of virulent phages with diverse receptors, in order to develop phage cocktails that will overcome resistant mutations. Two new non-type IV pilus dependent myophages, CCP569M and CCP594 that belong to two different unique host range groups, have been isolated, purified and partially characterized. Both with CCP569M and CCP594 exhibit long contractile tails of 130 nm and 215 nm, respectively, with the former having a capsid of 77 nm and latter a capsid of 121 nm in diameter.
Liberibacter crescens has been cultured under laboratory conditions and is considered a model system that can be used to develop a biocontrol for ‘Candidatus Liberibacter asiaticus’ (Las). The focus of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using L. crescens strain BT-1. We have developed an overlay assay for L. crescens and implemented the system to screen for phages, tailocins or antimicrobials active against the model organism. We have incrementally tested a large number of phage lysates, broad host-range tailocins, plant/psyllid extracts, water samples, and induced cultures, with no activity against L. crescens identified to date. While continuing the search for environmental phage(s) active against L. crescens, we have also initiated a recombinant approach to produce active hybrid tailocins. As proof of concept, we have deleted the tail fibers of a broad host range tailocin and complemented the deletion in trans to obtain active tailocins, in induced lysates, at concentrations similar to that of wild type. Our goal is to clone putative identified tail fibers genes from prophages in L. crescens strain BT-1 to obtain tailocins active against L. crescens BT-1. Identification of the proper protein junction will require an array of constructs. Once this system is optimized for L. crescens BT-1, the next step will be to complement with tail fibers from prophage identified in the Las genome. Previously we reported antimicrobial activity of several Actinomycetales against L. crescens. While we have been able to detect antimicrobial activity on solid medium, optimal conditions for production in liquid in quantities needed for structural analysis have not been achieved. Chromatographic analysis of organic extractions of Microbacterium strain TM 313 supernatants revealed three major components. Structural characterization of the active component(s) is an ongoing in collaboration with another group.
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. Six applications were conducted by foliar spray every 3 weeks. The disease incidence survey has been conducted once in June.
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. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. TALEN targeting the promoter of CsLOB1 is also being done using citrus protoplast.
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 have identified multiple small molecular ‘or’ peptide inhibitors against LipidA using pharmacophore based methods and are 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. We also identified multiple SecA inhibitors. We are testing their inhibitory effect against purified SecA right now. 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. For the field trial, 120 trees of four years old were selected. HLB disease severity was suveyed. The Las titers were measured for each tree using qPCR. We have compared spray and trunk injection. Trunk injection seems to have better efficacy. We have synthesized one compound at large amount for field trial. The field trial is ongoing.
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. Under room temperature, after a 20 month storage, the population reduced about 10E5 to 10E6 folds. 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 have completed 3 surveys of HLB disease severity and collected leaf samples for Las population analysis using PCR. 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. A population of 10E4 to 10E5 cfu/g root and 10E3 to 10E4 cfu/g of soil at one month was observed. We are evaluating plant immunity response by studying the expression of specific genes related to plant defense.
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