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 peptide addition to the toxin is expected to enhance both binding and toxicity against ACP. The proteolytic profiles of toxins from 46 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 a 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, one isolate showed promise based on statistical analyses with moderate ACP mortality relative to control treatments. This strain was selected for 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 and ACP gut binding peptides isolated as described in the previous report. Four peptide sequences have been cloned in the expression vector pBAD. The peptide produced is fused to a linker 2X(GGGS) and to mCherry. An autofluorescence analysis of the psyllid gut indicated that red fluorescent protein (mCherry) is the best option for analysis of in vivo binding with minimal background flourescence. mCherry will be used for assessment of peptide binding to the ACP gut. Two peptide sequences were randomly selected from the library Ph.D.-C7C for use as negative control peptides. Cloning of these sequences is underway.
Update on ACPS field trial with ‘Ray Ruby’ grapefruit at IRREC (B. Gruber): The overarching goal of this component is to develop ACPS and high-density plantings for commercial grapefruit on the east-coast. To date, the progress of this project is as follows: Irrigation installation was completed in October 2013. We are currently testing and evaluating the irrigation systems to fine-tune their performance and eliminate inefficiencies and any errors made during installation. The first experimental block of citrus (project A) was planted in November 2013. Ray Ruby grapefruit trees on Sour orange and US-897 rootstocks were planted on 8 acres on the citrus research grove at the UF-IFAS-IRREC station in Ft. Pierce. Ray Ruby / Sour orange trees were planted at a density of 152 trees/acre with microsprinklers and will be fertilized with granular, dry fertilizer: these plantings will serve as the grower standard control treatment. Ray Ruby trees on both Sour orange and US-897 rootstocks were planted in staggered-set, tramline configurations at a high-density of 421 trees/acre: half of these plots will be fertigated with microsprinklers and half will be fertigated with in-line drip tubing. Funds from this project were also used to partially support the construction of a 5-acre high-density block of Ray Ruby / Kuharske grapefruit at the IRREC (project B). In this block, all trees are being irrigated with microsprinklers but are planted at 3 different densities: 126, 189, and 421 trees/acre. Funds from other sources were used to cover the costs to complete this research block. These trees were also planted in November 2013. Monthly to bi-monthly measurements of tree growth (canopy size and trunk diameter) have been conducted since December 2013. A campaign of leaf samples for real time PCR analysis to confirm the presence of Candidatus Liberibacter asiaticus was conducted in March, 2015, for both projects A and B. A campaign of leaf and soil samples were collected in June 2014 for nutritional analyses. Another round of leaf and soil analyses are planned for May/June, 2015. For project A, funds from another source were used in to install permanent soil moisture sensors in project A to monitor volumetric water content as a function of microsprinkler and drip emitter irrigation conveyances. Products: Preliminary results from projects A and B were delivered in two separate oral presentations to citrus growers and allied industry members at the Arcadia Citrus Production School (24 February 2015) and the UF-IFAS-SWFREC Seminar Series (18 March 2015).
One of the newest ACPS field experiments was planted with ‘Valencia’ orange in August 2012 near Lake Alfred. Its primary objective was to test innovative super-high density citrus grove replanting configurations with a drip fertigation open hydroponics (OH) system for maximizing early return on investment for processed oranges in a canker and HLB-endemic disease environment. Treatments consist of the following: Main source of N: 1) calcium nitrate, 2) ammonium nitrate Rootstocks: 1) Swingle, 2) US897 Planting geometry / density: 1) 484 trees/acre in straight rows, 2) 538 trees/acre in dual tramlines The trees were established to receive all their fertilizer through drip fertigation (2×0.5 gph/tree), and a second microsprinkler irrigation system was installed for freeze protection. The ‘Valencia’ trees grew out very well and by the end of the second year already had a harvestable fruit crop, to be picked in about March 2015. In September 2014, two years after planting, the following tree canopy measurements were analyzed: Table 1: Tree canopy volume in cubic meters/tree showing significant rootstock x fertilizer response Rootstk Fert CaN AN US897 2.615 2.571 Swingle 3.277 2.921 Table 2: Tree canopy volume in cubic meters/tree showing significant effect of tree planting geometry Plant Straight Tram 2.744 3.020 The results show an improvement in tree canopy growth from using calcium nitrate versus ammonium nitrate for both rootstocks, but that the Swingle rootstock grew larger canopies than US897. The tramline configuration also produced slightly larger canopies than the straight hedgerows. We will continue monitoring these trends in subsequent seasons as the trees mature and fruit production increases. Unfortunately the incidence of HLB was established through visual symptoms at 30% in November 2014, after only 26 months. We could not detect differences in HLB severity or incidence that related to the treatments. Future management plans for HLB in this block will involve more intense foliar and root fertilization with, particularly the metal micronutrients.
A comprehensive factorial experiment was conducted to help explain the interactions between various spraying factors that are important in regulating the efficient uptake of foliarly applied nutrients. The spray solution was chosen to mimic an important citrus nutrient spray used particularly during the spring bloom period: calcium nitrate and sodium borate. However in order to improve the detection sensitivity of cations, the Ca was substituted with Strontium (Sr), which makes an extremely effective tracer to mimic Ca uptake in plant studies (we used strontium nitrate). The main experiment consists of 5 FACTORS arranged as two levels each, with two additional controls: 1. Concentration = half std(50%) versus std(100%) 2. Spray solution pH, 5.0/7.0 buffered with citric acid 3. Surfactant, nonionic versus nanoemulsion =Induce(R) / Biowash(R) 4. Leaf surface, adaxial / abaxial 5. Spray droplet charge, electrostatic / none Factorial treatments: 2x2x2x2x2 = 32 + 2 controls = 34 Controls: 1) Plain tap / well water, 2) Grower std = well water, nutrients, no surfactant, both leaf surfaces, no droplet charge Three replications: 34×3 = 102; Limit ANOVA to 3-way interactions Leaves were sampled 0) before spraying, 1) 3 days after spraying, 2) 7 days after spraying, and 3) 14 days after spraying. Analytes measured in the leaves for subsequent statistical analysis: Sr, N, B, Na. The results of the experiment are being analyzed and will be presented in the final report.
The objectives of this research are 1) to develop cost effective thermotherapy protocols for field application by optimizing temperature and relative humidity conditions in the tent; 2) to develop a mathematical model derived from our data and grower’s data which will be used to determine the best treatment duration in future applications; and 3) to study gene expression of HLB-affected citrus plants that received heat treatment, and identify critical citrus genes that may be induced by heat stress for the benefit of suppressing HLB. To reach our goals in objective 1, we have exposed both HLB-affected and healthy periwinkle (40’C) and citrus (42’C) to heat stress. DNA has been extracted and amplified for Las 16S rRNA and certain phage genes. A standard curve for a normalization gene has been established and data is being analyzed using the delta delta Ct method. Additionally, changes in expression levels for these genes are being monitored. As that Las cannot be cultured, a kill curve of Liberibacter crescens was determined. A dramatic decrease in viability was shown after L. crescens was exposed to 10 minutes of 46’C. For objective 2, we have conducted a comparison study between field heat-treated and non-heat-treated citrus plants. There were 31 consistent up-regulated genes and 47 down-regulated genes in the citrus trees treated with heating. Additionally, potted Las-positive and negative citrus was exposed to 4 hours for 4 days of 40’C, 85 % relative humidity (similar to heat exposure in field setting) in a controlled greenhouse. Comparison of Las positive trees before and after heat treated revealed 22 up-regulated and 20 down-regulated differentially expressed genes. In addition to performing RT-PCR verification of these genes, we are tracking at what specific time during heat exposure does large changes in gene expression occur. As for the third objective, over 3 years of prior data (tree Las Ct values, treatment procedures, and temperature logs from one location) have been summarized and are currently being used to determine an algorithm that relates environmental conditions with decreases in Las titer. This initial data indicates that longer durations of temperatures greater than 41-42’C is more effective with reduction of Las titer. Although, leaving the tent on the tree longer than a week to achieve this duration does not appear to improve thermotherapy results. Data analysis at this site indicates changes in Las titer were significant up to 18 months after treatment with the greatest positive changes of Las titer at 9 months. Additional data from three other locations tracked for the most recent 15 months are being added into the analysis to produce a treatment algorithm that will be tested in the upcoming warmer months. These three sites were monitored for fruit production, fruit drop, growth, and Las titer. Data will be complete in late Spring once the Valencia are ready to be harvested.
The project entitled ‘Further characterization of HLB resistant clones of selected citrus varieties’ (project no. 758) is currently in its second year. The aim of the project is to study tolerance/resistance of certain citrus relatives to Citrus huanglongbing. To corroborate the field study conducted earlier, we are now testing the resistance of psyllid-challenged plants under controlled greenhouse conditions. In the three month period from Oct1 to Dec 31, 2014, the PI traveled to Fort Pierce, Florida to monitor the psyllid-challenged experimental plants in the green houses, made observations on symptom expression, collected samples for DNA and RNA extractions. About 30 promising plants were chosen based on CLas titers recorded for one year. Conducted RNA extractions of these samples in Fort Pierce and the extracted RNA was brought to Riverside for further analysis. The plant samples were tested for the presence of Candidatus Liberibacter asiaticus by qPCR analysis. Plant samples with apparent resistance were checked by digital droplet PCR to detect low titers of bacteria. During the spring of 2014, we had conducted about 1000 pollinations using Eremocitrus and Microcitrus as pollen parents, certain mandarins and pummelos as female parents. The fruits are now ready for harvest. About 4-5% success was obtained from the 2014 pollination experiment. Considering the fact that these are wide inter-generic crosses, this is considered to be a successful experiment. At present we are collecting seed and germinating 1-2 seeds from each pollinated fruit so that DNA can be made from the seedling tissue for confirming the hybrid nature of the plants.
A JBrowse based genome viewer (http://hlbws.sgn.cornell.edu/jbrowse/JBrowse-1.11.4/) has been installed on the CG-HLB Genome Resources Website (http://www.citrusgreening.org/) to provide end users with a smoother interface for viewing seven Liberibacter genome sequences and to enhance user ability to visualize different categories of single nucleotide polymorphisms (SNPs). SNP locations can provide a basis for various analyses including strain and isolate tracking, and identification of sources of phenotypic difference. This is particularly valuable information for experimentally challenging organisms such as Liberibacter. Four sequences of CLas are now available including the reference strain CLas psy62, CLas gxpsy, CLas HHCA and CLas A4. Alignment in MAUVE (http://asap.ahabs.wisc.edu/software/mauve/) reveals an overall high level of sequence conservation among CLas strains, with the most extensive variation found in phage regions. SNP identification relative to CLas psy62 reveals 1766 SNPS in CLas gxpsy, 1410 SNPs in CLas A4, and 2910 SNPs in CLas HHCA. To characterize the potential impact of these SNPs on the biology of the different strains, SNPeff (http://snpeff.sourceforge.net/) has been used to discriminate among those SNPS resulting in synonymous changes and hence having no impact on protein sequence, those that result in nonsynonymous changes including introduction or deletion of stop codons, and those SNPs altering predicted intergenic regulatory regions. Preliminary analyses of the different SNP categories reveal that of the SNPs identified in CLas gxpsy relative to psy62, 277 result in nonsynonymous changes to predicted coding regions, and 615 are present in regions upstream of protein coding regions, potentially impacting protein expression. Nonsynonymous changes affect 71 of the 1109 predicted protein encoding genes with the majority clustered in a small number of genes. Different tools are being explored to predict the impact of these changes on protein structure. As additional strains are sequenced consistency of SNP locations will be evaluated and may inform regions of the original reference genome, CLas psy62 that could benefit from re-sequencing. One of the advantages of JBrowse over the older GBrowse viewer is am improved interface for visualizing different SNP categories. Different display options are currently being tested including color-coded mapping to distinguish synonymous and nonsynonymous changes.
The overall objective of this research program is to develop an effective and sustainable bacteriophage (phage) and/or phage components (tailocins) based biocontrol system for citrus canker. We have developed a pool of characterized virulent phages and identified five tailocins with activity against Xanthomonas axonopodis pv. citri (Xac). Second round therapeutic and prophylactic efficacy 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 representing four different host range groups was evaluated. Hamlin sweet orange plants grown in a citrus canker quarantine greenhouse were trimmed and fertilized to induce new growth. The abaxial surfaces of fully expanded, immature leaves of each plant were sprayed with a 20 ml aliquot of the following suspensions: (a) Xac (disease control), (b) phage cocktail (phage control), (c) Xac then treated 6 h later with phage cocktail (therapeutic: post-Xac treatment), (d) phage cocktail treatment then inoculated with Xac, 6 h post-cocktail treatment (prophylactic: pre-phage treatment) and (e) spray treated with sterile tap water (STW; negative control). Xac inoculations were done at 5 X 10^6 or 5 X 10^8 CFU/ml, and Xac inoculated plants were treated with the phage cocktail at a multiplicity of infection (MOI) of 20, as described. Phage control plants were treated with a suspension of 1 X 10^8 or 1 X 10^10 PFU/ml. Symptom development was monitored and the Xac population was quantified for a 21-day period. In two independent therapeutic experiments, when phage was applied at an MOI of 20 to plants at 6 h post-inoculation using 5 X 10^6 or 5 X 10^8 CFU/ml of Xac, there was an average reduction of 47% and 45% in leaf lesion formation as compared to the plants inoculated only with Xac, respectively. In two independent prophylactic experiments, when foliage was treated with cocktail phages at an MOI of 20 at 6 h pre-Xac inoculation, an average of 53% and 42% reduction in leaf lesion formation as compared to the plants inoculated only with Xac, was observed in plants inoculated at 5 X 10^6 or 5 X 10^8 CFU/ml of Xac, respectively. We have previously reported on broad host range activity of tailocins XT-1 and XT-4 against Xac. It was of interest to determine the efficacy of a cocktail consisting of tailocins XT-1and XT-4, to reduce lesion formation in Xac inoculated citrus plants. Hamlin sweet orange plants were cultivated as described above to induce new growth. The abaxial surfaces of fully expanded, immature leaves of each plant subjected to treatments. Negative control plants were spray treated with STW. In two independent experiments, when the tailocin cocktail (20 ml aliquot) was applied to foliage at a multiplicity of killing units of 16 at 6 h post- or pre-inoculation with 5 X 10^7 CFU/ml (20 ml aliquot) of Xac, there was a an average reduction of 49% and 53% in lesion formation as compared to the plants inoculated only with Xac, respectively. We observed a significant reduction in lesion formation when plants were treated with the phage cocktail both post- and pre- inoculation with 5 X 10^6 or 5 X 10^8 CFU/ml of Xac. Similar results were obtained with tailocin cocktail treatments, which indicate that the one hit killing kinetics of tailocins is a viable treatment that can reduce disease development in treated plants. The genome of the XT-1 producing strain has been sequenced and 61 contiqs were assembled. Our goal is to identify genes encoding for the tailocin.
Liberibacter crescens strain BT-1, has recently been cultured under laboratory conditions and is the model system for our studies. The focus of the project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) with activity against L. crescens strain BT-1. In a continued effort to isolate phages, tailocins and/or identify antimicrobials with activity against L. crescens strain BT-1, using the developed system we have screened a large number of phage lysates, tailocins, water filtrates, as well as plant and insect extracts with limited success. Our most recent studies have focused on members of the Rhizobiaceae since they may be a potential source of bacteriocins and/or prophages active against Liberibacter species because of their phylogenetic relationship to Liberibacter. Agrobacterium and Rhizobium species are members of the family Rhizobiaceae and produce inhibitory bacteriocins and harbor prophages. In particular, Agrobacterium radiobacter K84 produces three antimicrobial compounds, agrocins 84 and 434, which are encoded on plasmids pAgK84 and pAgK434, respectively, and a chromosomally encoded siderophore, ALS84. The specific permeases that transport agrocins have homologues in L. crescens. We evaluated potential of the agrocins to act as antimicrobials against L. crescens strain BT-1. In initial experiments, we observed a large zone of growth inhibition when strain K84 was used as the producer and L. crescens strain BT-1 as the indicator. However, using a series of plasmid deletion derivatives of strain K84, we determined that the agrocins 84 and 434 were not responsible for the observed zone of inhibition. When the assay medium was amended with increasing micromolar amounts of ferric chloride, no growth inhibitory zone was observed. The conclusion is that a siderophore ALS84, which is negatively regulated, was responsible for the observed inhibition of L. crescens in the in vitro assay. We are continuing our search to identify phages and tailocins active against L. crescens, and conducting experiments to construct tailocins active against L. crescens.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. During the recently completed quarter (November 1 to December 31, 2014), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Plant Improvement Team o Site prep continues for new planting of rootstock and scion cultivars being evaluated for HLB tolerance/resistance o Planted new NVDMC trees o Initiated renovation of pomegranates in the North Block for Dr. Castle ‘ Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance o Continue evaluating two new Dow herbicide trials ‘ Conducted fall fertilizer and pest management programs for the groves o Herbicide program on schedule o Psyllid management continued . Participated in coordinated area wide spray in early November ‘ Continue to maintain Dr. Bowman’s new USDA rootstock plantings ‘ Commercial Trials: o Eurofins evaluations on disease and insect management continue . Preparations for new plantings o Syntech evaluations on insect, weed and disease management (3) o Florida Ag Solutions continue to make applications to herbicide and insecticide evaluations (5) ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas ‘ Drs. Albrigo and Wong have continued to evaluate antibiotics to manage HLB o Dr. Wong increased size of trial in New Block o New trial conducted in 9 rows in Lake County Section ‘ International scientists visited on various occasions ‘ UF/IFAS Extension held a Field Day focusing on Plant Improvement on December 10, 2014 with approximately 35 growers and others in attendance.
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. For one field trial, we have compared the effect of different microbe products on fruit drop, yield, fruit quality, root density and disease index. The aforementioned analyses are being conducted for other field trials.
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. The application for the field trails have been completed as scheduled. Up to 12 applications have been done. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. the Las titer in lef samples were determined at multiple time points using qPCR assays. Lower Las titers were observed for some treatments. Yield data and fruit quality were also collected. The effect of different compounds on the area under the disease progress curve is investigated. 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. SA hydroxylase is being expressed and purified. Multiple SA hydroxylase inhibitors have been identified using molecular modeling. The inhibitory effect of the SA hydroxylase inhibitors have been tested using the purified SA hydroxylase. Three compounds showed inhibitory effect against SA hydroxylase. The three SA hydroxylase inhibitors will be used to control HLB in greenhouse or field trials. For the putative sec dependent effectors, we have identified some putative targets using yeast two hybrid assays. We are currently confirming the protein-protein interactions using immunoprecipitation method. We are verifying the interactions using tobacco as a model. Gene silencing of the tobacco homolog genes are being conducted.
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 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. Seven applications were conducted by foliar spray every 3 weeks. The disease incidence survey has been conducted in June (before treatment), October and December. The data showed that biofilm inhibitors, QS inhibitors and copper, individually or in combination, reduced cander disease incidence.
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. Transgenic Duncan and valencia transformed by Cas9/sgRNA has been established. Totally seven transgenic lines have been acquired and confirmed. The transgenic lines are being grafted to be used for test against citrus canker. We will continue to generate more transgenic lines to get biallelic mutations in the EBE region of the CsLOB1 gene.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We will mainly test the antagonistic effect of Bacillus, Paenibacillus, Streptomyces and Pseudomonas strains Agrobacterium tumefaciens, Sinorhizobium meliloti, and L. cresens; Control HLB using antibacterial-producing bacteria. For the field test, we will investigate how antibacterial-producing bacteria affect HLB disease severity, Las titres, and citrus yield, survival of the antibacterial-producing bacteria in the rhizosphere and expression of the antibacterial biosynthesis genes in vivo. We have isolated Streptomyces spp. Bacillus spp. Paenibacillus spp., and Pseudomonas spp. from Florida groves. Multiple isolates showed antimicrobial production activity. We tested 27 antibacterial compound producing bacteria. These strains had been recovered, purified and confirmed by 16S rDNA sequencing. The antagonistic activity against Agrobacterium, Sionrhizobium meliloti and Xanthomonas citri pv. citri was determined. 5 strains, belonging to Paenibacillus, Burkholderia, Paenibacillus, Streptomyces and Streptomyces showed good antagonistic activity. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi). In a separate experiment, two Burkholderia strains were tested and up to 10E5 cfu/g soil was recovered at five days post inoculation. To further monitor the colonization of beneficial bacteria, GFP reporter strains have been constructed and are being used to monitor the colonization of different bacteria in the soil and in the citrus roots.
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