Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1100 - Bacteriology | 60% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1150 - Toxicology | 20% |
| 102 - Soil, Plant, Water, Nutrient Relationships | 110 - Soil | 1000 - Biochemistry and biophysics | 10% |
| 206 - Basic Plant Biology | 999 - Citrus, general/other | 1040 - Molecular biology | 10% |
Florida growers have assumed a total infection (symptomatic or asymptomatic) of all producing groves across the state. The strategies used to reduce the effects of HLB have not been effective. Novel strategies directed to eliminate the bacteria from infected trees, and to protect new groves are needed.The goal of this proposal is to assess the efficacy of this antimicrobial therapy in active HLB-infected groves. We have designed an efficient treatment plan that is cost effective and scalable, two aspects that will be essential for any treatment discovery intended for large-scale application. Because recovery of HLB-infected plants in the greenhouse was rapid, we anticipate that the proposed therapy will deliver timely results in the mature, HLB-infected plants.. We will achieve this goal by (1) Optimizing the antimicrobial treatment in HLB-infected citrus seedlings in small scale field trials; (2) Evaluating the antimicrobial treatment in large-scale field trials. We will assess the economic, ecological, and environmental impact of each treatment; (3) Testing the antimicrobial activity of alternative organic compounds that have chemical scaffolds similar to those of the active compounds. We will have optimized the application of chemicals that are effective CLas antimicrobials.Our research team is working directly with citrus growers and groves in active production. The Stakeholder committee is comprised of large and small-scale growers, who will meet to discuss the progress and results of the project every 6 months. The results will be transferred to a broad audience through different publication media (such as the UF journal EDIS and industry publications) and presentations to grower associations.An effective antimicrobial treatment will have an immediate positive impact on the economic and social aspects of the citrus industry. The citrus industry, directly or indirectly, involves a large network of workers and companies currently operating in the state of Florida. Because the citrus industry represents a large portion of the Florida economy, the recovery of infected groves will have a substantial, positive impact on the economy in Florida as a whole.
The goal of this project is to develop an antimicrobial treatment to cure Huanglongbing disease (HLB) in the field. We have designed a plan to efficiently carry out treatments, thoroughly evaluate results on a laboratory scale, and quickly translate treatment discoveries to groves in production. Upon completion of this project, we will have optimized treatments for field-scale applications of HLB-specific antimicrobials, including the analysis of environmental impacts (if any) associated with each of the proposed treatments. We will achieve the goal of this proposal by completing the following objectives.
Objective 1. Determination and optimization of the antimicrobial treatment in infected citrus seedlings. For the proposed studies, we will use sweet orange (Citrus sinensis) "Valencia" seedlings. To accomplish this objective we will:1.1. Evaluate the toxicity of the selected chemicals on sweet orange seedlings. Different concentrations of each chemical will be tested.1.2. Evaluate the antimicrobial efficacy of the selected chemicals. The evaluation of the antimicrobial efficacy in plants will be performed by following the remission of HLB symptoms as well as the viability and titer of 'Ca. L. asiaticus' in tissue samples collected from new growth.1.3. Determine the stability of each compound within the citrus plant and identify residues in plant and fruit tissue as well as soil samples following treatment. The distribution, stability, and turnover of each compound will be determined in both plant tissues and soil samples. The data collected will be used to address environmental concerns and ensure compliance with EPA regulations.Objective 2. Field trials: application of the antimicrobial treatment and evaluation of environmental impact. This objective will be divided into two phases:2.1 Small scale field trial. Based on the results of the greenhouse experiments, we will test the most effective treatments on infected trees maintained by the Horticulture Science Department at the University of Florida main campus.2.2. Large-scale field trial. The chemicals found to be the most effective for treatment of HLB in the small scale field trial, will be tested in large orchards. Multiple orchards will be tested during the large-scale field trials with different varieties of HLB-infected citrus trees; sweet orange trees (Citrus sinensis, "Valencia") and grapefruit trees (Citrus paradisi, "Ruby red" or "Flame"). On each of those groves we will perform:2.2.1 Evaluation of antimicrobial efficacy. The antimicrobial efficacy of each compound will be evaluated by following the presence and viability of 'Ca. L. asiaticus', by real time PCR.2.2.2 Evaluation of the impact of each antimicrobial treatment on fruit production and quality. Oranges from each field will be collected before and after treatment, and sent to the Citrus Research and Education Center (CREC) for quality analysis.2.2.3 Determination of the stability of each compound within the citrus plant and identification of any residues in plant or fruit tissue following treatment.2.2.4 Evaluation of the impact of each antimicrobial on the host and its associated microbiota. To this end we will define the effect of each antimicrobial on the citrus microbiome and characterize plant responses to each compound. We will also elucidate the detoxification pathways for each compound.Objective 3. Identification and evaluation of antimicrobial activity of natural compounds that share a similar chemical scaffold to the initial lead compounds. We will perform an in silico identification of natural products that interact with the microbial target proteins and in vitro characterizations of protein/ligand interactions. The identified chemicals will then be evaluated (in vivo) in citrus seedlings as described in objectives 1 and 2.The data obtained on each of the objectives will be exhaustively analyzed using different statistical methods.We will also perform an economic impact analyses. The economic component will include two primary activities. First, a traditional cost-benefit analysis of the compounds will be conducted to determine which compound has the highest net benefit per tree. The second activity will involve the construction of a dynamic bioeconomic model to determine the optimal (profit-maximizing) use of each treatment in the field.
Target Audience
1. General Public: Online, through our website 2. Stakeholders: though planned meetings 3. Scientific community: -Poster presentations, in local, regional, national and international scientific meetings - Oral presentations, in local, regional, national and international scientific meetings - Publications in peer reviewed journals
Changes / Problems
We were unable to complete the analyses of the samples from the last timepoint of the field trial due to COVID-19. We expect to complete the analyses as soon as the UF is open for research activities.
Training & Professional Development
Over the 5 years of this project, training to 1 Master student, 4 PhD students, 6 Postdocs and several undergraduate students was provided. Master student: Pablo Moreno PhD students: Brian Blaustein, Lei Pan and Janelle Coyle graduated successfully defended their dissertation to earn the PhD degree. Kaylie Padgett has passed the qualifier and is in her way to earn her PhD. Posdoctoral training: This grant has provided postdoctoral training to Fernando Pagliai, Lei Pan, Marcelo Merli, Dan Zhang, Cecile Pereira and Ran Zuo. Undergraduate students: Six undergraduate students received field and experimental experiences. Two students completed Honors Thesis to the College of Agricultural Sciences.
Dissemination Streams
1. General Public: Online, through our website 2. Stakeholders: though planned meetings. We held two in person meetings at Fort Pierce. Meeting 1 was pre-field trial (received feedback on the approach and the growers agree to provide commercials groves for testing). Meeting 2 after year 1 of the trial, preliminary data was presented. The last meeting was planned for march 2020 however, due to COVID-19 we were unable to do it. 3. Scientific community: - Poster presentations, in local, regional, national and international scientific meetings - Oral presentations, in local, regional, national and international scientific meetings - Publications in peer reviewed journals
Next Reporting Steps
Nothing Reported
Target Audience
Our target audience are: 1. General Public 2. Stakeholders 3. Scientific community:
Changes / Problems
Nothing Reported
Training & Professional Development
This project is/has providing training to 3 PhD students, and 4 Postdocs. Several (2) undergraduate students are involved in the project. One undergraduate student is performing a Honors Thesis to the College of Agricultural Sciences. On 2018, one graduate student completed his graduate studies (Pan, Lei PhD).
Dissemination Streams
1. General Public: Online, through our website 2. Stakeholders: though planned meetings 3. Scientific community: -Poster presentations, in local, regional, national and international scientific meetings - Oral presentations, in local, regional, national and international scientific meetings - Publications in peer reviewed journals
Next Reporting Steps
We are on schedule to accomplish the goals proposed. <br><br>
<br>What was accomplished under these goals? Objective 1. Optimization of antimicrobial treatment in infected citrus seedlings. Over the last year we have tested efficacy of the second generation of compounds that were identified by completing Objective 3 of this application on small infected citrus trees. Based on the phytotoxicity of each chemical, the highest concentration possible was tested for efficacy. It was found that four of these chemicals significantly reduced titers of L. asiaticus after 3 and 6 months of treatment applied by foliar spray. For each of these chemicals, a LC-MS/MS method of extraction and detection was optimized, and subsequently used to analyze the stability and distribution of each chemical in large citrus trees, during field trials (see Objective 2). Similarly, we initiated phytotoxicity and efficacy testing on four additional compounds (generation three) that target the PrbP protein (see Objective 3). Objective 2. Field trials: application of the antimicrobial treatment and evaluation of environmental impact. Last year we collected the fruit from the year-long treatments that were performed with benzbromarone and tolfenamic acid foliar sprays. Based on initial visual inspection of the fields in Sebring and Vero Beach, we were expecting a record harvest (also indicated by the growers in our informal meetings). Unfortunately, our field trials were severely impacted by hurricane Irma; Sebring was hit by the center of the storm and the fields in Vero Beach were submerged by floodwaters (for 2 weeks). We were able to collect a small portion of fruit that remained after the hurricanes (less than 10% of the 2017 harvest), but unfortunately the results were inconclusive. Moreover, the Vero Beach grove was damaged beyond rehabilitation, thus the experiment was terminated. We initiated a new small-scale trial for the preventive treatment of HLB using foliar sprays. We are currently using tolfenamic/benzbromarone treatments in addition to the second generation of chemicals described in Objective 1. Objective 3. Identify and evaluate the antimicrobial activity of natural compounds that have chemical scaffolds similar to the two effective compounds we have already identified. Virtual docking in the ligand binding pocket of PrbP, combined with LC-MS/MS analyses of the stability of tolfenamic acid, allowed the identification of 3rd generation chemicals with improved stability in citrus trees. Four chemicals are currently being tested for efficacy against HLB in the greenhouse. Using a similar approach, several compounds were tested on purified YbeY protein to evaluate an agonist/antagonist mechanism of enzyme activation/inactivation. The role of these chemicals in the interactions of the pathogen with the citrus plant are currently under investigation. It is expected that after completion of these analyses we will have identified plant specific signals (agonists) that the pathogen senses in order to establish infection. Conversely, the antagonist signals identified may be used as antimicrobials. <br><br><b>Publications</b><br>
Target Audience
1. General Public: Online, through our website 2. Stakeholders: though planned meetings 3. Scientific community: -Poster presentations, in local, regional, national and international scientific meetings - Oral presentations, in local, regional, national and international scientific meetings - Publications in peer reviewed journals
Changes / Problems
Based on visual inspection of the fields in Sebring and Vero beach we were expecting a record harvest (also indicated by the growers in our informal meetings). Unfortunately, hurricane Irma hit the fields really bad. Sebring was under the center of the storm while the fields in Vero Beach were flooded (for at least 2 weeks). We are analyzing the results of the small harvest obtained from Vero Beach but we are not very optimistic on the results that we will be able to get.
Training & Professional Development
This project is/has providing training to 4 PhD students, 2 Master students and 2 Postdocs. Several (6) undergraduate students are involved in the project. One undergraduate student is performing a Honors Thesis to the College of Agricultural Sciences. On 2017, three graduate students have completed their graduate studies (Blaustein R. PhD, Coyle J. PhD and Moreno P. MS).
Dissemination Streams
1. General Public: Online, through our website 2. Stakeholders: though planned and informal meetings 3. Scientific community: -Poster presentations, in local, regional, national and international scientific meetings - Oral presentations, in local, regional, national and international scientific meetings - Publications in peer reviewed journals
Next Reporting Steps
We are on schedule to accomplish the goals proposed. <br><br>
<br>What was accomplished under these goals? 1.1.New compounds identified in Objective 3 are being tested for symptoms of phytotoxicity. 1.2.The new compounds/combinations were delivered to orange seedlings by trunk injection or foliar spray at the highest non-toxic concentration, as indicated above. Noteworthy, in all treatments we observed an initial decrease in on Ca. L. asiaticus after three months of the treatment. Experiments are underway to optimize the dose and treatment interval of foliar applications. 1.3.We developed a new LC-MS/MS method for the detection of benzbromarone and tolfenamic acid in plant tissue. The method is applicable to leaves, fruit rind, juice and root tissue. The recovery of the chemicals from orange juice and orange peel was 96% and 92.5% for benzbromarone, respectively, while tolfenamic acid was recovered at 96.7% and 98.6%, respectively. The results obtained indicated the chemicals are able to efficiently move throughout the plant over time. 2.1 Small scale field trial. Based on the results of the initial greenhouse experiments, we performed the administration of two treatments (benzbromarone and tolfenamic acid) plus a control group using trunk injections in infected trees maintained in the orange grove at the University of Florida main campus. We have evaluated the effect of the chemicals on the microbial titers using mRNA-based RT-PCR in leaf and root tissue. The statistical analyses of leaf tissue indicated that there is a significant decrease (p<br><b>Publications</b><br>
Target Audience
1. General Public:Online, through our website 2. Stakeholders: though planned meetings 3. Scientific community: -Poster presentations, in local, regional, national and international scientific meetings - Oral presentations, inlocal, regional, national and internationalscientific meetings - Publications in peer reviewed journals ?
Changes / Problems
Based on our meetings with the stakeholder committe, we decided to include field tests where the chemicals are delivered by spray in addition to the trunk injections originally planned.
Training & Professional Development
This project is providing training to 4 PhD students, 2 Master students and 2 Postdocs. Several (6) undergraduate students are involved in the project. One undergraduate student is performing a Honors Thesis to the College of Agricultural Sciences.
Dissemination Streams
1. General Public:Online, through our website 2. Stakeholders: though planned meetings 3. Scientific community: -Poster presentations, in local, regional, national and international scientific meetings - Oral presentations, inlocal, regional, national and internationalscientific meetings - Publications in peer reviewed journals ?
Next Reporting Steps
We are on schedule to accomplish the goals proposed. <br><br>
<br>What was accomplished under these goals? Objective 1. 1.1 During this period, we evaluated the phytotoxicity of a combination of the effective compounds identified during Year 1 delivered by trunk injections. Additionally, the phytotoxicity of these compounds via spray delivery was determined in presence or in absence of adjuvants and penetrants. It was found that 47 compounds/combination of compounds were not toxic to citrus seedlings when tested at up to 250 uM (due to the solubility of some of the compounds this was the highest concentration that we were able to test), while 2 compounds did not show toxic effects when administered at concentrations up to 20 mM 1.2 The compounds/combinations at concentrations non-toxic for the orange seedlings were administered by trunk injections or by spray applications. A total of 47 treatments were conducted. We identified 4 compounds/compound mix that resulted effective (p<br><b>Publications</b><br>
Target Audience
Stakeholders are being continuously engaged in project development and evaluation. We have made our first meeting with all the members of the stakeholders committee. The Stakeholder Advisory Committee included producers, academic scientists, environmental specialists and extension agents. The Stakeholder Advisory Committee will receive research updates twice a year, and extensive progress evaluations will be conducted yearly. These evaluations will include self-assessment of progress, assessment by the Stakeholder Advisory Committee, and feedback we will receive during presentations and manuscript submissions. Keep growers informed of our progress and ensure all of our research and results are readily available to the public. We have developed a website where we post monthly news about HLB research. It can be found at: http://ufgi.ufl.edu/other-news-in-citrus-greening-research/ http://ufgi.ufl.edu/research/citrus-greening-update/resources-for-citrus-growers/ http://ufgi.ufl.edu/research/citrus-greening-update/contact-the-researchers/ http://ufgi.ufl.edu/gout-treatment-may-solve-citrus-greening/ Train graduate and undergraduate students in the treatment of HLB. Multiple graduate and undergraduate students are currently involved in the project. The students are involved in the analyses of bacterial survival, plant and environmental responses to the chemical compounds. Students are being trained on statistical analyses and economical impact of the antimicrobial treatment on the field.
Changes / Problems
Nothing Reported
Training & Professional Development
This project is providing training to 4 PhD students, 2 Master students and 2 Postdocs. At least 4 undergraduate students are involved in the project, two of them have recently completed and submited their Honors Thesis to the College of Agricultural Sciences.
Dissemination Streams
The results obtained have been diseminated in the form of: - Online, through our website - Meetings with stakeholders - Poster presentations, in diverse scientific meetings - Oral presentations, in diverse scientific meetings - Publications in peer reviewed journals
Next Reporting Steps
We are on schedule to accomplish the goals proposed. <br><br>
<br>What was accomplished under these goals? Objective 1. Determination and optimization of the antimicrobial treatment in infected citrus seedlings. Evaluate the toxicity of the selected chemicals on sweet orange seedlings. We have completed the evaluation of 16 compounds or combinations of compounds. Different concentrations of each chemical were tested. We found that only three compounds showed toxic effects when administered at concentrations higher that 20 mM. Evaluate the antimicrobial efficacy of the selected chemicals. The evaluation of the antimicrobial efficacy in plants is currently being tested. The compounds/combinations at concentrations non-toxic for the orange seedlings have been administered by trunk injections. We are currently following the remission of HLB symptoms as well as the viability and titer of 'Ca. L. asiaticus' in tissue samples collected from new growth. Determine the stability of each compound within the citrus plant and identify residues in plant and fruit tissue as well as soil samples following treatment. We are currently optimizing a LC-MS method for detection of the distribution, stability, and turnover of each compound. Determinations will be performed in both plant tissues and soil samples. Objective 2. Field trials: application of the antimicrobial treatment and evaluation of environmental impact. This objective is divided into two phases: 2.1 Small scale field trial. Based on the results of the greenhouse experiments, we started the treatments on infected trees maintained by the Horticulture Science Department at the University of Florida main campus. We have collected samples at T0, T2, T6 and T12 will be collected next week. We have performed RT-PCR assays to evaluate the effect of the chemicals on the microbial titers. The results are under analyses. 2.2. Large-scale field trial. The chemicals found to be the most effective for treatment of HLB in the small scale field trial, are being tested in large orchards. Three orchards were selected: one with sweet orange trees (Citrus sinensis, "Valencia") and two with grapefruit trees (Citrus paradisi, "Ruby red" or "Flame"). We have completed the administration of the treatment by trunk injections this week. On each of those groves we will perform: 2.2.1 Evaluation of antimicrobial efficacy. The antimicrobial efficacy of each compound will be evaluated by following the presence and viability of 'Ca. L. asiaticus', by real time PCR. 2.2.2 Evaluation of the impact of each antimicrobial treatment on fruit production and quality. Oranges from each field will be collected before and after treatment, and sent to the Citrus Research and Education Center (CREC) for quality analysis. 2.2.3 Determination of the stability of each compound within the citrus plant and identification of any residues in plant or fruit tissue following treatment. 2.2.4 Evaluation of the impact of each antimicrobial on the host and its associated microbiota. To this end we will define the effect of each antimicrobial on the citrus microbiome and characterize plant responses to each compound. Objective 3. Identification and evaluation of antimicrobial activity of natural compounds that share a similar chemical scaffold to the initial lead compounds. We have performed an in silico identification of natural products that interact with the microbial target proteins and in vitro characterizations of protein/ligand interactions. The identified chemicals will then be evaluated (in vivo) in citrus seedlings as described in objectives 1 and 2. We have also initiated the economic impact analyses. The economic component includes two primary activities. First, a traditional cost-benefit analysis of the compounds is being conducted to determine which compound has the highest net benefit per tree. The second activity is the construction of a dynamic bioeconomic model to determine the optimal (profit-maximizing) use of each treatment in the field. <br><br><b>Publications</b><br>