Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1000 - Biochemistry and biophysics | 100% |
Oranges, tangerines, lemons and limes provide nutrition and food flavorings, and are an important source of vitamin C for healthy immune systems, something needed now more than ever during our current global health crisis.Globally, the US is one of the top three countries for consumption of citrus per capita. Citrus production in the United States is worth $3 billion and in Florida and California provides nearly 100,000 jobs. However, the future of the citrus industry producing orange juice and fresh fruit is threatened worldwide by a devastating disease known as Huanglongbing (HLB for short). It is also referred to as "citrus greening."The disease originated in China and has spread rapidly across the globe, leading to the loss of more than one million citrus trees. The disease is difficult to stop because the causal bacterium lives in the tree's phloem, which is a part of the plant's circulatory system lying just beneath the bark, that is very difficult to reach.The disease is carried by a flying insect called a psyllid, which is smaller than a sesame seed. The psyllid becomes a transmitter of the disease by first feeding on the leaves of an infected tree, ingesting HLB bacterium. Once the bacterium is in the psyllid, the psyllid conveys the disease to other trees it feeds upon. In this way, the disease is transmitted from tree to tree.Suppression of the psyllid was tried in Florida but failed. It is now assumed that virtually every tree of fruit-bearing age in Florida is infected.HLB affects citrus trees from the roots up, clogging the tree's vascular system and disrupting carbohydrate production in the leaves and nutrient and water flow throughout the tree. What results is debilitation of the tree with fewer leaves and small, misshapen fruit that develops a bitter taste. Infected trees decline in production a few years after infection.Over the past ten years, hundreds of millions of dollars have been spent on research in the United States on this problem. While we now know more about the disease than ever, and while enhanced nutritional programs and other horticultural practices have improved yield and fruit quality, not a single cost-effective therapy has resulted from these efforts. Why is this? There are several reasons, but one of the major ones is that this bacterium can only propagate in the citrus tree's circulatory system or psyllid's gut, making traditional screening and discovery in the laboratory difficult. Detecting the disease in citrus trees is also tricky because signs of the disease are not manifested quickly; it can take years for an infected tree to show visual symptoms. And since nearly all mature trees in Florida and many in Texas are infected, there is a huge reservoir of inoculum being spread by the insect carriers. In California, more than 1,500 trees in residential settings have been found to be infected, causing them to be removed to keep the disease out of commercially producing groves.To address a problem of this complexity and magnitude, a concerted effort is needed which combines the expertise across several academic disciplines, the agrochemical industry and citrus industry experts. New approaches are needed to identify potential products, quickly detect the disease, and test trees for the ability to suppress or kill HLB. Two approaches are being investigated in parallel to speed development.In one case, the agrochemical industry partner is looking for naturally occurring microbes that could fight the disease. Microbes protect both humans and plants against many diseases and are important for our health. In fact, many of our important human therapies, like erythromycin and penicillin, are produced by microorganisms. A library of more than 100,000 isolated microbial strains is being searched for one that could naturally control citrus greening.In a second approach, more than 200,000 synthetic compounds are being screened to determine if they can boost the plant's defense system. Think of it as developing a vaccine for plants. Combining different ways to stop citrus greening can help keep HLB from developing immunity to the therapies.After laboratory testing on model organisms, it is important to begin testing on plant systems. At Texas A&M, a researcher has developed a way to use root tissue from infected plants raised in the greenhouse to provide a rapid method for screening microbes and chemicals for their potential in killing HLB. The ones found to be active are then sent to a researcher at the University of Florida where a greenhouse citrus assay has been developed for testing therapies for control of HLB. While this test takes several months, it provides valuable information on how therapies can be applied to citrus to reach the pathogen and how often treatment is needed. Finally, the best performing microbes and compounds are tested in field trials in Florida. Since the disease is everywhere there, it is certain that the trees will eventually become infected, making it possible to determine if the microbes and compounds are effective. To track the activity of the therapies, testing is carried out on citrus transplants coming from nurseries without disease. As they are exposed to the population of psyllids carrying the bacterium, the progress of disease development can be monitored to see how the therapies are performing.Traditional methods for following disease are challenging because the bacterium is not evenly spread throughout the tree itself, and bacterial distribution varies with the time of the year. For example, in certain seasons, the bacterium may move into the roots so sampling leaves for the disease will give a negative result even if the tree is infected. To address this, a researcher from the University of California Davis has developed a way to look at the tree's response to the infection, like monitoring the level of white blood cells in an infected human. Here, infected citrus trees produce an immune response that can be seen in any tissue of the tree, so sampling is not a problem. Also, this response occurs very quickly after infection even while the trees do not show any disease symptoms. This method is being compared to the more traditional disease detection method to see if bacterial infection is detected sooner and more accurately.In the end, it is hoped that by combining the screening capabilities and product development experience of the agrochemical industry partner with the expertise found in top agricultural research universities in the United States, while at the same time maintaining good communication with the citrus industry, we will make progress in identifying treatments that will slow down or halt this disease. The result will be healthier trees with more production to sustain US citrus production, while at the same time protecting the jobs associated with a vibrant citrus industry.We are determined to succeed because it is hard to imagine a world without citrus.
The goal of the project is to develop therapeutic leads identified in a previous three-year screening campaign into viable commercial product candidates that demonstrate efficacy in citrus fields against citrus Huanglongbing (HLB) disease for delaying or halting disease development to maintain productivity.Objectives:To provide integrated disease management strategies, two approaches to address HLB disease management have been included: (1) synthetic Plant Defense Inducers (PDI) with an indirect mode of action, and (2) anti-bacterial microbial strains (AM) that could be developed into biopesticides with direct activity against 'Candidatus Liberibacter asiaticus', the bacterium associated with HLB. These approaches can result in registration timelines for both short- and medium-term product development.
To measure efficacy of synthetic analogs, two bacterial strains - Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. - will be used for screening analogs on a fully automated platform in 96 plates. In the primary greenhouse test, compounds will be evaluated using an automated spraying platform to screen hundreds of compounds every year in a highly reliable manner. The mode of action of newly synthesized analogs is checked using plant reporters on the pathogenesis-related protein I (PR1) Vivo Reporter screening tool (vRS). This assay relies on Arabidopsis thaliana plants containing the green fluorescent protein (GFP) linked to the salicylate responsive promoter sequence of the PR1 gene (AT2G14610). Xylem translocation of compounds on tomato and citrus will be evaluated by assessing the presence/quantity of analogs in each plant tissue area, analyzed by liquid chromatography/mass spectrometry (LCMS). The uptake and movement will indicate bioavailability and will be used to select the best compounds for further evaluation as well as potential for transfer of efficacy in field. Similar methodology will be developed to assess phloem translocation. Phytotoxicity will be measured to eliminate analogs with undesirable effects. The citrus molecular diagnostic test assesses citrus immune response to PDI leads. Salicylate and ethylene pathways are monitored with WRKY50 and ACS12/ERF105 genes respectively. Callose deposition is checked with callose synthase 1 gene. WRKY70 gene is also a node of convergence for salicylic acid and jasmonic acid mediated defense. Both WRKY50 and WRKY70 are important genes in response to CLas infection. Abiotic stress is monitored with MYB38 citrus marker. Absolute quantification of messenger RNA is performed by digital droplet PCR, and data are normalized to the level of expression of housekeeping genes to select best leads. The Cuticular Penetration Test allows the comparison of adjuvants to increase mobility of analogs in plant systems. The uptake of analogs across the cuticle is quantified using LCMS, allowing the selection of the best systems for greenhouse testing or field trials.Microbial strains will be prioritized for Biomarker Identification. Once in planta hits have been selected, they are promoted into our biomarker identification (BMID) platform. This is a series of multidisciplinary experimental processes to identify and validate the genes, proteins, or metabolites (biomarkers) produced by an active strain that directly contribute to the efficacy. Complete genome sequence assemblies are created for each hit strain and run through an internal annotation pipeline to characterize pathways and predict biosynthetic gene clusters (BGC). BGCs encode secondary metabolic pathways that often result in metabolites that contribute to the efficacy of the strain and can be used to identify toxins, clinical antibiotics, and antibiotic resistance markers that might affect registration. Comparative metabolomics, proteomics, and molecular networking are used to identify biomarkers from an active strain. These methods do not require the laborious process of fractionation, and allow rapid identification of potential biomarkers of interest that align with our genomic prioritizations before more targeted bioassay-guided fractionation is needed to confirm and validate the active biomarkers. Biochemical components of an active strain are fractionated using chemical separatory means. Fractions are tested in bioassays to determine which retain active biomarkers against the research target. To confirm the mode of action, molecular biology techniques are used, including targeted knock-outs and heterologous expression, to generate strains with eliminated or inserted genetic biomarkers, thereby removing from or introducing to the engineered strain the desired activity. Once Biomarker Identification is complete and analytical assays developed using LCMS, process optimization is necessary to determine the optimal media and fermentation conditions needed to increase production of bioactive secondary metabolites. Small-scale evaluations can be carried out using both a Pall microreactor and an Ambr250 bioreactor system which allows for comparing an array of media components and a multitude of growth conditions quickly. Scale up to 20L Sartorius fermenters is important for identifying candidates with potential to be grown at industrial scale and for producing material to support early field trial evaluation.The pilot plant is equipped with several options of downstream processing and concentration, centrifugation and filtration, for developing promising microbial strains into viable product candidates. After development of an optimized fermentation process, stabilization and prototype formulations are needed for delivering stable materials to be tested in field.Hairy root assay: Selected citrus tissues will be used as explant sources for hairy root induction. CLas-citrus hairy root cultures are periodically subjected to multiple quality control diagnostics to confirm and quantify CLas titers within the hairy root cultures. The CLas-citrus hairy root cultures will be treated with the respective therapies by vacuum infiltration for penetration into hairy root matrices and incubated for 72 hours. The effectiveness of the therapies will be determined by estimating relative CLas titers among the controls and treatments by qPCR amplification of ribonucleotide reductase β-subunit gene marker of CLas (RNR/nrdB, RNR-F/RNR-R).Greenhouse assay: The CLas titer will be monitored in plants, using the TaqMan PCR assay for detection, identification and quantification with the quantitative real time polymerase chain reaction (qPCR) tests and primers approved by USDA. Standard curves with plasmid carrying CLas sequence and normalized DNA templates will be developed for samples for titer calculations. This will be achieved using a plate reader and automated liquid handler to eliminate operator errors, which will further allow us to ensure that results from qPCR sample processing will be consistent and comparable within and between microbial metabolite screening experiments.Field trials: Leaves will be collected for metabolomics analysis and qPCR analysis. Visual assessments such as vigor, canopy size, trunk diameter and phytotoxicity will be recorded.Metabolomics: Leaf tissue samples collected from field and greenhouse are frozen and shipped to UC Davis under an existing USDA permit. Leaves are weighed, extracted with methanol then dried and resuspended with methanol:water and stored at -20°C until analysis using LCMS with a tandem quadrupole-time-of-light (qTOF) analyzer. Data analysis is performed to determine if a therapy is causing metabolomic changes to the trees. Different statistical models will be tested using untargeted metabolomic data.?Evaluation of project progress: Advancement of the project will be evaluated against the timeline and objectives every 6 months. The advancement to meet the final objective of selecting a candidate that meets regulatory, performance, and manufacturing feasibility will be evaluated at the end of each year when field trial results are available to the team.Year one will evaluate:Delivery of hits showing planta activity against HLB or significant reduction of HLBIdentification of starting points for development candidates while screening and optimization continuesSecond year advancement on:Optimization of selected candidatesEarly development studies (toxicology assessment, e-fate, metabolism, extended field trials, formulation)The third year will have the objective of defining the product concept for early development as well as determining the probability of technical and regulatory success of the candidates selected.
Target Audience
The target audience for this reporting period included the CAP Project Advisory Group, citrus industry stakeholders (CRDF, CRB), growers, advisors, research centers (SWFREC), Florida Department of Agriculture, International academic collaborator groups, International Industry collaborators, International HLB researchers and International Horticulture researchers with presentations and publications.
Changes / Problems
Nothing Reported
Training & Professional Development
Two undergraduate student assistants, Cierra Shelton and Trishla Mehta, were hired in January 2024 at UC Davis, to assist with work on this project. Under guidance from Dr. Rojas, these students are learning basic laboratory techniques related to citrus sample preparation, such as pipetting, weighing, extracting, labeling, and data logging. At the University of Florida SWFREC, one international intern, two high school interns, and a graduate student were trained in 2024.
Dissemination Streams
The results were broadly disseminated to the stakeholders through a Commercialization Update to the Citrus Commission. Research updates were provided at multiple international scientific congresses, for grower groups and at research institutions. A website was created for public access to results.
Next Reporting Steps
Objective 1: Further advanced in vivo Human Safety studies will be carried out to identify new front-runners and back up plant defense compounds to ensure providing a sustainable solution against HLB and to identify a potential development candidate. The team will also carry on the complete profiling of potential back-up compounds. A broad range of fruit and vegetable field trials will be initiated to improve the methodology required for PDI. Objective 2: Work on this objective has been completed. A viable microbial product candidate has not yet been identified that meets regulatory and commercial requirements. Screening in HLB greenhouse candidates is ongoing with the last lead strains that have been delivered to UF. Objective 3: Completed Objective 4: HLB greenhouse screening assays will continue to assess additional PDI leads and remaining microbial extracts for their potentials on reduction of CLas infection or to delay the infection. We will further fine tune our application concentration, timing and frequency to establish more consistent and reliable control effect. We will continue to monitor these compounds' efficacy on ACP colonization and behavior. Objective 5: The OTC-PDI integrated program trial at UF will be continued for a second year of applications. A second trial initiated to repeat first results will be carried out to yield and quality. Two trials large scale initiated in late 2023 on infected trees will continue to receive applications of test compounds and will be monitored for results. Harvest yields and brix measurements along with vigor and disease ratings will be collected in early 2025. This will help confirm the preliminary result showing that PDI compounds can suppress HLB of OTC injected infected trees and determine if yield and quality are improved to support growers with additional tools. Objective 6: We will continue to make HLB predictions for provided leaf samples through the end of the project. In addition to making our metabolomics-based HLB prediction, the plant vigor screening will provide additional biochemical information as to the response to plants from treatments and preventions. While no treatment or prevention seems to be reversing a positive HLB status, some treatments are showing improvements to plant vigor, based on visual inspection of trees. Through our plant vigor metabolite assessment, the Davis Lab will quantitatively detail the impact of the experimental treatments as to their ability to help citrus trees survive HLB and remain agriculturally productive. In collaboration with Dr Batuman, we are also working to develop a metabolomics assay predictive of fruit yield in citrus samples. Objective 7. This goal has been completed.
Target Audience
The target audience for this reporting period included the CAP Project Advisory Group, citrus industry stakeholders (CRDF, CRB), growers, advisors, research centers (SWFREC), Florida Department of Agriculture, International academic collaborator groups, Texas academic collaborator groups, International Industry collaborators, and International Horticulture researchers with presentations and publications.
Changes / Problems
Nothing Reported
Training & Professional Development
The project provided research training and mentoring opportunities to multiple Hispanic-minority undergraduate students and research technicians, as well as early career postdocs and research scientists at Texas A&M AgriLife research. We are happy to welcome Dr. Dante Rojas, PhD to the team at UC Davis through funding from this award. Dr. Rojas is learning new analytical approaches, such as sample analysis through a high-resolution mass spectrometer (LC-qTOF), and multivariate statistical analyses techniques (PLS-DA, XGB-DA) to chemometrically assess metabolomics datasets. Additionally, Blake Bryant, a PhD candidate that started in April 2023, is contributing to this project. Through this work, Blake has been trained on sample extraction techniques for the development of citrus metabolites related to HLB detection and plant vigor status. He is also being trained on how to analyze samples using the LC-qTOF mass spectrometer. Two undergraduate student assistants, Nick Gregan and Dominic LeLouis, were hired in June and October 2023, respectively, to assist with this work. Under guidance from Dr. Rojas, these students are learning basic laboratory techniques related to citrus sample preparation, such as pipetting, weighing, extracting, labeling, and data logging. At the University of Florida, SWFREC, we were able to train two interns, a graduate student, and a postdoc in 2023 At Bayer, the project provided the opportunities to develop new competencies and attract new talents within the company. Among the CRDF temporary contracts, 4 additional collaborators were hired internally in permanent position in 2023. Information related to results, knowledge, knowhow, and expertise were exchanged with our partners through regular meetings and brainstorming sessions.
Dissemination Streams
Batuman, O. New SAR inducers for HLB control. SWFREC Open House Demos and Presentations. November 8, 2023 Batuman O. Citrus Pathology Program for Huanglongbing (HLB) management. Presentation to USDA/Cornell Research Group. June 1, 2023 (Virtual) Batuman, O. IPCs' beneficial effects on citrus diseases and challenges they create. UF-IFAS In-service training to extension Agents. May 3, 2023; Immokalee, FL Batuman, O. Emerging viral diseases of citrus and management of their insect vectors. UF-IFAS In-service training to extension Agents. Mar 28, 2023; Immokalee, FL Batuman, O. Citrus Pathology Program and Projects. Presentation to specialty crop county extension agents. Apr 27, 2023; Clewiston, FL Batuman, O. Plant pathogens that may exacerbate the HLB effects on citrus trees. UF-IFAS Citrus Seminar Series for citrus stakeholders Jan 10, 2023; Immokalee, FL
Next Reporting Steps
Objective 1: Further advanced in vivo Human Safety studies will be initiated with front-runner and back up plant defense compounds to ensure providing a sustainable solution against HLB and to identify a potential development candidate. The team will also carry on the complete profiling of potential back-up compounds. A broad range of fruit and vegetable field trials will be initiated to improve the methodology required for PDI. Objective 2: Work on this objective has been completed. A viable microbial product candidate has not yet been identified that meets regulatory and commercial requirements. Screening in HLB greenhouse candidates is ongoing with the last lead strains that have been delivered to UF. Objective 3: Most of the CLas-citrus hairy root screening activities are completed. All pending assays will be completed in the next few months. We will also continue to disseminate the project results to the stakeholders and communities of interest. Objective 4: HLB greenhouse screening assays will continue to assess additional PDI leads and remaining microbial extracts for their potentials on reduction of CLas infection or to delay the infection. We will further fine tune our application concentration, timing and frequency to establish more consistent and reliable control effect. We will continue to monitor these compounds' efficacy on ACP colonization and behavior. Objective 5: The OTC-PDI integrated program trial at UF will be repeated as well as setting up an additional field trial including additional potential therapies. Two trials initiated in late 2022 will continue to receive applications of test compounds and will be monitored for results. Two new large scale trials will be initiated on 2-3 year old HLB infected trees to determine if a combination of injections of oxytetracycline and treatment with PDI leads will protect trees during the 180 day period before harvest as suggested by the preliminary results from the smaller trial at UF. Objective 6: We will continue to make HLB predictions for provided leaf samples through the end of the project. By December 2023, we anticipate having finalized our plant vigor metabolomics test. We will apply this test to samples from ongoing field trials. In addition to making our metabolomics-based HLB prediction, the plant vigor screening will provide additional biochemical information as to the response to plants from treatments and preventions. While no treatment or prevention seems to be reversing a positive HLB status, some treatments are showing improvements to plant vigor, based on visual inspection of trees. Through our plant vigor metabolite assessment, the Davis Lab will quantitatively detail the impact of the experimental treatments as to their ability to help citrus trees survive HLB and remain agriculturally productive. In collaboration with Dr Batuman, we are also working to develop a metabolomics assay predictive of fruit yield in citrus samples. Objective 7. This goal has been completed. <br><br>
<br>What was accomplished under these goals? Goal: develop therapeutic leads into viable commercial product candidates that demonstrate efficacy in citrus fields against Huanglongbing (HLB) for delaying or reducing disease to maintain grower productivity. Objective 1. Optimizing a lead class of synthetic plant defense inducers (PDIs) Exploration of the H class has progressed with the aim to combine best efficacy with the best regulatory profile. Synthesis of 680 additional compounds in the prioritized H-class 37 additional internal batch productions for Field testing and Human safety studies Evaluation of 900 compounds on vivo reporter plants for biochemical profile and in greenhouse for activity against bacteria in greenhouse Production of 3600 citrus plantlets for the molecular diagnostic Characterization of 11 compounds: foliar bioavailability, metabolization pattern and distribution of parents & metabolites in tomato model plant Profiling of 39 best compounds in citrus diagnostic test New in vivo Human safety studies on 8 compounds New advanced in vivo Human safety studies on 2 compounds Selection of 11 new candidates for greenhouse evaluation against HLB OUTCOMES AND IMPACTS: Consistent efficacy against HLB in greenhouse was observed for several H class leads. Additional efficacy against citrus canker was also established. Two lead compounds were prioritized and pushed forward for turning on the citrus plant defense system, ability to move through plants, quick penetration of citrus leaf, soil degradation, impact on human safety models. These have the highest chance of surviving the tough regulatory process to get new synthetic products to growers. Objective 2. Developing promising microbial strains into viable product candidates 30 strains were grown to complete selection for hairy root and greenhouse assays and processed for shipment 34 samples were sent to Texas A&M from last of hits from in vitro cascade 36 samples were sent to UF for greenhouse testing in CLas seedlings from last of hits from in vitro cascade Microbial fermentations prepared and shipped to the field for final applications in field trials Supported biomarker workflow for 2 field compounds with in vitro testing Focus on single strain, tested in cytological profiling assay (by Linnaeus Biosciences, San Diego), showed that this supernatant has compounds that can (1) disrupt membranes (2) disrupt DNA synthesis and (3) enhance membrane permeability in AGRBRH (and E. coli) At least 10 compound clusters and 30 molecular features are correlated to bioactivity Additional experiments would need to be done to conclude which compounds cause antibacterial activity in AGRBRH and LIBECR, and if this activity translates to activity against HLB in citrus trees OUTCOMES AND IMPACTS: From the last wave of active strains selected based on in vitro antibacterial activity, fermentations were prepared and will be tested in the final round of hairy root assays and in on going greenhouse assays at UF. Objective 3. Determining relevance of hairy root plant tissue culture in predicting activity on HLB CLas-citrus hairy root cultures were prepared, and QC conducted to determine CLas titers in hairy roots CLas-citrus hairy roots were then used in the HR assays with leads, in replicate plate design/layouts, followed by molecular diagnostics/data analysis to determine efficacy In this reporting period, we completed >1500 HR assays Among them, >20 hits reduced CLas in citrus root tissues, signifying promising leads OUTCOMES AND IMPACTS: Using this relatively quick plant tissue assay allows for screening larger numbers of leads before testing in greenhouse assays with citrus plants and helps build confidence in the screening process developed. Objective 4. Using greenhouse citrus assays to determine best conditions for field testing leads A total of 56 PDI and BLX leads and 2 external compounds were tested for phytotoxicity followed application using NATI on HLB positive plants (BLX leads) or foliar applications before infecting citrus (PDI leads) ACP preventative assays were developed to screen leads on HLB infected citrus Several PDI leads were tested for canker control in greenhouse experiments Grafting with CLas-infected material was carried out to provide ~600 HLB positive plants for testing OUTCOMES AND IMPACTS: Several compounds were identified that substantially delay infection with a single application and also control canker. The knowledge gained about doses and phytotoxicity will guide efforts to translate these results to field conditions. This is a key component of the project for moving leads towards possible commercialization. Objective 5. Field testing of leads to determine efficacy against HLB development Two field trials in grower fields with trees planted and netted in the previous year were set up (9 tree plots, six replications=54 trees for each treatment) to test the most advanced PDI and microbial leads Two field trials initiated in April and June 2021 had final foliar applications of PDI leads applied in December 2022 with disease ratings taken into 2023 Two trials established in July 2022 to test new PDI candidates were continued through mid 2023 Two additional trials started in October 2022 including a microbial lead, alone and in combination with a PDI lead and an external synthetic compound were continued through 2023 A new trial was started at UF to create an integrated program with OTC injections using PDI or BLX applications made during the pre-harvest interval timing when OTC cannot be used OUTCOMES AND IMPACTS: There was indication that a PDI lead could delay disease development in field in two trials and first results that PDI following OTC provided significant HLB suppression compared to OTC alone. This could provide an alternate mode of action to slow resistance development to OTC and protect trees in season when OTC cannot be applied. Objective 6. Investigating metabolomics as an early detection method for HLB in field conditions Since May 2022, Davis Lab has carried out ~ 9,000 sample injections and analyses, totaling more than 19,500 Plant samples are analyzed twice (for HLB prediction and a second for plant vigor rating) Metabolomics results on HLB status still align with PCR findings. To date, no field treatments are reversing or preventing HLB infections in citrus, though field experiments with new treatment regimes are pending Due to observed differences in plant vigor with different treatment options (in spite of HLB positive status), Davis Lab developed a new metabolomics approach to assess plant vigor Tree vigor can be predicted, as measured by canopy height and width, through measurement of select metabolites in citrus leaf samples This helps determine impact of treatments on overall tree health Davis Lab is working with Batuman Lab to develop a metabolomics assay to predict fruit yield Batuman lab has ongoing experiments where fruit yield data will be captured Batuman Lab will send samples to UCD to analyze and develop AI/ML models to map metabolomics signatures to fruit yield This could have major implications for citrus growers, should we develop this new tool to help them predict yield OUTCOMES AND IMPACTS: Faster disease detection and ability to predict yield despite HLB infections will benefit growers in implementing new technologies to address HLB. Objective 7. Define a registration pathway for moving leads towards commercialization An extensive evaluation of potential regulatory approaches was carried out to provide a roadmap for regulatory data requirements, timelines and costs the information for conventional, biochemical and microbial pesticides. This 41 page report also included the potential for an emergency registration and for state registrations specific to citrus. OUTCOMES AND IMPACTS: A clear path forward for moving research leads to commercial products is through the regulatory process and this analysis will help in the process. <br><br><b>Publications</b><br>
Target Audience
The target audience for this reporting period included the CAP Project Advisory Group, citrus industry stakeholders (CRDF, CRB), growers, advisors, research centers (SWFREC), Florida Department of Agriculture, International academic collaborator groups, Texas academic collaborator groups, International Industry collaborators, and International Horticulture researcherswith presentations and Field Days
Changes / Problems
Nothing Reported
Training & Professional Development
The project provided research training and mentoring opportunities to multiple Hispanic-minority undergraduate students and research technicians, as well as early career postdocs and research scientists at Texas A&M AgriLife research. ? We were happy to welcome Dr. Michael Eze, PhD to the team through funding from this award. Dr. Eze is a Postdoctoral scholar who joined Prof Davis' group in UC Davis in April 2022. Through this project, Dr. Eze is learning new analytical approaches, such as sample analysis through a high resolution mass spectrometer (LC-qTOF), and multivariate statistical analyses techniques (PLS-DA, XGB-DA) to chemometrically assess metabolomics datasets. Additionally, Katie Hamera, a Junior Specialist staff member hired in April 2022, is contributing to this project. Through this work, Katie is being trained on additional sample extraction techniques for the development of citrus metabolites related to plant vigor status. She is also being trained on how to analyze samples using the LC-qTOF mass spectrometer. Two undergraduate student assistants, Chloe Barry and Christian Soto, were hired in October 2022 to assist with this work. Under guidance from Dr. Eze, these students are learning basic laboratory techniques related to citrus sample preparation, such as pipetting, weighing, extracting, labeling, and data logging. In 2022, we hired and trained one postdoc and one new technician, who all were able to learn and improve their skills and gain new knowledge about screening techniques and methods as part of this project. We also trained an intern (Veteran) in the summer at the University of Florida. At Bayer, the project provided the opportunities to develop new competencies and attract new talents within the company. Among all the CRDF temporary contracts, 4 additional collaborators were hired internally in permanent position. The project also provided research training to undergraduate students. Information related to results, knowledge, knowhow, and expertise were exchanged with our partners through regular meetings and brainstorming sessions.
Dissemination Streams
The results were broadly disseminated to the stakeholders during the CRDF Project Advisory Board meetings on December 15, 2021 and June 23, 2022. The overall approaches and strategies of antimicrobial screening cascades were discussed/presented at multiple workshops and symposia. Mandadi, K.K (2022). High-throughput antimicrobial testing using microbial hairy root system. Antimicrobial Summit/Workshop, Citrus Research Board. May 23-24, San Francisco, CA. Mandadi, K.K. (2022). High throughput therapeutic screening using hairy root system. Joint Annual Meeting of the American Phytopathological Society Caribbean Division (APS-CD) and Entomological Society of America South-Eastern Branch (SEB). March 26-30, San Juan, PR. An extension/outreach article discussing the project activities and findings was prepared and published in a leading citrus growers' magazine (Citrograph). Manker, Denise. (2022) Collaborative approach to developing therapies for HLB, Citrograph, 13. Dr. Batuman gave five local presentations to growers and additional two to Advisory Board members in 2022. Part of the results were also presented at the International Horticultural Congress in Angers, France and, an accompanying proceeding paper has been published. Additionally, Dr. Batuman also presented and demonstrated results of this project to individual and groups of lab and greenhouse visitors including students, visiting scientists, homeowners, industry representatives and growers. Presentations McCartney MM, Eze MO. "Metabolomics: a window into cellular and biochemical processes". Bayer Crop Sciences Meeting. July 18 2022. Virtual presentation Davis CE. (Plenary Speaker) "Volatiles in Biological Systems Shift with Disease". International Association of Breath Research Conference. June 13 2022. McCartney MM. "VOC sensing of produce quality." Sensing Quality in the Produce Industry. May 24 2022.
Next Reporting Steps
Objective 1: Further Environmental and Human Safety studies will be initiated with front-runner synthetic plant defense compounds to ensure providing a sustainable solution against HLB and to identify a potential development candidate. The team will also carry on the exploration of the H class to identify potential back-up compounds. The complete profile of those compounds will be established. A broad range of fruit and vegetable field trials will be initiated to better understand the methodology required for PDI. Such test will also include treatment program combining PDI and BLX on citrus. Objective 2: Focused effort will be taken to identify bioactive compounds from a lead strain. Preparation of sample materials for collaborator assays and field trials has been moved from Chemistry to Fermentation clearing the way for chemical analysis. Objective 3: We will continue the CLas-citrus hairy root screening activities in the next reporting periods with additional leads. We will also continue training and mentoring the students and staff and disseminate the project results to the stakeholders and communities of interest. Objective 4: We will continue with our screening assays to assess these and additional new compounds for their potentials on reduction of CLas infection or to delay the infection. We will further fine tune our application concentration, timing and frequency to establish more consistent and reliable control effect. We will continue to monitor these compounds efficacy on ACP colonization and behavior. Objective 5: Four additional trials initiated in 2022 will continue to receive applications of test compounds and ongoing trials from 2021 will be monitored for results. Work in the field to determine if a combination of injections of oxytetracycline and treatment with PDI leads will protect trees during the 180 day period before harvest are being planned for 2023. Objective 6: By December 20222, we anticipate having finalized our plant vigor metabolomics test. We will apply this test to samples from ongoing field trials. In addition to making our metabolomics-based HLB prediction, the plant vigor screening will provide additional biochemical information as to the response to plants from treatments and preventions. While no treatment or prevention seems to be reversing a positive HLB status, some treatments are showing improvements to plant vigor, based on visual inspection of trees. Through our plant vigor metabolite assessment, the Davis Lab will quantitatively detail the impact of the experimental treatments as to their ability to help citrus trees survive HLB and remain agriculturally productive. Objective 7. A project has been set for the first half of 2023 to map out requirements for EPA registration for plant defense synthetic compounds, microbial extracts and an external synthetic antibacterial mixture to determine toxicology testing required, approximate costs and timelines for a registration for HLB in the US. <br><br>
<br>What was accomplished under these goals? Objective 1. Optimizing a lead class of synthetic plant defense inducers (PDIs) Exploration of the H class has progressed with the aim to combine best efficacy with the best regulatory profile. Synthesis of 700 additional compounds in the prioritized H-class 39 additional internal batch productions for Field testing and Human safety studies 1 radiolabeled compound for Soil studies Evaluation of 900 new compounds for biochemical profile and activity against bacteria in greenhouse Production of 3600 citrus plantlets for the molecular diagnostic Characterization of 4 compounds: foliar bioavailability, metabolization pattern and distribution of parents & metabolites in tomato model plant Development of a citrus phytotoxicity test in greenhouse Environmental Safety soil studies on radiolabeled compound to evaluate soil behavior of a parent molecule and its metabolite. New in vivo studies on 6 compounds validated the Human Safety screening process to prevent loss of candidates due to regulatory hurdles Selection of 11 new candidates for greenhouse evaluation against HLB Two front runners selected for new field trials in Florida OUTCOMES AND IMPACTS: Lead compounds were prioritized by testing many synthetic analogs for turning on the citrus plant defense system, ability to move through plants, quick penetration of citrus leaf, soil degradation, impact on human safety models and safety for citrus plants. These have the highest chance of surviving the tough regulatory process for getting new synthetic products to growers. Objective 2. Developing promising microbial strains into viable product candidates Primary Screening was completed with selection and testing of 3100 strains including Xenorhabdus, Photorhabdus, and Streptomyces 50 organic extract samples from the hit selection were sent to be tested in Hairy Root assay and 14 were confirmed positive with the direct pathogen 14 samples were tested at the University of Florida for phytotoxicity and to qualify for field testing 4 samples (2 positive & 2 negative) from hairy root assay were tested to see translation effect in UF's Greenhouse assay on CLas infected plants, only 1 was efficacious. 11 samples are in the process of testing A protocol for identifying biomarkers in lead strains has been developed and preliminary work is underway Thirty strains have been fermented to be tested in greenhouse assays. From those hits, 2 strains were successfully scaled up to 20 liter fermentation for concentration for field trial testing and to support biomarker research Support on external leads from collaborators was performed with BOOST and PT-159 OUTCOMES AND IMPACTS: The outcome is focus on the most active strains to advance them to testing in systems with HLB in plant tissues: hairy root assay (Obj 3) and greenhouse citrus assay (Obj 4) and begin identifying active natural compounds to move the best microbial strains towards eventual candidates for registration. Objective 3. Determining relevance of hairy root plant tissue culture in predicting activity on HLB CLas-citrus hairy root cultures were prepared, and QC conducted to determine CLas titers in the hairy roots. Next, CLas-citrus hairy roots were used in the HR assays with Bayer leads, in replicate plate design/layouts, followed by molecular diagnostics/data analysis to determine efficacy. In this reporting period, we focused on screening Bayer microbial leads, and third-party samples completing >460 HR assays. Among them, >12 leads reduced HLB in citrus root tissues, signifying promising leads. OUTCOMES AND IMPACTS: Using this relatively quick plant tissue assay allows for screening larger numbers of leads before passing on to the greenhouse assay with citrus plants and helps build our knowledge for how to predict activity based on relating various screens in our cascade. Objective 4. Using greenhouse citrus assays to determine best conditions for field testing leads In 2022, 130 microbial and synthetic leads were screened in greenhouse assays on young citrus seedlings by foliar spray, soil drench, or NATI. Some were screened at multiple concentrations to find the least phytotoxic dose in advance of field trials. We completed 7 experiments with preventive PDI treatments for HLB and an additional 6 for canker. Screening is ongoing with new microbial leads. Preventive assays, treated HLB negative plants with synthetic PDI's to protect young citrus trees from infection by CLas by protecting flushes. Curative assays, treated HLB-positive plants with microbial leads for controlling (or reducing) the HLB levels. Multiple doses of ~30 PDIs were assessed determining flushing patterns & application timing to protect new shoots and observing insect repellent properties. Several leads substantially delayed CLas infection by 3-6 months compared to untreated plants and reduced the numbers of ACP on colonized plants. All PDIs screened against citrus canker in greenhouse provided significant protection, much better than copper (grower standard) and equal to/better than commercial standard Actigard. Thus far no microbials have consistently performed as well as OTC in reducing HLB titers in citrus. There is a substantial visible improvement in BLX-treated plants compared with water-treated control plants, and equal to OTC-treated plants. OUTCOMES AND IMPACTS: Several compounds were identified that can substantially delay infection with a single application. The knowledge gained about doses and phytotoxicity will guide efforts to translate these results to field conditions. This is a key component of the project for moving leads towards possible commercialization. Objective 5. Field testing of leads to determine efficacy against HLB development Field trials in newly planted grower fields were set up (9 tree plots, six replications=54 trees for each treatment) to test the most advanced PDI and microbial leads. Two field trials initiated in April and June 2021 are on-going with foliar applications of PDI leads applied at two doses and a treatment combining both PDI chemistry and a soil application of a microbial strain. Two trials were established in July 2022 to test new PDI candidates Two additional trials were started in October including a microbial lead, alone and in combination with a PDI lead and an external synthetic compound OUTCOMES AND IMPACTS: Demonstrating activity of lead candidates that can delay or reduce HLB infections in citrus trees under grower conditions is paramount. The results of these trials will determine if leads can be progressed towards registration. Objective 6. Investigating metabolomics as an early detection method for HLB in field conditions Since May 2022, Davis Lab has carried out ~ 2,500 sample injections and analyses, totaling > 13,000 during this award period. Metabolomics results regarding HLB status still align with PCR findings. No treatments or preventions are reversing or preventing HLB infections in citrus in field. In view of the observed differences in plant vigor with the different treatment options (in spite of HLB positive status), Davis Lab is developing a new metabolomics approach to assess plant vigor The new approach accurately predicted plant vigor using previous leaf samples in a pilot investigation Davis Lab is currently developing and optimizing new metabolomics method for vigor assessment New metabolomics method focuses on phytohormones and sugar metabolism The new method will enable the prediction of fruit quality in addition to plant vigor Once finalized, plant samples will be analyzed twice (one for HLB prediction by metabolomics, and a second for plant vigor rating) Objective 7. Define a registration pathway for moving leads towards commercialization This activity will be carried out in 2023 <br><br><b>Publications</b><br>
Target Audience
The target audience for this reporting period included the CAP Project Advisory Group,citrus industry stakeholders (CRDF, CRB), growers, advisors, research centers (SWFREC), Florida Department of Agriculture, International academic collaborator groups, Texas academic collaborator groups, International Industry collaborators, Rotary club and a middle school career day with presentations and Field Days
Changes / Problems
Nothing Reported
Training & Professional Development
The project provided the opportunities to develop new competencies and attract new talents within Bayer Crop Science in Lyon, France. Among all the CRDF temporary contracts, 4 collaborators were hired internally in permanent positions. The project also provided research training to undergraduate students. During the first year in Bayer Crop Science, West Sacramento California,the project hired 3 temporary contractors that had just graduated from college. Working in the company and in the project allowed these scientists tostarttheir careers and gain experiencein the agricultural field.The interdisciplinary natureof the project allowed them to gain experience not only in their fieldbut also in other areas of the project that needed advancement. This mindset greatlyformedthe mindset of the team to develop more flexibly,where they specialized in one area but canalso support in other areas to advance the project.This project alsoprovidedhands-on experienceforour research project managertoqualify to apply for her Project Management Professional certificate. At Texas A&M AgriLife Research, the project provided research training and mentoring opportunities to multiple Hispanic-minority undergraduate students and research technicians, as well as early career postdocs and research scientists. University of Florida SWFREC has hired and trained one postdoc, one bio scientist and one technician that all were able to learn and improve their skills and gain new knowledge about screening techniques and methods as part of this project. At UC Davis, the project has provided training and mentoring opportunities to two undergraduate researchers, a postdoctoral scholar, and two R&D Engineer staff members, including persons from ethnic minorities, and two LGBTQ+ individuals.
Dissemination Streams
Two Project Advisory Group meetings have been held (December 2020 and June 2021) and will continue every six months. The PAG includes industry stakeholders (CRDF, CRB) and academic advisors. Presentations made available to citrus industry stakeholders and available on their websites. Written updates are provided to CRDF quarterly and CRB annually, as requested, and are made available to their stakeholders Summaries of work on this project were provided in the Booklet of the Citrus Expo meeting in August 2021 Outreach presentations have been made by Dr. Batuman in 2021 to Industry Collaborators, Rotary club, Growers, SWFREC, International Academic Collaborators, Florida Dept of Agriculture, Texas Academic Collaborators, International Industry Collaborators and to Immokalee Foundation's Middle School Career day, totaling 17 presentations. Dr. Mandadi presented results in the CLas Culturing Panel/Workshop for CRDF in Nov 2020
Next Reporting Steps
Objective 1. Optimizing a lead class of synthetic plant defense inducers Complete characterization of lead compounds including efficacy on HLB and other bacterial diseases, as well as environmental, plant and animal safety to select best compound for commercial development Submit patents for IP protection of product candidates Objective 2. Developing promising microbial strains into viable product candidates Complete biomarker identification, fermentation scale up and optimization and prototype formulation development of top microbial leads Submit patents for IP protection of product candidates Objective 3. Determining relevance of hairy root plant tissue culture in predicting activity on HLB Continue screening leads to identify best microbial candidates to progress to greenhouse studies Publish findings in peer reviewed journals and present results at conferences Objective 4. Using greenhouse citrus assays to determine best conditions for field testing leads Screen top leads and determine optimal doses and application methods and intervals of application for testing in field Publish findings in peer reviewed journals and present results at conferences Objective 5. Field testing of leads to determine efficacy against HLB development Set up 2-3 trials each year, collecting disease data to identify leads active against HLB and determine integrated programs with both plant defense compounds and microbials for best efficacy Objective 6. Investigating metabolomics as an early detection method for HLB in field conditions Develop predictive models from field data for early detection of HLB Publish findings in peer reviewed journals and present results at conferences Objective 7. Defining a registration pathway for moving leads towards commercialization Once lead compounds are selected, use toxicology data to develop US registration plan to provide a route to commercialize products for use in citrus Determine regulatory path for other citrus producing countries to slow the spread of HLB Objective 8. Developing an effective extension and outreach program Develop public webpages and continue presentations and publications of research findings to stakeholders in citrus community including growers, advisors and commodity groups Develop and provide best use guidelines for employing new therapies to fight HLB Publish peer-reviewed journal articles and present at scientific and industry conferences on research results <br><br>
<br>What was accomplished under these goals? The future of the citrus industry producing orange juice and fresh fruit is threatened worldwide by a devastating disease known as Huanglongbing (HLB). It is also referred to as citrus greening or CLas. The bacterial infection is difficult to stop because it lives in the tree's circulatory system lying just beneath the bark, out of reach. The disease is carried by a flying insect called a psyllid, which is smaller than a sesame seed. The psyllid transmits the disease by feeding on the leaves of an infected tree, ingesting HLB bacterium. Once the bacterium is in the psyllid, the psyllid conveys the disease to other trees it feeds upon. This disease threatens citrus production in the US, which is worth $3 billion and in Florida and California provides nearly 100,000 jobs. New approaches are needed to identify potential products, quickly detect the disease, and test trees for the ability to suppress or kill HLB. Two approaches are being investigated in parallel to speed development. In one case, the agrochemical industry partner is looking for naturally occurring microbes that could fight the disease. A library of more than 150,000 isolated microbial strains is being searched for one that could naturally control citrus greening. In a second approach, more than 200,000 synthetic compounds are being screened to determine if they can boost the plant's defense system in the same way a vaccine can protect against disease. Combining different ways to stop citrus greening can help keep HLB from developing immunity to the therapies. Objective 1: Optimizing a lead class of synthetic plant defense inducers, 30% complete Many synthetic compounds in the H-class were tested for turning on the citrus plant defense system, ability to move through plant material, penetrate of the citrus leaf, how they degrade in soil, what impact they have on human safety models and how safe they are for citrus plants. This has allowed for selection of compounds with the best profiles for controlling bacterial diseases in plants and safety to plants, animals and the environment. These compounds have the highest chance of making it through the tough regulatory process required to get new synthetic products to growers through EPA registration Objective 2. Developing promising microbial strains into viable product candidates, 20% complete Primary screening was completed for2490 strains from 326 different species on bacterial surrogates for HLB which cannot be grown in a lab. Results prioritized 200 strains which were grown in shake flasks and tested for potency. Of these 50 were chosen for testing in plant assays (hairy root and greenhouse HLB). Objective 3. Determining relevance of hairy root plant tissue culture in predicting activity on HLB, 30% complete To understand the translation of activity seen in screening carried out in Objectives 1 and 2 to the root tissue infected with HLB (hairy root assay), around 288 assays were carried out, and ~3 leads showed efficacy against CLas in citrus root tissues, on par with reference bactericides (e.g., oxytetracycline) and are of potential interest as therapeutics for HLB. Objective 4. Using greenhouse citrus assays to determine best conditions for field testing leads, 30% complete 54 compounds were screened, (microbial extracts and plant defense inducers), in gh assays applied to young citrus seedlings by foliar spray, soil drench or NATI (trunk injection) in 7 completed and 6 ongoing experiments Several leads were screened to find the least phytotoxic dose before use in field trials Two screening approaches were used: Preventive, treating 1-year old, healthy (HLB negative) plants used to test plant defense compounds in a strategy to protect young citrus trees from CLas infection by protecting flushes Curative, treating HLB positive plants to determine efficacy CLas titer in plants infected before treatment, used to test microbial extracts Several leads were identified to have a substantial preventive property and were able to delay the CLas infection for 3-5 months with a single application compared to untreated plants Reduction of CLas titer was seen with microbial extracts but was not consistent. These will be repeated. Objective 5. Field testing of leads to determine efficacy against HLB development, 30% complete Field trials in newly planted grower fields were set up on Valencia Orange on US942 rootstock using 9 tree plots with 6 replicates (54 trees) to test the most advanced plant defense and microbial leads Two field trials initiated in May and June 2020 were continued in this grant year, including 4 treatments of PDI leads and 3 microbial extracts. Foliar applications of PDI leads were made in Sept, Nov, Dec, Jan and Mar and trunk injections of microbial extracts were made in Oct, Jan, and Mar Leaf samples were collected for metabolomics analysis and qPCR at 6 week intervals with HLB first detection 9 months after exposure to psyllids with 13% and 7% of trees HLB positive in the two trials by qPCR and no apparent effect of any treatment Two field trials were planted in April and June 2021. Foliar applications of 3 new PDI leads were made at two doses and 1 treatment combined PDI chemistry and a microbial soil application. Foliar applications will be made every 2 months and the microbial injections every 4 months. Leaf samples are collected at 8-week intervals for HLB detection A phytotoxicity field trial was started with 3 PDI leads at three rates, with and without adjuvant Objective 6. Investigating metabolomics as an early detection method for HLB in field conditions, 30% complete Non-volatile compounds were measured from leaves by organic extraction using liquid chromatography separation techniques coupled with mass spectrometric (MS) instruments for detection. This data should provide prediction values of the infected or healthy state of the tree. Results from gh and field trials will be compared with the standard qPCR assay used to detect HLB today. Over 8,000 leaf samples from field trials have been processed A library of over 700 metabolites was built, to aid in biochemical understanding of HLB mechanisms, preventions and cures Early field time points (weeks to several months) do not show HLB infection, so priority has shifted to analyzing later timepoints where we might expect HLB activity to compare efficacy of treatments/preventions. This metabolomics assay was able to identify HLB positive trees several months before disease was detected by qPCR, allowing for faster decision making in field trials In greenhouse studies, it was determined that application methods such as foliar sprays and trunk injection do not disrupt prediction of infected or non HLB infected trees Objective 7. Define a registration pathway for moving leads towards commercialization, 0% Complete This objective relies on identifying candidates for development and can be expected to be needed in Years 2 and 3 Objective 8. Develop an effective extension and outreach program, 30% completed Two Project Advisory Group meetings have been held (December 2020 and June 2021) and will continue every six months. The PAG includes industry stakeholders (CRDF, CRB) and academic advisors. Presentations made available to citrus industry stakeholders and available on their websites. Written updates are provided to CRDF quarterly and CRB annually, as requested, and are made available to their stakeholders Four summaries of work on this project were provided in the Booklet of the Citrus Expo meeting in August 2021 Outreach presentations reaching ~850 peoplehave been made by Dr. Batuman in 2021 to Industry Collaborators, Rotary club, Growers, SWFREC, International Academic Collaborators, Florida Dept of Agriculture, Texas Academic Collaborators, International Industry Collaborators and to Immokalee Foundation's Middle School Career day, totaling 17 presentations. Dr. Mandadi presented results in the CLas Culturing Panel/Workshop for CRDF in Nov 2020. <br><br><b>Publications</b><br>