Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1160 - Pathology | 100% |
Citrus greening or Huanglongbing (HLB) disease, caused by Candidatus Liberibacter asiaticus (CLas) in the U.S. is the most devastating citrus disease today, and has invaded all production areas in the U.S. Developing effective and practical (field deployable and commercially viable) HLB control strategies is critical for the survival of the U.S. citrus industry. In this CAP, by leveraging public-private partnerships between State agencies, Universities, USDA-ARS, regulatory affairs consultants, and the citrus industry, we will pursue advanced testing and commercialization of promising HLB therapies and extend outcomes to stakeholders. Thegoal is to find one or more effective therapies to kill CLas and control HLB. For this, we will utilize high throughput and innovative technologies such as CLas-citrus hairy root system to screen potent anti-CLas peptides and small molecules, followed by rigorous multi-location, multi-year field evaluations of the therapies and delivery systems.Promising therapies will be moved into a commercialization pipeline for regulatory approvals. Economic feasibility studies will be conducted to determine the extent of benefits to the citrus growersand a multi-state extension and outreach team will use diverse multi-media outlets to disseminate project information to the stakeholders.
Obj. 1. High throughput hairy root pre-screening. Two classes of therapies will be evaluated: a) defensins/antimicrobial peptide (AMP) and b) small molecules/chemistries. Each AMP and small molecule will be evaluated using established methods of hairy root bioassays. Hairy roots will be subjected to multiple QC diagnostics followed by relative CLas titer estimation using qPCR and CLas-specific primers, to determine effectiveness of the treatments when compared to untreated controls (vector-alone or mock). Statistical analysis will be performed using ANOVA/Post hoc and/or Student's t-test as appropriate to determine significant differences. Promising therapies will be moved to the in-planta evaluation using the Citrus tristeza virus (CTV) viral vector delivery system (Obj. 2) and vascular injection/delivery systems (Obj. 3).Obj. 2. Evaluation of antimicrobial peptides using CTV vectors. AMPs will be cloned into the CTV viral vectors and used to inoculate citrus trees in the greenhouse. After confirmation of systemic spread of the CTV-AMP in the trees, the material will be used as a source for inoculum to graft field-grown citrus trees. The field trials will be conducted in FL under an EPA EUP and USDA APHIS/BRS permits. The trials will be in a randomized complete block design under natural HLB/ACP pressure. Standard grove caretaking procedures are used to manage the plots. Multiple tree health traits will be assessed semi-annually. These include quantifying CLas incidence/titers and HLB disease symptoms, CTV titer (by ELISA), AMP stability (by RT-PCR), canopy volume and density (by measuring photosynthetically active radiation [PAR] penetrating the canopy), canopy volume, and flushing ratio. We will employ both ground truthing and high-throughput technologies (e.g., UAS) for data collection. CLas titers within the leaves and fine root samples will be estimated semi-annually by qPCR. Pre-harvest fruit drop will be assessed monthly starting in October. Finally, fruit yield (boxes/tree) and quality traits (e.g., fruit size and color, % juice, brix, acid, etc.) will be determined using standard procedures annually during harvest season. All fruits will be destroyed after harvest as per crop destruct and APHIS/EUP permit protocols. Statistical analyses will be performed using ANOVA/post hoc tests and/or Student t-test, as appropriate, to determine significant differences.Obj. 3. Evaluation of small molecule therapies. For the potential new anti-CLas candidates, first we will evaluate: i) systemic translocation in the plants, ii) suitable adjuvant/carrier combinations, iii) potential phytotoxicity, and iv) residual levels in plant tissues. The goal is to optimize effective delivery dosages, adjuvant combinations, and obtain information on residual levels and phytotoxicity to optimize approaches for field trials in FL and TX. For field trials, we will use a randomized block design. Briefly, we will compare efficacy of new compounds, alongside antibiotic controls, mock (e.g., solvent/adjuvant) and untreated plants as negative controls. All injections will be performed with Chemjet injectors above the graft union, two per tree on opposite sides. Multiple tree health traits will be assessed biannually or annually as described in Obj. 2 such as disease ratings, canopy volume and density, CLas titers biannually, pre-harvest fruit drop, fruit yield (boxes/tree), and quality traits annually during harvest season. All fruits will be destroyed after harvest as per crop destruct protocols. Statistical analyses will be performed using ANOVA/post hoc tests and/or Student t-test, as appropriate, to determine significant differences. Selected compounds with highest efficacy in year 1 will be evaluated in the second year.Obj. 4. Pursue data collection for regulatory approvals of the therapies. This work is NOT funded by this project; however, it will be complementary to the R&D activities and pursued in close collaboration with the citrus industry and a dedicated regulatory affairs team. We will initiate various required data collections and waivers to seek EPA approvals for promising therapies. A suite of good laboratory practice (GLP) data collection will be pursued, using agency guidelines, and included in a registration application package to support pesticide registration regulatory requirements for approval. Including, but not limited to, market basket study, health/dietary risk assessment, non-target effects on beneficials, pollinator/honeybee toxicity, and product characterization.Obj. 5. Evaluate the economic feasibility of deploying new HLB therapies and stakeholder surveys. To evaluate the profitability of adopting new HLB therapies on new and existing groves we will use investment analysis, which is used to assess the profitability of long-term capital investment, such as a citrus grove. The Net Present Value (NPV) is the preferred method for such evaluation because it considers the time value of money as well as the size of the stream of cash flows. In general, investments that yield a positive net present value should be accepted, otherwise rejected. For our purposes, therapies with higher NPVs will be preferred over alternatives with lower NPV because it would mean they are more profitable. The data generated from the project's trials will be inputs to the economic model along with the relevant market prices. The enterprise citrus budgets developed by FL, TX and CA University systems will serve as the baseline to estimate the cost of production in each citrus production region. We will also engage collaborating growers and other citrus growers in FL, TX, and CA, as needed, to seek various farm-level input data. In addition to determining the economic feasibility data, we will garner grower/industry feedback via surveys to understand the potential use/adoption rates. We will poll citrus growers using paper and electronic questionnaires as described before, to assess their willingness to adopt the novel HLB management strategies presented. Gathered data will be subjected to parametric and non-parametric econometric valuation models to examine the factors that could affect the adoption of the proposed HLB therapies by growers.Obj. 6. Extension and outreach to inform stakeholders. We will leverage every possible means to engage the stakeholders in the project by selecting multiple growers, industry, and commodity group members in our project advisory board, who will be directly informed of the project findings. Furthermore, our extension team is affiliated with regional citrus research and extension centers in TX, FL, and CA, and have continuous interactions with citrus growers, industry, and other stakeholders. They will interact with growers in TX, FL, and CA to provide HLB disease management recommendations resulting from this study and will organize extension programs and/or workshops to disseminate the results and economic benefits. Multiple engagement/outreach streams will be pursued such as: a) direct stakeholder engagement at industry and advisory board meetings, workshops, and field days. b) dissemination of project results via. presentations and talks. c) dissemination of project results via. factsheets, brochures, and research/extension flyers. d) dissemination of project results via. online websites related citrus research and HLB, and Facebook and twitter handles.
Target Audience
The following target audience was reached by the efforts and activities of this reporting period. • Plant pathology, microbiology, and biotechnology scientific research communities. • Students, postdocs, and early career scientists. • Agricultural community, including the citrus industry, growers, and consumers.
Changes / Problems
Nothing Reported
Training & Professional Development
• The project has provided research training and mentoring opportunities to several undergraduate students, graduate students, research technicians, as well as early career postdocs and research scientists. • The students and staff were routinely mentored by the PIs in laboratory techniques and scientific inquiry. • The students and staff were also given opportunities to attend and present their research at professional conferences. • Additionally, senior staff was provided opportunities to junior mentor staff, thus enhancing their teaching and mentoring skills.
Dissemination Streams
• As detailed in Obj. 6, project updates and results were periodically disseminated by the team members to various groups of stakeholders in TX, FL, and CA through a variety of avenues, including in-person meetings, workshops, and various social media channels, as detailed in the extension/outreach accomplishments above. • The results were also periodically disseminated to the broader scientific community through research publications and presentations at regional and international conferences.
Next Reporting Steps
• We will continue the proposed research activities in the laboratory, greenhouse, and field trials. Any additional candidate small molecules and AMPs that demonstrated promising activity in the hairy root assays will be moved to the greenhouse and field testing pipeline. • Results from the efficacy/field trials will be utilized to initiate economic feasibility studies in order to evaluate the profitability of adopting new HLB therapies on new and existing groves. • We will continue our outreach and extension activities to stakeholders in TX, FL, and CA and to the broader scientific community through a variety of avenues such as meetings, conferences, publications, and online media.
Target Audience
The following target audience was reached by the efforts and activities of this reporting period. • Plant pathology, microbiology, and biotechnology scientific research communities. • Students, postdocs, and early career scientists. • Agricultural community, including the citrus industry, growers, and consumers.
Changes / Problems
Nothing Reported
Training & Professional Development
• The project has provided research training and mentoring opportunities to several undergraduate students, graduate students, research technicians, as well as early career postdocs and research scientists. • The students and staff were routinely mentored by the PIs in laboratory techniques and scientific inquiry. • The students and staff were also given opportunities to attend and present their research at professional conferences. • Additionally, senior staff was provided opportunities to junior mentor staff, thus enhancing their teaching and mentoring skills.
Dissemination Streams
• Project updates and results were periodically disseminated by the team members to various groups of stakeholders in TX, FL, and CA through a variety of avenues, including in-person meetings, workshops, and various social media channels, as detailed in the extension/outreach accomplishments above. • The results were also periodically disseminated to the broader scientific community through research publications and presentations at regional and international conferences.
Next Reporting Steps
• We will continue the proposed research activities in the laboratory, greenhouse, and field trials. Any additional candidate small molecules and AMPs that demonstrated promising activity in the hairy root assays will be moved to the greenhouse and field testing pipeline. • Results from the efficacy/field trials will be utilized to initiate economic feasibility studies in order to evaluate the profitability of adopting new HLB therapies on new and existing groves. • We will continue our outreach and extension activities to stakeholders in TX, FL, and CA and to the broader scientific community through a variety of avenues such as meetings, conferences, publications, and online media. <br><br>
<br>What was accomplished under these goals? The proposed objectives/activities are underway and on track. Briefaccomplishments are below: Obj. 1. High throughput hairy root pre-screening and selection of effective HLB therapies. For this reporting period, after screening multiple classes of therapies (defensins/antimicrobial peptides and small molecules/chemistries) in hairy root assays, we identified ~6-8 promising candidates that were advanced to field trials (Obj. 2-3). Additionally, we have started screening new libraries of chemistries (agrochemicals and natural products) to gain more hits. We have also optimized the efficiency of the hairy root transformation and efficacy testing by evaluating new explant sources based on citron. The new sources/methods enabled testing the efficacy of AMPs in as little as 2 months. Additionally, we have developed new methods to regenerate stable citrus transgenic plants from hairy roots, and the results are published in the Plant Biotechnology Journal. Together, these continuous developments have been helpful in further speeding up the AMP and therapeutic testing pipeline. Obj. 2. Evaluation of antimicrobial peptides using CTV vector in citrus trees. For this reporting period, CTV-AMP vector optimization and cloning were completed with the selected AMPs. The performance of the citrus trees expressing the first batch of CTV-AMPs was also evaluated in the experimental field sites of Southern Gardens Citrus in FL. The preliminary data show promising yield improvement compared to controls. These updates were presented at the 2023 Annual Project Directors meeting in D.C. The evaluation of the new batches of AMPs will be pursued similarly for the remainder of the project's timeframe and will be reported accordingly. Briefly, all the field trials are being conducted under approved EPA EUP/BRS and USDA permits. The experimental design comprises a randomized complete block design with 50-60 tree replicates under natural HLB/ACP pressure. As per the workflow, the respective CTV-AMP expressing mother citrus plants were first generated in the greenhouse. Subsequently, the CTV-AMP expressing inoculum was graft inoculated onto citrus nursery trees ready to be planted in the field. The stability and CTV expression of the CTV-AMP inserts is monitored and periodically verified by RT-PCR in both the mother and the field plants. The HLB incidence in the field is also monitored by qPCR. Fruit yield and other plant health traits (canopy volume, trunk diameter, etc.) will be analyzed annually. Obj. 3. Evaluation of small molecule therapies in citrus trees. For this reporting period, we completed testing of the first batch of small molecule (~3 candidates) treatments along with Oxytetracycline (OTC) in HLB-affected citrus trees in TX by trunk injections. Preliminary data shows promising yield (quantity and quality) improvement in the trees injected with the new molecules compared to controls. The effects were on par or better than OTC. These updates were presented at the 2023 Annual Project Directors meeting in D.C. We have also initiated a second year of injections of the same trees to determine the cumulative effects. As before, the molecules were delivered into trees using trunk injections with Chemjet injectors above the graft union, two per tree on opposite sides. Multiple tree health traits will be assessed periodically by ground truthing and unmanned aerial vehicles (UAVs). Examples include measurement of disease index, flushing rate, canopy volume/density, CLas titers biannually, pre-harvest fruit drop, and ultimately fruit yield (boxes/tree), and quality traits annually during harvest season. Obj. 4. Pursue data collection for regulatory approvals of the new therapies. This objective is NOT funded by NIFA. It isindependently pursued by Southern Gardens Citrus (SGC) and partners. SGC has developed a plan and initiated various required data collections and waivers to seek EPA approvals for promising defensin/AMP therapies. A suite of good laboratory practice (GLP) data collection will be pursued using agency guidelines and included in a registration application package to support pesticide registration regulatory requirements for approval. These included, but were not limited to, a market basket study, health/dietary risk assessment, non-target effects on beneficial, pollinator/honeybee toxicity, and product characterization. Obj. 5. Evaluate the economic feasibility of deploying new HLB therapies. Initiated. Recent results from the efficacy/field trials of OTC and new chemistries were provided to the economists to initiate economic feasibility studies. The goal is to evaluate the profitability of adopting new HLB therapies on new and existing groves. The work is ongoing, and future reports will report outcomes to the stakeholders. Obj. 6. Extension and outreach to inform stakeholders Team members organized citrus greening workshops/symposiums at the 2023 Annual Meeting (2023) of the American Phytopathological Society-Caribbean Division (APS-CD) at South Padre Island, TX. Attendees: ~60. Team members organized and presented multiple talks at the 2nd Congress of the International Society for Citrus Huanglongbing and Phloem-Colonizing Bacterial Pathosystems (IS-CHPP), October 25-28, Clearwater Beach, FL. Attendees: ~100 Team members presented a poster entitled "Advanced Testing of Promising HLB Therapies and Extending Outcomes to the Citrus Industry" to showcase the project highlights at the Plant Health 2023 annual meeting of the American Phytopathological Society (APS), August 12-16, Denver, CO. Team members organized and presented at the 76th Annual Meeting of Subtropical Agriculture and Environments Society, Weslaco, TX. Attendees: ~50. A NIFA ECDRE research project snapshot was published on the Science for Citrus Health website. Attendees: World Wide Web. Team members were invited as speakers at the 2023 Florida Citrus Mutual and the 2023 Texas Citrus Mutual annual conferences. We also leveraged several means to engage stakeholders by publishing news releases on the project findings and/or individual meetings with citrus stakeholders in TX, FL, CA, and the wider public. These were prepared and disseminated through regional and national news outlets and on social media channels. Our extension team, affiliated with regional citrus research and extension centers in TX, FL, and CA, continuously interacted with citrus growers, industry, and other stakeholders. Team members also participated in citrus growers' and organizations' meetings (e.g., CRDF, TCM, TCPDMC, TAMUK-Citrus Center), where the project activities/updates were provided to the stakeholders. Lastly, our project advisory board included multiple growers and industry and commodity group members, who were periodically informed of the project activities both at the annual advisory board meeting and by engaging with them individually. <br><br><b>Publications</b><br>
Target Audience
The following target audience was reached by the efforts and activities of this reporting period. Plant pathology,microbiology, and biotechnologyscientific research communities. Students, postdocs, and early career scientists. Agricultural community, including the citrus industry,growers, and consumers.
Changes / Problems
Nothing Reported
Training & Professional Development
The project has provided research training and mentoring opportunities to severalundergraduate students, graduate students, research technicians, as well as early career postdocs and research scientists. The students and staff were routinely mentored by the PIs in laboratory techniques and scientific inquiry. The students and staff were also given opportunities to attend and present their research at professional conferences. Additionally, senior staff was provided opportunities to junior mentor staff, thus enhancing their teaching and mentoring skills.
Dissemination Streams
Project updates and results were periodically disseminated by the team members to various groups of stakeholders in TX, FL, and CAthrough a variety of avenues, including in-person meetings, workshops, and various social media channels, as detailed in the extension/outreach accomplishments above. The results were also periodically disseminated to the broader scientific community throughresearch publications andpresentations at regional and international conferences.
Next Reporting Steps
We will continue the proposed research activities in the laboratory, greenhouse, and field trials. Any additional candidatesmall molecules and AMPs that demonstrated promising activity in the hairy root assays will be moved to thegreenhouse and field testing pipeline. Results from the efficacy/field trials will be utilized to initiate economic feasibility studies in order to evaluate the profitability of adopting new HLB therapies on new and existing groves. We will continue our outreach and extension activities to stakeholdersin TX, FL, and CA and to the broader scientific community through a variety of avenues such as meetings,conferences, publications, and online media. <br><br>
<br>What was accomplished under these goals? The proposed objectives/activitiesare underway and on track. Briefaccomplishments forthis reporting period are below: Obj. 1. High throughput hairy root pre-screening and selection of effective HLB therapies. Two classes of therapies were evaluated: a) defensins/antimicrobial peptides (AMPs) and b) small molecules/chemistries. For evaluating AMPs, each of the coding regions was cloned into binary expression vectors and transformed into CLas-citrus hairy roots using the optimized hairy root transformation methods. Hairy root tissue samples were collected for molecular diagnostics at ~90-120 days to confirm the expression of the candidate AMP using RT-qPCR, followed by CLas molecular diagnostics/data analysis to determine efficacy. For small-molecule therapeutic screening, CLas-citrus hairy root cultures were initiated as per established protocols, and QC was conducted periodically to determine CLas titers in the hairy roots. Subsequently, the CLas-citrus hairy roots were used for the HR assays with several candidate molecules, followed by molecular diagnostics/data analysis to determine efficacy. Statistical analysis was performed using ANOVA/Post hoc and/or Student's t-test as appropriate to determine significant differences. Together, these experiments identified multiple AMPs and small molecules that were advanced to in planta trials. Obj. 2. Evaluation of antimicrobial peptides using CTV vector in citrus trees. For AMP evaluation, CTV vector optimization and cloning were initiated with the selected AMPs. A batch of CTV-defensin testing is also underway under greenhouse conditions. After confirmation of the systemic spread of the CTV-AMP in the trees, the material will be used as a source of inoculum to graft citrus trees for field planting. The field trials will be conducted in FL under EPA EUP, and USDA APHIS/BRS permits. These trials will be performed in a randomized complete block design under natural HLB/ACP pressure. Obj. 3. Evaluation of small molecule therapies in citrus trees. Phytotoxicity assays with the small molecules were initiated and completed in the greenhouse citrus plants. Based on the results of the phytotoxicity, appropriate dosages for field treatments were formulated. Field tree scouting and diagnostics in TX and FL locations were completed, and the first round of trials was initiated in the two locations. Delivery of the molecules into trees was achieved using trunk injections with Chemjet injectors above the graft union, two per tree on opposite sides. Multiple tree health traits will be assessed periodically by ground truthing and by using unmanned aerial vehicles. Examples include measurement of disease index, flushing rate, canopy volume/density, CLas titers biannually, pre-harvest fruit drop, and ultimately fruit yield (boxes/tree), and quality traits annually during harvest season. Obj. 4. Pursue data collection for regulatory approvals of the new therapies. This objective is NOT funded by NIFA. It isindependently pursued by Southern Gardens Citrus (SGC). SGC has initiated various required data collections and waivers to seek EPA approvals for promising defensin/AMP therapies. A suite of good laboratory practice (GLP) data collection was being pursued using agency guidelines and included in a registration application package to support pesticide registration regulatory requirements for approval. These included but were not limited to, a market basket study, health/dietary risk assessment, non-target effects on beneficial, pollinator/honeybee toxicity, and product characterization. Obj. 5. Evaluate the economic feasibility of deploying new HLB therapies. Results from the ongoing efficacy/field trials will be utilized in the future to initiate economic feasibility studies. The goal is to evaluate the profitability of adopting new HLB therapies on new and existing groves. Obj. 6. Extension and outreach to inform stakeholders Team members have been active in organizing and presenting at several symposia, workshops, and meetings. Specifically, team members organized and presented a citrus greening workshop/symposium at the Joint Annual Meeting of the American Phytopathological Society Caribbean Division (APS-CD) and Entomological Society of America South-Eastern Branch (SEB), San Juan, Puerto Rico. Its titled "Citrus greening or Huanglongbing (HLB): Strategies for mitigation and control." The goal was to provide an update on the HLB status and ongoing R&D efforts on new strategies and therapies to control the vector and the pathogen. We covered HLB epidemiology, insect vector management, RNAi, and new therapeutic screening and delivery systems to control HLB. Attendees: ~30. Team members also organized and presented at the symposium of the 76th Annual Meeting of Subtropical Agriculture and Environments Society, Weslaco, TX. Relevant topics covered included ongoing R&D efforts on new strategies and therapies to control HLB, as well as perspectives on the state of affairs of the citrus industry after the freezes. Attendees: ~50. Team members published a Research Snapshot article that is available for download on the Science for Citrus Health website. Attendees: World wide web. Mandadi, K., Irigoyen, S., Ramasamy, M. (2021). Novel tools for antimicrobial testing and discovery of new HLB therapies. Science for Citrus Health. We also leveraged several means to engage stakeholders by publishing news releases on the project overview aimed to inform citrus stakeholders in TX, FL, and CA and the wider public. These were prepared and disseminated through regional and national news outlets, as well as on social media channels. Our extension team, affiliated with regional citrus research and extension centers in TX, FL, and CA, had continuous interactions with citrus growers, industry, and other stakeholders. Team members also participated in citrus commodity and growers' board meetings where the project activities/updates were provided to the stakeholders. Lastly, our project advisory board included multiple growers, industry, and commodity group members, who were periodically informed of the project activities both at the annual advisory board meeting, as well as engaging with them individually. <br><br><b>Publications</b><br>