Source: FLORIDA INTERNATIONAL UNIVERSITY submitted to NRP
ENDOPHYTES AS SOURCES OF ANTIMICROBIALS TO CONTROL HUANGLONGBING (HLB)
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1029427
Grant No.
2022-70029-38526
Cumulative Award Amt.
$500,000.00
Proposal No.
2022-06729
Multistate No.
(N/A)
Project Start Date
Sep 15, 2022
Project End Date
Sep 14, 2025
Grant Year
2022
Program Code
[ECDRE]- Emergency Citrus Disease Research and Extension Program
Recipient Organization
FLORIDA INTERNATIONAL UNIVERSITY
(N/A)
MIAMI,FL 33199
Performing Department
(N/A)
Non Technical Summary
Huanglongbing (HLB), also known as citrus greening is the most devastating citrus diseases in the U.S. HLB is caused by the bacterium Candidatus Liberibacter asiaticus (CLas) and is transmitted by a tiny insect, called the Asian citrus psyllid. Since HLB was detected in Florida in 2005, Florida's citrus production has fallen by 75%. The disease also threatens other citrus producing states. There is no cure for HLB, effective disease prevention and treatment methods have proven elusive. There is a high priority need for developing anti-microbial treatments that could reverse the disease impacts, specifically by reducing bacterial inoculum in infected citrus trees. The project proposes to first purify and characterize active anti-CLas compounds derived from bacterial endophytes isolated from survivor citrus trees. Next, uptake and anti-CLas activity within the citrus root system will be evaluated using CLas-citrus hairy root bio-assay. Finally, the protective and curative effects of purified antimicrobial compound treatment against CLas will be demonstrated under greenhouse conditions. Promising results from the project can readily be translated into products for the benefit of stakeholders through the agency of Marrone Bio Innovations, Inc. Initial information about the project will be shared with growers, promising results will be shared with growers through seminars, extension website, social media, newsletters, etc.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21509991160100%
Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1160 - Pathology;
Goals / Objectives
This research project is designed to fast-track the discovery and development of novel anti-CLas natural compounds with unique mode of action so that the citrus growers can have diverse, effective HLB prevention and mitigation tools. This two-year project would comprise four main objectives: 1. Extract, fractionate, purify, and characterize most active anti-CLas components from selected endophytes; Objective 2. Pre-screen most active component/s for Anti-CLas activity using CLas hairy root assay; Objective 3. Evaluate protective and curative effects of purified antimicrobial compound/s treatment against CLas under greenhouse conditions; Objective 4. Stakeholder outreach and engagement at different stages of the project through cooperative extension.
Project Methods
(Obj. 1) To identify compounds that are responsible for the anti-CLas activity found during the previous work (psyllid homogenate assay) we will use a bioassay-guided fractioning and purification scheme. we will analyze the crude extract using GCMS and UHLPC-MS/MS to identify the major groups of compounds found in the extracts. After, we will start initial fractioning of the crude extracts using C18 solid phase separation. This will yield four fractions based on the compound's polarity, the anti-CLas activity of these fractions will be tested once more. The most active fraction will be further separated via Automated Flash Chromatography paired with an ELSD detector. Each of the fractions obtained via flash chromatography will be teste once more for anti-CLas activity. If the chromatography yields more than 20 fractions, some of the fractions will be "pooled" together. Biologically active fractions will be analyzed via UHPLC-MS/MS to determine their composition. If multiple compounds are present, preparative HPLC will be performed. Pure compound fractions will be analyzed and identified via Fourier Transform Ion Cyclotron Resonance (FT-ICR) and Nuclear Magnetic Resonance (NMR). (Obj. 2) We will utilize the CLas-citrus hairy root bioassays to evaluate the efficacy of anti-CLas active fraction derived from endophytic bacteria and determine their inhibitory activity against CLas within the citrus root tissue. CLas-citrus hairy root cultures are periodically subjected to multiple quality control diagnostics to confirm and quantify CLas titers within the hairy root cultures. Next, the CLas-citrus hairy root cultures will be treated with the respective fraction/s, at multiple dosages (2%, 5%, 10% v/v), using the in vitro multi-well plate format, alongside untreated and DMSO/solvent alone controls. All the treatments are vacuum infiltrated to facilitate the penetration into the hairy root matrices and incubated for 72 hours. The hairy root tissues are subsequently sampled for molecular diagnostics. The effectiveness of the treatments 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). (Obj. 3) The most promising pure compound/s found to have activity against CLas in the CLas-ctirus hairy root assay will be tested in planta. We plan to test the compound/s on nursery trees that are approximately 1 year from budding. Scion and rootstock will depend on what is available from nurseries 3 to 6 months prior to initiation of the greenhouse studies but we will aim for sweet orange scions like 'Valencia'. Common rootstocks include 'Swingle' citrumelo, X-639, Cleopatra mandarin, and US-897. Two types of trials will be undertaken: 1) to determine the curative effect of compounds and 2) to determine the protective effect of compounds. Whether a compound is tested for a curative or protective effect will depend on the results from previous analysis and the likely mode of action. To investigate whether there is a curative effect on CLas in planta, trees will be inoculated with CLas by grafting 4 months prior to trial initiation with budwood that is known to contain active populations of CLas. The trees will be tested monthly after graft inoculation to confirm whether they have become CLas positive. Only trees that are confirmed CLas positive will be used in the trial. The compounds will be applied one of three ways, spray application, soil drenching, or possibly via injection. Soil uptake will be evaluated by seedling assay where 6-month-old seedlings in containers will be drenched with the compounds and the foliage tested to see if the compound can be detected. Similarly, the concentration range will also be determined prior to starting the greenhouse trial based on experiments with HLB-affected cuttings. These cuttings will contain one angular stem node with a leaf attached of approximately 10 cm in length. The cuttings will be placed in tubes containing a solution of the compound at multiple concentrations for 48 hours. The cuttings will then be transferred to water for a week. After a week they will be tested for the presence of live CLas cells with the RNA-based CLas viability assay. In the greenhouse experiment. there will be a minimum of five trees per treatment and preferably there should be 10. The leaf titer of CLas and viability will be taken at the time 0 just before the applications and 2 weeks, 4 weeks and 4 months post-application from two newly mature leaves per tree. At 6 months, the trees will be harvested. The total leaf area, trunk diameter, root mass, dry weight will be collected along with a disease rating. The titer will be measured in the leaves and roots. Two control treatments will be included in each trial, an untreated CLas inoculated and an untreated uninoculated control. The protective trial will be very similar with the exception of the inoculation timing to the compound application. In this case, the compound will be applied 2 to 3 weeks prior to inoculation with CLas. The trees will be monitored for CLas titer. The number of repetitions will need to be larger as there is a probability that some trees will not become CLas positive. The statistical analysis will be conducted with generalized linear mixed model methods and the distributions used will be dependent on the data. (Obj. 4) We understand outreach and extension of the project results to citrus growers is a key component and will be executed by all PIs. Members of the project team will communicate with stakeholders to identify needs and obstacles through surveys and grower forums. It is also essential to disseminate information rapidly as it arises from this proposed research. This will be accomplished efficiently via websites, presentations at industry and scientific meetings as well as through our stakeholder members. The information will be shared through regular dissemination of research reports, blogs, webinars (i.e., www.citrusresearch.ifas.ufl.edu), and participation in UF/IFAS hosted podcasts and newsletters focused on citrus. Findings and recommendations from this project will be conveyed to growers using these and other resources. Recommendations will be quickly delivered through multiple extension events through posters, displays, and handouts. Dissemination of information is also expected to take place via peer-reviewed scientific journals, extension publications, trade periodicals, oral presentations at field days held at the project sites, citrus industry events, professional society meetings, and citrus industry governing board meetings to be determined.

Progress 09/15/23 to 09/14/24

Outputs
Target Audience:U.S. Citrus growers, U.S. citrus industry, Extension agents, Pesticide and Ag. biologicals industry, HLB research scientists, USDA-NIFA Changes/Problems:Regrettably, the progress on isolation of pure active compounds has been very slow due to technical reasons and challenges with our personnel. On the technical side, the nature of the target compounds has made challenging their isolation using standard approaches, both silica and C18 columns do not seem to have enough separative power to produce pure quantities of the target compounds. Additionally, our equipment has had some issues that have further delayed our progress. On the personnel side, one of our FIU Co-PD moved to a different institution and the graduate student in charge of developing the isolation protocols has had some family-related leaves. What opportunities for training and professional development has the project provided?One doctoral student (fellowship supported by another USDA grant) and threeundergraduate students. The project has provided research training and mentoring opportunities to a Hispanic minority undergraduate student and technician. The students and staff were routinely mentored by the PI in laboratory techniques and scientific inquiry. How have the results been disseminated to communities of interest?Project updates and results were periodically disseminated to the team members, as well as to stakeholders in TX, FL, and CA, and the broader scientific/citrus stakeholder community through publications, regional and national news outlets directly and indirectly through FIU news article and FIU Agroecology Program website. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, FIU lab will focus on completing the isolation and characterization of target metabolites. We will also supply our collaborators with 95% pure active compound for lab and greenhouse-based experimental evaluations. At UF we are waiting for the anti-CLas components to conduct our greenhouse and cutting assays. We will hopefully be ready to go with inoculated and CLas-infected trees. The testing for CLas titer in December 2024 will let us know how soon we can proceed with the assays. The active metabolites will also be evaluated first for anti-CLas activity in citrus using hairy root assays (TAMU), where CLas is being co-cultured/maintained in citrus-derived hairy roots, and finally in planta activity in greenhouse studies

Impacts
What was accomplished under these goals? The major goals of the project are to: (1) Extract, fractionate, purify, and characterize most active anti-CLas components from selected endophytic bacteria (FIU); (2) Pre-screen most active component/s for Anti-CLas activity using citrus CLas hairy root assay (TAMU); (3) Evaluate protective and curative effects of purified antimicrobial compound/s treatment against CLas under greenhouse conditions (UF); and (4) Stakeholder outreach and engagement at different stages of the project through cooperative extension (UF, TAMU, FIU) At FIU, the focus was on objective 1, geared towards (1a) Isolating the active compounds (or compounds) present in the endophyte extracts, (1b) Scaling the isolation procedure of this compound to purify quantities that could allow experimental manipulation of live plants, (1c) Identifying and characterizing the active compounds in the extract. We are at the final stages of objectives 1 and 2, and we have made progress on objective 3. For objective 1, we have determined the specific fraction of the extracts that confers biological activity against the Huanglongbing (HLB) pathogen. We have developed a scalable isolation protocol that uses silica gel columns and a progressive sequence of three solvents. Using HPLC-DAD, GCMS, and UHPLC-MS, we have confirmed that this fraction contains no more than 9 putative compounds. Using analytical scale isolations, we have identified a set of closely eluting compounds that are responsible for the biological activity of our extracts. At this point we are working towards developing a scalable protocol to isolate the individual compounds. We have some putative identification of the compounds we are targeting for isolation. Two of the compounds have been tentatively identified as carbazole alkaloids and have already been shown to have strong antibacterial activity in other systems. Most notably, it has been suggested that carbazole alkaloids might play a role in HLB disease resistance in Murraya koenigii (L.). The similar structures of the two carbazole alkaloids have made it difficult to isolate and purify the compounds. It is possible they both have biological activity against the HLB pathogen. Characterization of other focus compounds are in progress. At the University of Florida (UF) trees were inoculated with 2 buds per tree from Ca. Liberibacter asiaticus positive trees in July to prepare for greenhouse assays with the anti-CLas components that will hopefully occur early in the new year. We are planning to test if the trees if there is a detectable quantity of CLas cells in the trees in December 2024. It may be a little early, but we hope to see an 80 to 90% inoculation success. We have also worked with citrus cuttings to determine how much water up take occurs in a week for the cutting assays. We measured that the cuttings used between 0.5 to 1 ml of water and through food coloring could see it was distributed consistently in the cuttings.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Shetty, K. G. Endophytes as sources of antimicrobials to control huanglongbing (HLB).Citrus Disease Subcommittee (CDS) and NIFAs ECDRE (Emergency Citrus Disease Research Extension) Program Joint Meeting, November 12  15, 2023, Washington D.C.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mandadi, K.K. (2024). Novel strategies to screen and evaluate genetic- and chemical-based solutions for crop improvement. Annual Conference of the Society for Experimental Biology (SEB), July 2-5. Prague, CZ.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mandadi, K.K. (2024). Citrus CAP: A NIFA Center of Excellence for Discovery and Development of Citrus Greening Therapies. Invited Webinar for USDA APHIS PPQ-S&T-Domestic & Emergency Scientific Support (DESS), May 10, Washington DC.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Mandadi, K.K. (2024). Identification and evaluation of new chemicals and antimicrobial peptides for HLB management. International Research Conference on Huanglongbing (IRCHLB VII). March 26-29. Riverside, CA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Dominguez, J., Jayachandran, K., Bhat, M., Stover, E., Salazar, D., and Shetty, K. G. The Use of Endophytes and their Antimicrobial Extracts to Control Citrus Greening. Earth & Environment Graduate Research Symposium February 16th, 2024, Florida International University, Miami, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Dominguez, J., Jayachandran, Bhat, M., Stover, E., Salazar, D., and Shetty, K. G. Characterizing Novel Antimicrobial Compounds to Control Citrus Greening. 2024 GSAW Scholarly Forum April 1st, 2024, Florida International University, Miami, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Dominguez, J., Jayachandran, Bhat, M., Stover, E., Salazar, D., and Shetty, K. G. Characterizing Novel Antimicrobial Compounds to Control Citrus Greening. Agroecology Research Symposium April 5th, 2024, Florida International University, Miami, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Rezazadeh, Amir, Dewdney, Megan M., Dominguez, Jessica, Shetty, Kateel G., Jayachandran, Krish. Salazar, Deigo, Mandadi, K. Kranthi. Utilizing Research and Extension Collaboration to Save Florida's Citrus Industry: Endophytes as Sources of Antimicrobials to Control Citrus Greening. 2025 Extension professional associations of Florida /Extension symposium conference, August 26  29, 2024, Daytona Beach, FL.


Progress 09/15/22 to 09/14/23

Outputs
Target Audience:U.S. Citrus growers, U.S. citrus industry, Extension agents, Pesticide and Ag. biologicals industry, HLB research scientists, USDA-NIFA Changes/Problems:The first assays were very inconsistent due to variations in the raw extract. After some experimentation, these issues were identified as problems with the significant ingredients of the growth media. The significant challenges to isolating the active compounds are the complexity of the original growth media and subsequent extracts. The media used to culture the endophytes are rich in carbohydrate proteins and contain many secondary metabolites that do not derive from the endophytes but come from the complex components used for the "broth". The project also slowed down significantly because of the need to remove large quantities of water from the raw extracts (48-72 hours of freeze-drying per batch). What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Team members were actively engaged in preparing project summaries and topics related to our citrus stakeholders in TX, FL, and CA. These were prepared and disseminated through regional and national news outlets directly and indirectly through FIU news article and FIU Agroecology Program website. Attendees: World wide web "Scientists believe weapon against citrus greening already exists in nature" FIU News, February 1, 2023 https://news.fiu.edu/2023/scientists-believe-weapon-against-citrus-greening-already-exists-in-nature Team members published a Research Snapshot that is available for download on the Science for Citrus Health website. Attendees: World wide web Kateel G. Shetty (2023). Endophytes as sources of antimicrobials to control Huanglongbing (HLB) UC Agriculture and Natural Resources - Science for Citrus Health - Research Snapshots https://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/https___ucanr.edu_sites_scienceforcitrushealth_Research_Snapshots_NIFA_I_938_/Shetty1/ Team members organized and presented a citrus greening workshop/symposium at the citrus greening workshops/symposiums at the 2023 Annual Meeting (2023) of the American Phytopathological Society-Caribbean Division (APS-CD) at South Padre Island. Attendees: ~60. https://www.apsnet.org/members/community/divisions/carib/Pages/MeetingHistory.aspx Team members organized and presented in multiple sessions of 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 https://crec.ifas.ufl.edu/ISCHPP/ Team members also participated actively in various citrus growers' and organizations' meetings (e.g., Texas Citrus Pest and Disease Management Corporation, Texas Citrus Mutual, and TAMUK-Citrus Center), where the citrus project activities/updates were periodically updated to the stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We are making progress towards isolating single compounds, escalating the isolation procedure, and characterizing the active metabolites. The active metabolites will be evaluated first for anti-CLas activity in citrus using hairy root assays (TAMU), where CLas is being co-cultured/maintained in citrus-derived hairy roots, and finally in planta activity in greenhouse studies (UF).

Impacts
What was accomplished under these goals? The major goals of the project are to: (1) Extract, fractionate, purify, and characterize most active anti-CLas components from selected endophytic bacteria (FIU); (2) Pre-screen most active component/s for Anti-CLas activity using citrus CLas hairy root assay (TAMU); (3) Evaluate protective and curative effects of purified antimicrobial compound/s treatment against CLas under greenhouse conditions (UF); and (4) Stakeholder outreach and engagement at different stages of the project through cooperative extension (UF, TAMU, FIU) At FIU the focus was on objective 1 geared towards (1a) Isolating the active compounds (or compounds) present in the endophyte extracts, (1b) Scaling the isolation procedure of this compound to purify quantities that could allow experimental manipulation of live plants, (1c) Identifying and characterizing the active compounds in the extract. During the year-1 of the project following objectives/activities supported by the grant were accomplished. Our approach uses a bioassay-guided fractionation scheme combining liquid-liquid extraction and reverse and normal phase flash chromatography. Based on the initial trials, it was realized that the active compound is highly polar and cannot be extracted from the aqueous broth media using an organic solvent via direct liquid-liquid. Thus, the entire broth extract is processed by removing the protein (protein precipitation) and the water (Freeze Drying). The dry extract is reconstituted into 100% Methanol, and it is prepared for Dry-Loading using chromatography-grade silica at a ratio of 10:1. This silica is loaded into cartridges to be used for flash chromatography. In the initial step normal-phase chromatography (Silica with a two-solvent system: hexane and 3:1 Ethylacetate:2-propanol) was used to identify the significant fraction where the active compounds are present. We have narrowed down the activity of the extracts to two significant fractions. These fractions show 100% inhibition in the bioassays.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Dominguez, J., Jayachandran, K., Stover, E., Krystel,J., Shetty, K.G. Endophytes and Plant Extracts as Potential Antimicrobial Agents against Candidatus Liberibacter Asiaticus, Causal Agent of Huanglongbing. Microorganisms 2023, 11, 1529. https://doi.org/10.3390/microorganisms11061529
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Potential antimicrobial agents against Candidatus Liberibacter asiaticus, causal agent of huanglongbing. Jessica Dominguez, Jayachandran, K., Stover, E., Krystel, J., and Shetty, K.G. The Florida Phytopathological Society (FPS) 18th biennial meeting, May 17 - 19, 2023. University of Florida - Southwest Florida Research & Education Center (SWFREC), Immokalee, FL.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Citrus greening. Jessica Dominguez. Agroecology Symposium March 16th, 2023, Florida International University, Miami, FL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Mandadi, K. K. (2023). New technologies to control HLB and promote tree health., Texas Citrus Mutual Annual Meeting. Sep 12, Weslaco, TX.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Mandadi, K. K. (2023). Evaluation of novel antimicrobials for HLB management. Florida Citrus Industry Annual Conference, Florida Citrus Mutual. June 14-16, Bonita Springs, FL.