Source: UNIVERSITY OF FLORIDA submitted to
SP: A NOVEL THERAPEUTIC STRATEGY FOR HLB-INFECTED TREES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
EXTENDED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1024608
Grant No.
2020-70029-33195
Project No.
FLA-MCS-006012
Proposal No.
2020-08446
Multistate No.
(N/A)
Program Code
ECDRE
Project Start Date
Sep 1, 2020
Project End Date
Aug 31, 2024
Grant Year
2020
Project Director
Mou, Z.
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
Microbiology and Cell Science
Non Technical Summary
Huanglongbing (HLB)-resistant or tolerant citrus trees are the long-term solution for this devastating disease. Our on-going collaboration has generated transgenic citrus lines that provide robust tolerance to HLB. These transgenic lines are already in the field tests as a potential management possibility for HLB. However, these trees are genetically modified (GM) and will have to go through the approval process. We are reproducing this phenotype in non-GM plants by gene editing using CRISPR/Cas9. This is a better long-term approach, but will take more time. Our first goal here is to develop an interim treatment using CTV-based plant-mediated RNA interference (RNAi) to silence negative regulators of the citrus immune system to create HLB tolerance. This approach will piggyback on the progress that Southern Gardens Citrus has made in field testing and permitting of the use of the CTV vector to express defensins. The synergism between RNAi-triggered tolerance and defensin-mediated CLas reduction is expected to provide better control of HLB. This non-transgenic approach will allow treatment of already infected mature trees and development of new groves in Florida and can also be used in other citrus production areas including California and Texas. Our second goal is to educate the citrus communities about the CTV technology and its benefits to the citrus industry and consumers. This will help establish market acceptance for CTV-based citrus products.
Animal Health Component
0%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21209201090100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
0920 - Orange;

Field Of Science
1090 - Immunology;
Goals / Objectives
The overall goal of this project is to develop effective citrus tristeza virus (CTV)-based therapeutic measures for Huanglongbing (HLB) control and to educate the public (including citrus growers, consumers, and regulators) about the CTV technology and its benefits to the citrus industry. The central hypothesis of this project is that enhancement of host immunity by RNA interference (RNAi) and reduction of the pathogen by defensin act synergistically in keeping HLB at bay and the resulting products provide increased consumer acceptance and lower the regulatory hurdles. We plan to accomplish the goal of this application by pursuing six specific objectives:(1) Identify candidate targets based on information in well-studied plants(2) Silence the candidate targets by CTV-RNAi in citrus(3) Evaluate the protein levels of a citrus major defense regulator in the RNAi lines(4) Define the efficacy of potential CTV-defensin-RNAi vectors in commercial citrus(5) Field trial of promising CTV-defensin-RNAi vectors(6) Engage stakeholders in project development and execution process
Project Methods
(1) Putative citrus negative immune regulators will be identified using the protein sequences of the negative immune regulators reported in Arabidopsis and other plant species as the queries for BLASTp in the sweet orange (C. sinensis) genome database.(2) RNAi inducer sequences targeting the citrus negative immune regulators will be cloned into the CTV vector. The resulting CTV vector plasmids will be checked for the right insert size via restriction enzyme digestion followed by sequencing. The CTV vectors will be inoculated into Nicotiana benthamiana using an Agro-infiltration approach. Recombinant virions of CTV vectors isolated from systemic leaves of N. benthamiana will be concentrated by centrifugation in a step gradient followed by a cushion gradient. Inoculation of citrus plants will be done by bark flap inoculation into C. macrophylla seedlings. Establishment of systemic infection in the plants will be validated. Silencing of the target genes will be assessed by quantitative PCR (qPCR) analysis.(3) Total protein will be extracted from bark tissues of the RNAi lines, separated by SDS-PAGE, transferred onto nitrocellulose, and probed with an antibody against a citrus major defense regulator. The antibody-bound proteins will be detected using a horseradish peroxidase-conjugated anti-rabbit secondary antibody followed by chemiluminescence detection and quantification.(4) The promising RNAi inducer sequences will be inserted between the 3'-most gene and the non-translated 3' sequence of the CTV-defensin constructs. The resulting vectors will be inoculated into N. benthamiana and then C. macrophylla. After systemic infection is established in C. macrophylla, the CTV-defensin-RNAi constructs will be graft-transmitted into sweet orange or grapefruit seedlings for HLB phenotype screening. Both uninfected and HLB-infected seedlings will be included. After systemic CTV infection is established, the citrus trees will be molecularly characterized. Silencing of the negative immune regulators and expression of the defensin gene will be confirmed by qPCR, and the RNAi-induced increase in the protein levels of the citrus major defense regulator will be verified by western blot analysis. For the HLB-infected trees, the therapeutic effect of CTV-defensin-RNAi constructs will be assessed via monitoring HLB symptom development and CLas titer reduction. For preventative effect, citrus trees will be inoculated with HLB by incubating them in a containment plant growth room with CLas-infected psyllids. Only plants with ideal new flush will be placed into the room and plants on which psyllids reproduce will be labeled. Approximately 10 replicates of each construct will be tested.(5) A large number of C. macrophylla trees will be inoculated with two selected CTV-defensin-RNAi constructs. After systemic CTV infection is established and validated, stem or bark pieces from the C. macrophylla trees will be grafted onto both HLB-infected and uninfected trees in the field to evaluate the therapeutic and preventative effects of the constructs, respectively. CLas titers will be determined in the second year of the project and beyond the project. Moreover, border trees will be included at the end of rows and in adjacent rows as needed. Additionally, tree management including fertilization and irrigation will follow good practices in the grove.(6) The field trials will be setup in commercial groves and results will be validated with several grower collaborators who will be involved in trial development and validation process by providing tree management and feedback throughout the project. Teaching materials including PowerPoint presentations, extension articles, and website will be prepared for broad audiences including citrus growers, industry representatives, consumers, and state/federal regulators. PowerPoint presentations will be used as the basis for an online course, which will be posted online. A website associated with this project will be specifically designed to provide citrus communities information on HLB, CTV, defensin, RNAi, and progresses of the project. Flyers, handouts, and fact sheets will be created with lay language to cover two topics: the citrus HLB crisis and its potential solutions, and the state-of-the-art CTV-defensin-RNAi method that will be used in this project to treat infected trees and protect healthy trees from infection. Workshops, seminars, and meetings will be organized to engage with and educate citrus growers, industry representatives, students and public in large about how the CTV-defensin-RNAi strategy used in our research is really going to be useful to the citrus industry in combating HLB in the field.

Progress 09/01/22 to 08/31/23

Outputs
Target Audience:Batuman, O., May 15-17, 2023: Injecting Oxytetracycline: An Effective Approach for Managing HLB Disease in Citrus. 18th Biennial Florida Phytopathological Society Meeting. Immokalee, FL. Batuman, O., May 15-17, 2023: New Antimicrobials and Systemic Acquired Resistance (SAR) Inducers for Managing Citrus Canker Disease. 18th Biennial Florida Phytopathological Society Meeting. Immokalee, FL. Batuman, O., May 15-17, 2023: The Asian citrus psyllid as a bioindicator of citrus tristeza virus in Florida groves. 18th Biennial Florida Phytopathological Society Meeting. Immokalee, FL. Batuman et al. Novel strategies for HLB resistance/tolerance in citrus by gene editing. Citrus Expo. August 16-17, Tampa, FL. Batuman, O. HLB and its management in citrus groves. Presentation to citrus Advisory Board. June 9, 2023 (Virtual). 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. Mou, Z.,Dawson, W.,Grosser, J., Orbovic, V., Dutt, M., Levy, A., El Mohtar, C., Batuman, O., Irey, M.pproaches toward Huanglongbing tolerance. Citrus Expo, August 16-17, 2023, Abstract. Mou, Z., Dawson, W.Generation of transgenic Huanglongbing tolerant citrus varieties. Citrus Expo, August 16-17, 2023, Abstract. Sarkar, P., Mou, Z., Levy, A. Overexpression of AtNPR1 enhances tolerance to Liberibacter infection by elevating basal immune responses. 2023 IS-MPMI Congress, July 16-20, Poster. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project helped train two postdocs in project design, experimental design, advising graduate students, presentation, grantsmanship, and progress report. It also trained two graduate students in experimental design, basic molecular biology techniques including vector construction, Agrobacterium-mediated transformation, and CTV inoculation, as well as collection and analysis of data. One undergraduate student was involved in general laboratory works including preparation of various media and buffers, tissue culture, bacterial cell culture, and citrus plant maintenance and was trained in these techniques. How have the results been disseminated to communities of interest?We have been communicating since the start of project with stakeholders including citrus growers, industry, state/federal regulators, as well as students and the general public through various approaches including publications, meeting abstracts, talks, presentations, and demonstrations. We do our best to inform the stakeholders the project progresses and the potential strategies to mitigate the HLB disease. What do you plan to do during the next reporting period to accomplish the goals?We will continue conducting the proposed experiments in the proposal to achieve the project objetcives.Our priority is still to compare the CTV constructs combining antimicrobial peptides and RNAi sequences targeting citrus negative immune regulators in the greenhouse to identify the best CTV constructs that can provide robust tolerance to HLB.

Impacts
What was accomplished under these goals? (1) Identify candidate targets based on information in well-studied plants This objective has been completed. In total, we have identified 44 homologs of negative immune regulators that have been reported in other plant species. We have been using CTV-RNAi to silencethese homologs in citrus. (2) Silence the candidate targets by CTV-RNAi in citrus In last year, we infected Citrusmocrophylla plants with 20 CTV-RNAi constructs and tested their stability in the infected C. microphylla seedlings. These constructs were all stable at the time of the RT-PCR assay. Eighteen of the 20 CTV RNAi vectors stable in the infected C. macrophylla plants were graft-transmitted into at least five plants of Pineapple sweet orange to screen for HLB phenotypes.We monitoredthe different graft-inoculated CTV-RNAi sweet orange plants for the establishment of CTV infection using ELISA with antibodies against the coat protein. In this year, ELISA positive plants was screened for the HLB phenotypes by inoculation with CLas-positive ACP. We generated at least fiveprogeny plants from eachmother plantfor each CTV RNAi construct. However, not all progeny plants were CTV positive based on ELISA analysis. The plants that were CTV positive often grew poorly, which is consistent with constitutive immune responses in the RNAi plants. Additionally, we attempted to speed up the screening process by growing sweet orange plants on their own rootstocks, which turned out to be challenging. The plants that were CTV positive and large enough were screened by infection with CLas positveACP for HLB tolerance. We have found another gene(two have been found in previous years)that, when silenced, will likely provide tolerance to HLB,since some of its RNAiplants were positive for HLB but did not havesymptoms. We are increasing the number of plants for this CTV RNAi vector. (3) Evaluate the protein levels of a citrus major defense regulator in the RNAi lines We tested the protein levels of the major citrus defense regulator in the C. microphylla plants infected witht the second batch of twelveCTV-RNAi vectors. Among these twelvevectors, threesignificantly increased the proteins levels of the defense regulator in the RNAi plants. In total, we found six genes that, when silenced, significantly elevated the protein leves of the defense regulator in citrus. (4) Define the efficacy of potential CTV-defensin-RNAi vectors in commercial citrus We previously grafted leaf pieces from CTV-infected C. macrophylla into Pineapple sweet orange plants. Most grafts stayed alive (green); however, ELISA results of the leaves from the sweet orange plants remained negative in multiple assays.In last year, we tested the roots of sweet orange seedlings by ELISA because roots are easier to be infected than shoots by different strains of CTV. ELISA from roots of the CTV-infectedsweet orange plants were positive for CTV. To confirm these results, we reinfectedsweet orange with different constructs that simultaneously expressing defensins and the truncated mRNA fragments targeting citrus genes using budwoods rther than leaf pieces. After a longincubation period, some of the plants became CTV positive in the shoots. Wescreened these plants for CTV stability by RT-PCR with primers upstream and downstream of the insertion sites. The RT-PCR assays revealed the stability of the CTV vectors within these plants. Unfortunately, some of the CTV positive plants were lost due to mealybug and snow scale infestations. We are reinfecting sweet orange plants with threeconstructs that combine the expression of antimicrobial defensin peptides and RNAi. (5) Field trial of promising CTV-defensin-RNAi vectors Nothing to report. (6) Engage stakeholders in project development and execution process We designed outreach activities to inform stakeholders including citrus growers, industry, state/federal regulators, as well as students and the public about theprogress of the project and on HLB control strategies. As in previous years, using the CTV vector to simultaneously deliver defensinpeptides and RNAito silence genes encoding citrus negative immune regulators is still an exciting topic for a wide range of audience. This topic, together with other HLB management topics, was discussed, presented, and demonstrated virtually or in person in multiple events held in Florida and elsewhere in 2022-2023. Various potential strategies for mitigating HLB in Florida citrus groves were also discussed with the stakeholders.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sanju Kunwar, Ana Redondo, Leigh Archer, Fernando Alferez, Ute Albrecht, Ozgur Batuman (2023). Injecting Oxytetracycline: An Effective Approach for Managing HLB Disease in Citrus. 12th International Congress of Plant Pathology. Lyon, France. August 20-25, 2023.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Ozgur Batuman, Sanju Kunwar, Ana Redondo, Denise Manker, Meagan Iott, Thomas Knobloch, Stephane Brunet, and Jeremy Dufour (2023). Novel Plant Defense Inducers for Managing Huanglongbing (Citrus Greening) and Citrus Canker Diseases. 12th International Congress of Plant Pathology. Lyon, France. August 20-25, 2023.


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:Batuman, O. LB Strategies From Zoom'okalee. Growers Meeting. January 12, 2022. Virtual Presentation. Batuman, O.Development of a citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. International industry collaborators meeting (France and Germany). January 24, 2022. Virtual Presentation. Batuman, O.Huanglongbing (aka citrus greening) Disease of Citrus. Industry Presentation. April 6, 2022. Virtual. Mou, Z. Establish early-stage field trials for new HLB-tolerant canker-resistant transgenic scions. April 26, Lake Placid, FL. Batuman, O. ANovel Method and Screening Antimicrobial Compounds. California Research Board Antimicrobial Summit. May 23-24, San Francisco, CA. Mou, Z. New HLB-tolerant canker-resistant transgenic scions. Florida Citrus Research and Development Foundation special meeting. May 31, 2022, Wauchula, FL. Batuman, O.Florida citrus growers' potential 'toolbox' for Huanglongbing (HLB) management: an alphabet soup (ISVs, PDIs, IPCs, NATI, etc.). 15th International Symposium of Plant Virus Epidemiology. Madrid, Spain. June 5-8, 2022. Mou, Z., Dawson, W., Grosser, J., Orbovic, V., Dutt, M., Levy, A., El Mohtar, C., Batuman, O. and Irey, M. Approaches towards Huanglongbing tolerance. August 17-18, 2022, Abstract. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two postdocs were trained in project design, experimental design, advising graduate students, presentation, grantsmanship, and progress report. Two graduate students were trained in experimental design, basic molecular biology techniques including vector construction, Agrobacterium-mediated transformation, and CTV inoculation, as well as collection and analysis of data. One undergraduate student was trained in general laboratory techniques including preparation of various media and buffers, tissue culture, bacterial cell culture, and citrus plant maintenance. How have the results been disseminated to communities of interest?The overall project goal and results obtained so far have been communicated to stakeholders including citrus growers, industry, state/federal regulators, as well as students and the general public through various approaches including publications, meeting abstracts, talks, presentations, and demonstrations. What do you plan to do during the next reporting period to accomplish the goals?We will follow the well-designed experiments described in the proposal to accomplish the project goals. Our current priority is to test the CTV constructs combining antimicrobial peptides and RNAi sequences targeting citrus negative immune regulators in the greenhouse to identify the CTV constructs that can provide robust tolerance to HLB.

Impacts
What was accomplished under these goals? (1)Identify candidate targets based on information in well-studied plants This objective has been accomplished in the last year. In total, we have identified 44 homologs of negative immune regulators that have been reporetd in other plant species. We are working on these homologs. (2)Silence the candidate targets by CTV-RNAi incitrus In the previous year, we bulit 21 CTV RNAi vectors (constructs 623-632 and 639-649) by inserting truncated mRNA fragments of the 21 citrus negative immune regulators identified in this project and tested the stability of eight CTV RNAi constructs inCitrus macrophylla plantsby RT-PCR using primers within CTV upstream and downstream of the insertion site. In this year, we infected C. mocrophyllaplants with the remaining CTV RNAi constructs (except 626, which is currently under testing) and tested their stability in the infected C. microphylla seedlings. These constructs were all stable at the time of the RT-PCR assay. Eighteen of the twenty CTV RNAi vectors stable in the infected C. macrophylla plants were graft-transmitted into at least fiveplants of Pineapple sweet orange to screen for HLB phenotypes. The C. macrophylla plants infected with the other two CTV RNAi vectors died. We re-inoculated a new set of C. macrophylla seedlings with these two vectors and confirmed theirstability. Currently, we are monitoring the different graft-inoculated CTV RNAi sweet orange plants for the establishment ofCTV infection using ELISAwith antibodies against the coat protein. ELISA positive plants will be screened for the HLB phenotypes by inoculation with CLas-positive ACP. (3)Evaluate the protein levelsof a citrus major defense regulator in the RNAi lines Protein levels of the major citrus defense regulator in theC. microphylla plants infected witht the first batch of eight CTV RNAi vectors were evaluated by western blotting analysis. Results showed that the proteins levels varied significantly in these plants. To determine whether these differences are caused by RNAi silencing, we are testing the silencing effects of the CTV RNAi in the CTV-infected C. microphylla plants. (4)Define the efficacy of potential CTV-defensin-RNAi vectors in commercial citrus In the previous year, we built seven CTV vectors, namely, 603, 604, 606, 620, 621, 650, 651 and 652, to combinethe expression of the most promising spinach defensin AMPs and truncated mRNA fragments (RNAi)targeting citrus genes. We successfully infected four of these constructs into citrus and screened them for stability by RT-PCR, followed by sequencingconfirmation. In this year, we worked on inoculating citrus with the other three CTV vectors, namely 620, 650 and 652 that previously failed to infect citrus. On the second attempt, we successfully infected citrus with the new constructs. As the four previous vectors, RT-PCR confirmed the stability of the three CTV vectors in citrus and Sanger sequencing confirmed theseqences of the defensin and the truncated mRNA fragmentsin a subset of the plants.To screen the different CTV vectors simultaneously expressing the antimicrobial peptides and the truncated mRNA targeting citrus genes,we graftedleaf pieces from CTV-infectedC. macrophylla into Pineapple sweet orange plants. Most grafts stayed alive (green);however, ELISA results of the leaves from the sweet orange plants remained negative in several assays. It prompted us to test the roots of sweet orange seedlings by ELISA because roots are easier to be infected than shoots by different strains of CTV as well as deletion mutants. For example, T36 infects the roots of Swingle citrumelo and Sun Chu Sha mandarin but not the shoots. ELISA from roots of these sweet orange plants were positive for CTV. To confirm the results obtained in these experiments, we are reinfecting sweet orange with different constructs that simultaneously expressingAMPs and the truncated mRNA fragments targetingcitrus genes using budwoods rather than leaf pieces. Budwoods are expected to provide better CTV transmission. (5)Field trial of promising CTV-defensin-RNAi vectors Nothing to report. (6)Engage stakeholders in project development and execution process Outreach activities are designed to provide information on progress of the project and on HLB control strategies to stakeholders including citrus growers, industry, state/federal regulators, as well as students and the public. In this year, potential strategies for controlling HLB in Florida citrus groves were further discussed with the stakeholders. As in the previous year, the use of the CTV vector to simultaneously deliver defense peptides and truncated mRNA fragments to silence genes encoding citrus negative immune regulators was still the most exciting topic for a wide range of audience. This topic was again discussed, presented, and demonstrated virtually or in person in multiple events held in Florida and elsewhere in 2021-2022.

Publications

  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Kunwar, S. P, Redondo, A.&, Manker, D., lott, M., Knobloch, T., Brunet, S., Dufour, J., and Batuman, O. (2022). Novel systemic acquired resistance (SAR) inducers for managing Huanglongbing (citrus greening) and citrus canker diseases. (Accepted: Acta Horticulture; Proceeding Research Paper).
  • Type: Other Status: Published Year Published: 2022 Citation: Citrus Industry Magazine article on web HLB Strategies From Zoomokalee https://citrusindustry.net/2022/01/14/hlb-strategies-from-zoomokalee/
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kunwar, S.P, Redondo, A.&, Manker, D., Iott, M., Knobloch, T., Brunet, S., Dufour, J. and Batuman, O. (2022). Novel systemic acquired resistance (SAR) inducers for managing Huanglongbing (citrus greening) and citrus canker diseases. 31st International Horticultural Congress. Angers, France. August 14-20, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kunwar, S.P, Redondo, A.& and Batuman, O. (2022) Copper-Alternative Tools to Manage Citrus Canker (Xanthomonas citri subsp. citri) in Florida Groves. The Annual American Phytopathology Society (APS) Meeting. Pittsburgh, Pennsylvania, August 6-10, 2022.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Batuman O., Britt, K.G, Kunwar, S.P, Redondo, A.& and Alferez, F. (2022). Florida citrus growers potential toolbox for Huanglongbing (HLB) management: an alphabet soup (ISVs, PDIs, IPCs, NATI, etc.). 15th International Symposium of Plant Virus Epidemiology. Madrid, Spain. June 5-8, 2022.
  • Type: Websites Status: Published Year Published: 2021 Citation: Transgene-free citrus using CRISPR CAS9 (project website; Updated April 7, 2021): https://swfrec.ifas.ufl.edu/programs/citrus-path/transgene-free/


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:Outreach activities in Florida are designed to provide information on progress of our project and on HLB control strategies to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public. Talks, presentations and demonstrations given in 2021: Batuman, O. Development of a citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. International industry collaborators meeting (France and Germany). January 5, 2021. Virtual Presentation. Batuman, O. Needle assisted trunk infusion (NATI) and Automated Delivery System (ADS). The California Subtropical Work Group Seminar. January 15, 2021. Virtual presentation. Batuman, O. Development of a novel automated delivery system for citrus trees. Ag Tech Expo. May 11, 2021. Virtual presentation. Batuman, O. Citrus disease trends we should heed: Phytophthora, HLB and Leprosis. Growers Meeting. January 20, 2021. Virtual Presentation. Batuman, O. What is the main cause of fruit drop in the groves in HLB era? SWFREC Advisory Board Meeting. January 26, 2021. Virtual Presentation. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing (Candidatus Liberibacter asiaticus) and Citrus canker (Xanthomonas citri subsp. citri) in Florida Citrus groves. Industry collaborators meeting. January 26, 2021. Virtual presentation. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing (Candidatus Liberibacter asiaticus) and Citrus canker (Xanthomonas citri subsp. citri) in Florida Citrus groves. Industry collaborators meeting. February 2, 2021. Field Demonstration. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing in Florida Citrus groves. Rotary Club Invited Seminar. February 9, 2021. Virtual presentation. Britt, K. A new Diaphorina citri-associated reo-like virus in Asian citrus psyllid populations in Florida citrus groves. Florida Phytopathological Society Meeting. May 13-14, 2021. Virtual Presentation. Batuman, O. Citrus HLB and Blight. Growers Meeting. March 14, 2021. Field Demonstration. Batuman, O. SWFREC Citrus Pathology program overview. The Immokalee Foundation's middle school Career Day Program. March 16, 2021. Virtual Presentation. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing in Florida Citrus groves. Growers Meeting. April 7, 2021. Virtual presentation. Batuman, O. Novel management methods of Huanglongbing in Florida Citrus groves. Quarterly SWFREC Foundation Board of Directors meeting. April 13, 2021. In person presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. International academic collaborators meeting (Germany). April 21, 2021. Virtual Presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. SWFREC Citrus Advisory Board Meeting. April 27, 2021. In person presentation. Batuman, O. Impact of HLB and Hurricane Irma on citrus groves. Growers Meeting. May 1 and 3, 2021. Virtual presentation and Field Demonstration. Batuman, O. Citrus tree decline. Growers Discussion Meeting. May 18, 2021 Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing (Candidatus Liberibacter asiaticus) and Citrus canker (Xanthomonas citri subsp. citri) in Florida Citrus groves. Industry collaborators meeting. May 27, 2021. Field Demonstration. Batuman, O. Needle-assisted trunk infusion (NATI) of therapeutic material for controlling HLB and its psyllid vector. Meeting with Division of Agricultural Environmental Services, Florida Department of Agriculture and Consumer Services. June 2, 2021. Virtual presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. Texas Academic Collaborators Meeting. June 3, 2021. Virtual presentation. Batuman, O. Development of a citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. International industry collaborators meeting (France). June 7, 2021. Virtual Presentation. Batuman, O. Needle-assisted trunk infusion (NATI) of therapeutic material for controlling HLB and its psyllid vector. Industry collaborators meeting. June 30, 2021. Virtual Presentation. Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing (Candidatus Liberibacter asiaticus) and Citrus canker (Xanthomonas citri subsp. citri) in Florida Citrus groves. Texas Academic collaborators meeting. July 2, 2021. Virtual presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. International academic collaborators meeting (Germany). July 6, 2021. Virtual Presentation. Batuman, O. Using citrus screening technique and evaluation of various substances to prevent infection and spread of CLas in citrus. SWFREC Foundation Board Meeting. July 13, 2021. In person presentation.Batuman, O. Novel application methods and therapeutic treatments for management of Huanglongbing. SWFREC Citrus Advisory Board Meeting. July 27, 2021. In person presentation. Mou, Z., Dawson, W., Grosser, J., Orbovic, V., Dutt, M., Levy, A., El Mohtar, C., Batuman, O. and Irey, M. Seeking long-term solutions to the Huanglongbing disease. Citrus Expo. August 18-19, 2021, Abstract. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One postdoc was trained in project design, experimental design, advising graduate students, presentation, and progress report. Two graduate students were trained in experimental design, basic molecular biology techniques including vector construction, Agrobacterium-mediated transformation, and CTV inoculation, as well as collection and analysis of data. One undergraduate student was trained in general laboratory techniques including preparation of various media and buffers, tissue culture, bacterial cell culture, and citrus plant maintenance. How have the results been disseminated to communities of interest?Results of the project have been disseminated to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public through publications, meeting abstracts, talks, presentations, and demonstrations. What do you plan to do during the next reporting period to accomplish the goals?We will follow the well-designed experiments described in the proposal to accomplish the project goals. Specifically, CTV constructs that are able to provide HLB tolerance will be identified and field trials will be initiated as soon as possible.

Impacts
What was accomplished under these goals? (1) Identify candidate targets based on information in well-studied plants A total of 23 citrus homologs of plant negative immune regulators that have been studied in other plant species have been identified previously. In this project, another 21 citrus homologs of reported plant negative immune regulators were identified. (2) Silence the candidate targets by CTV-RNAi in citrus Truncated mRNAs from the 21 new citrus homologs of plant negative immune regulators were cloned into the CTV vector. The new constructs were successfully identified by restriction digestion and confirmed by sanger sequencing. The newly built constructs were Agroinfiltrated into Nicothiana benthamiana where they went systemic. Currently, semi-purification of the virions is ongoing. (3) Evaluate the protein levels of a citrus major defense regulator in the RNAi lines N/A (4) Define the efficacy of potential CTV-defensin-RNAi vectors in commercial citrus In an earlier project, three citrus genes where identified, which when downregulated by CTV RNAi increased the level of a major defense protein, and one has shown an HLB-tolerant phenotype. Similarly, field screening of two defensin peptides expressed from CTV revealed a HLB tolerant phenotype. In this project, five different constructs that pyramid the peptide strategy and the RNAi strategy were constructed. Citrus plants were successfully inoculated with four of the five constructs. (5) Field trial of promising CTV-defensin-RNAi vectors Field trial permit applications have been submitted to USDA. (6) Engage stakeholders in project development and execution process Outreach and extension activities in Florida are designed to provide information on progress of the project and on HLB control strategies to stakeholders including citrus growers, industry, state and federal regulators, as well as students and the general public. During this period, efforts were focused on introduction of the strategies that are being developed for controlling HLB in citrus groves in Florida. Among the strategies that are being communicated across, the use of CTV vector to silence genes encoding citrus negative immune regulators was the most intriguing topic for a wide range of audience. This topic was discussed, presented and demonstrated virtually or in person in multiple events held in Florida in 2021.

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Merritt, B.A., Zhang, X., Riplett, E.W., Mou, Z., Orbovic, V. (2021). Selection of transgenic citrus plants based on glyphosate tolerance conferred by a citrus 5-enolpyruvylshikimate-3-phosphate synthase variant. Plant Cell Reports (https://doi.org/10.1007/s00299-021-02760-y)