CAP: DEVELOPMENT, EVALUATION, AND DELIVERY OF CITRUS HLB MANAGEMENT APPROACHES BY TARGETING ITS NATURE AS A PATHOGEN-TRIGGERED IMMUNE DISEASE

Citrus HLB is a pathogen-triggered immune disease similar to sepsis in humans. Recent work led by PD Wang demonstrated that CLas stimulates a systemic and chronic immune response in citrus phloem including reactive oxygen species (ROS) production, which causes systemic phloem cell death and subsequent HLB disease symptoms. Our central hypothesis is that HLB can be controlled by managing CLas-triggered ROS. We will control HLB with three approaches:1) mitigating the production of ROS in HLB-affected groves with integrated horticultural measures. 2) Protecting citrus plants from CLas-triggered ROS via CTV-mediated expression of antioxidant enzymes and silencing of key genes involved in CLas-triggered ROS production. 3) Generating non-transgenic HLB resistant/tolerant citrus varieties. We demonstrated previously that RBOHD is the main producer of ROS triggered by CLas. Multiple genes activating RBOHD were identified and CTV-mediated gene silencing of one of RBOHD activating gene RLKO1 abolished CLas-triggered ROS production and HLB symptoms. Our ultimate goal is to leverage the breakthrough discovery that HLB is a chronic immune disease to develop shovel-ready HLB management approaches for existing groves and non-transgenic HLB resistant/tolerant citrus varieties for long-term, sustainable HLB control. We expect our interdisciplinary approach will increase production efficiency in existing HLB-affected groves and protect citrus production in all growing regions in the country by developing the first non-transgenic gene-edited citrus varieties resistant/tolerant to HLB.

Objective 1.Methods: 1.1. Optimization of micronutrient, GA, and uric acid application. We will test soil and foliar applications of BFeZnMoNi at different concentrations to obtain approximately B (2 μM), Fe (3 μM), Mo (2 μM), Ni (6 μM), and Zn (12 μM) in the phloem sap. GA and uric acid will be applied via foliar spray. All tests will be conducted individually with water as control. We will use 3- and 6-year-old Valencia/US-942 (scion/rootstock) in FL and 9-year-old Rio Red/sour orange in TX, as well as 11-year-old Tango mandarin/Carrizo and 30-year-old Navel orange/Trifoliate in CA in field trials. Exudates will be extracted from the phloem-enriched bark tissues at day 1, 5, 10, 20, and 30 after each treatment to determine BFeZnMoNi, GA, and uric acid concentrations. B, Fe, Mo, Ni, and Zn will be analyzed by inductively coupled plasma-mass spectroscopy (ICP-MS).1.2. Test the effect of different combinations of micronutrients, GA or uric acid on ROS levels, phloem cell death, HLB symptom development, and others. Once we have determined the suitable concentrations of micronutrients, GA, and uric acid for each application method we will assess the effects of each treatment individually and in combination with Valencia/US-942 in FL and Rio Red/sour orange in TX in the field. In CA, the tests will be conducted in greenhouse with Tango mandarin/Carrizo because HLB is absent in CA commercial groves. Treatments will be applied every 1, 2, or 3 month(s) in a randomized block design. To avoid the negative effect of GA on fruit color, we will stop GA treatments 3 months prior to fruit harvest.1.3. Field trials. Based on the results from Objective 1.2, we will conduct large-scale field trials in collaboration with citrus growers (see support letters). We plan to select the two best-performing treatments from Objective 1.2 and will include a no-treatment block as control. We will replicate these trials in six locations with the same scion/rootstock combination. We will use Valencia/US-942 in FL and Rio Red/sour orange in TX. Standard grove management practices will be followed, including pest control, fertilization, and irrigation. Copper will not be applied within two weeks of micronutrient application to avoid potential interference. We will monitor HLB symptoms, canker and greasy spot by randomly surveying 200 trees/block and will collect yield and fruit quality data annually.Economic analysis. We will identify and quantify the potential costs and benefits of treatment with micronutrients, GA, and uric acid.Objective 2.Methods: 2.1. CTV-mediated expression of antioxidant enzymes.Expression of antioxidant enzymes using the CTV vector. A CTV vector developed by co-PD EL Mohtar will be used for expression of antioxidant enzymes. We will express copper-zinc SOD (CuZnSOD), APX, CAT, and GPX homologs of tomato and Swinglea glutinosa, a distant citrus relative, using the CTV vector.Evaluate the effect of CTV constructs on CLas-triggered ROS, phloem cell death, HLB symptoms, and other traits. First, we will evaluate the effect of CTV constructs overexpressing antioxidant enzymes (hereafter CTV-antioxidant) on HLB positive plants in greenhouse and field trials. Next, we will evaluate whether CTV-antioxidant constructs can prevent CLas-triggered ROS, cell death of phloem tissues and HLB symptoms.2.2. CTV-mediated silencing of key genes involved in CLas-triggered ROS production. We will use CTV constructs to silence key genes involved in CLas-triggered ROS production as described previously. The effect of CTV constructs will be investigated as described above.Objective 3.Methods: 3.1. Generate non-transgenic genome edited citrus varieties. For genome editing of the coding and promoter regions of target genes, we will use Cas9/sgRNA DNA or RNP or Cas12a/crRNA RNP (see preliminary data 1.2.4). In addition, we will edit the seven phosphorylation sites of RBOHD using base editors.3.2. To evaluate the genome edited lines on CLas-triggered ROS production, phloem cell death, HLB symptoms, and other traits.Greenhouse assays. The genome edited lines (20 plants/line) and wild type (20 plants) of 12-month-old plants will be graft-inoculated with CLas in greenhouse. In addition, the genome edited lines (5 plants/line) and wild type plants (5 plants) of 12-month-old will be mock-inoculated as a control. We will investigate ROS and phloem cell death every 3 months for a period of 24 months. We will monitor HLB symptoms monthly, investigate tree growth (trunk diameter, height, canopy) annually, and analyze callose deposition and starch accumulation annually. We will also evaluate whether the genome edited lines are affected in disease resistance to Xcc and M. citri.Field trials. To test whether field performance is affected in genome edited plants we will evaluate the growth (height, trunk diameter, canopy), rate of photosynthesis, gas exchange, leaf chlorophyll content, root density, carbohydrate metabolism, and phytohormone analysis of the genome edited and wild-type plants in field trials.Objective 4. Deliver HLB management approaches/products through extension and outreachMethods: All participating PDs of this project will participate in the extension and outreach activities led by co-PDs TV, MK, and AEK in FL, TX, and CA, respectively. PDs of this team have routine, frequent interactions with citrus growers and local industry organizations and have been engaged with stakeholders to address the needs and hurdles associated with HLB in the corresponding states. We will organize workshops, grower meetings, field day events regarding using optimized application of BFeZnMoNi, GA, and uric acid, CTV constructs, and non-transgenic genome edited citrus varieties to control HLB. Information will be published in industry magazines such as "Citrograph", "Citrus Industry", "Fruit Gardner", and Cooperative Extension newsletters, and disseminated to the stakeholders at extension events such as Citrus Expo and Citrus Show. This information will also be present on multiple websites including the citrus agents and TX citrus grower portal. Co-PD Coltrane and cooperator Irey will lead our effort to acquire regulatory approval, registration, and commercialization of CTV constructs, non-transgenic genome edited HLB resistant/tolerant citrus varieties, and uric acid. We will take into the consideration the collection of required data for regulatory approval in our experimental plan.