Location: Subtropical Plant Pathology Research
Project Number: 6034-22000-045-024-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2023
End Date: Sep 14, 2025
Objective:
Objective 1: Establish and optimize methods for rapid selection, assessment, and delivery of HLB-resistant/tolerant citrus bud-sports and seedling variants. Objective 2: Identify genetic variants and genes related to HLB tolerance/resistance. Objective 3: Validate gene-edited citrus mutants for HLB resistance and generate novel non-transgenic HLB-resistant gene-edited selections of major commercial cultivars. Objective 4: Multistate field evaluations of HLB-resistant/tolerant citrus scion and rootstock cultivars. Objective 5: Outreach and dissemination of project results to stakeholders and the public.
Approach:
Objective 1: Our previous work demonstrated that it is possible to select HLB-resistant/tolerant citrus variants and identified transposon activities associated with HLB resistance. Here, we will develop methods to accelerate the selection, assessment, and delivery of HLB-resistant/tolerant citrus variants. We have collected multiple strains of CLas bacterium. We will develop an assay to evaluate the resistance of citrus variants using a simultaneous multi-strain inoculation strategy. Furthermore, we found that transposons are the main driver of citrus mutation. We will optimize a transposon-based assay for fast selection and stabilization of the HLB-resistant/tolerant citrus variant progenies. We will use our methods to continue selecting HLB-resistant/tolerant natural mutant citrus plants or bud sports of commercially grown cultivars. Objective 2: We will profile the dual transcriptomes of 20 resistant/tolerant citrus plants and their susceptible siblings before and after CLas infection using RNA-seq. We will re-sequence five tolerant/resistant and five susceptible sibling plants and their parents. Bioinformatics and functional genomic analysis will be used to predict a list of HLB tolerance/resistance-related genes and structural variants. Expressions of selected genes will be verified by RT-qPCR on HLB-challenged citrus varieties. The identified variants will also be further verified experimentally using RT-qPCR. Objective 3: We have edited five citrus genes and produced novel mutants that have shown strong resistance to citrus canker and possess potential for HLB resistance. They will be inoculated with CLas and evaluated for HLB resistance to confirm the best gene targets. Field trials will be planted to validate greenhouse results, then commercial cultivars will be edited using the latest mature tissue transformation techniques to produce non-transgenic, HLB-resistant mutants as future citrus cultivars. Objective 4: We have selected over 24 HLB-resistant/tolerant citrus cultivars in FL and transferred more than 12 of them to CA through the California Clonal Protection Program and produced certified stock plants. These cultivars/selections are ready for propagation and real-world field evaluations in multiple states. They will be grafted on different rootstocks and established in replicated trials in FL, CA, and TX. Citrus trees will be evaluated for foliage and canopy health, yield, and fruit quality, and HLB resistance and CLas titers will be assessed over multiple years. Healthy and profitable scion and rootstock combinations will be identified for the industry. The costs and benefits of adopting new resistant cultivars will be evaluated using investment analysis. Decision-making tools will be developed. Objective 5: Field days and demonstration events will be organized in FL, CA, and TX to showcase new cultivars to the citrus industry. Presentations, an active web presence, trade magazine articles, social media, and other outreach activities will disseminate findings in a timely manner to the industry and support the industry’s adoption of new cultivars.
