Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
Huanglongbing (HLB) represents a significant existential threat to the citrus industry in the United States, particularly impacting major citrus-producing states such as Florida, California, and Texas. The disease, severely diminishes citrus yield and quality by disrupting nutrient uptake and compromising tree health. Current commercially cultivated citrus rootstocks and scions lack inherent resistance to HLB, necessitating urgent research into alternative strategies for mitigating its devastating effects. The objective of this project is to assess HLB tolerant rootstocks and interstocks, achieved through conventional breeding and developed through crosses utilizing HLB-resistant Australian lime germplasm. Specifically, the project aims to achieve the following goals: (1) Establishing and evaluating field trials of novel HLB-tolerant rootstocks and interstocks in geographically diverse environments across California and Florida; (2) Rapid validation of genetic factors underlying HLB tolerance using advanced molecular techniques, including the deployment of the CTV-T36 vector system for targeted gene expression manipulation; (3) Employing a comprehensive multi-omics approach to identify and validate genetic markers associated with HLB tolerance to facilitate accelerated breeding programs and precise selection of superior cultivars and (4) Engaging stakeholders through robust outreach initiatives to disseminate research findings, educate growers on the benefits of adopting HLB-tolerant citrus varieties, and foster industry-wide adoption of sustainable citrus production practices. This project is relevant to the Citrus Disease Subcommittee recommendations for priority development of commercial citrus varieties (rootstocks and scions) with genetic resistance to HLB using traditional breeding techniques.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
0%
Goals / Objectives
Huanglongbing (HLB) is a highly destructive disease causing significant economic losses in citrus industries globally. Citrus, with a varied genetic background, has been shown to be more adaptive to environmental pressures. There is abundant genetic variation in citrus relatives, such as in the Australian lime species, which has evolved to be HLB resistant and can pass on the resistance trait to their progeny.The critical need being addressed here is the development and testing of a diverse germplasm that includes the HLB resistance genes from the Australian lime species. The project aims to establish a solid foundation for long-term economic sustainability, stabilize acreage, and maintain the livelihood of many families in multiple states who directly or indirectly depend on the citrus industry. The major goals of this project are to gain a comprehensive horticultural and pathological understanding of how Australian lime hybrids resist HLB in the field, detailing the underlying mechanisms and provide citrus stakeholders with specific recommendations on which Australian lime hybrids are best suited for their needs..
Project Methods
Rootstock, Interstock and Inarching trial: HLB-free and certified cuttings from eight Australian lime hybrids will be propagated through mistbed or tissue culture derived cuttings. The resultant liners will be budded with a standard sweet orange and grapefruit in the Florida trials. In California, rootstocks will be budded with a navel orange and mandarin scions.These trees will be planted out in the grove for evaluation. Trees will be planted both on University owned land as well as on stakeholder property ensuring coverage across various soil types and agroclimatic environment. For field evaluation, interstock trees with similar scion will be developed on Carrizo rootstock for California and Swingle (HLB-sensitive) and US942/X639 (HLB-tolerant) rootstocks for Florida.Inarching experiments will also be conducted with the best hybrids identified in previous studies onto Swingle or US942 rootstock trees.. Monitoring growth parameters such as tree height, trunk diameter, canopy width, and shoot growth over time will track the development and vigor of the trees. Root architecture will also be evaluated to understand the impact of various rootstocks and interstocks on root development, including length, density, and distribution. After the fruit set, fruit yield and quality metrics will be assessed to compare the performance of trees budded with different combinations. The severity of CLas infection (qPCR) will be monitored to investigate the progression of the disease. Each of these parameters will also be recorded bi-annually. Additionally, compatibility between interstock and scion will be evaluated through graft union formation and overall vigor assessments. Sampling will be systematically conducted at regular intervals across all trial sites. At each assessment point, a random subset of trees from each treatment group will be selected for detailed measurements and observations. Statistical analysis will be applied to the collected data to perceive significant differences between treatments.Quick testing of genes using the CTV vector system: CTV vectors based on 35S CTV9-47R will be designed to target specific genes that have been identified to play a role in Australian lime's HLB tolerance process-either through overexpression or downregulation using RNAi. Agro-inoculation into N. benthamiana and inoculation of citrus will be done. After systemic infection is established in C. macrophylla according to ELISA results, we will graft transmit these constructs into sweet orange or grapefruit seedlings for HLB phenotype screening. These trees will be assessed for HLB tolerance by inoculating them in a containment plant growth room with CLas viruliferous psyllids, typically for a duration of 4 weeks.Identify differentially expressed genes and differentially expressed metabolites and develop PCR markers: All metabolic experiments will be conducted in a completely randomized design. 'Valencia' sweet orange will serve as the control. In all experiments, ten biological replicates (trees) and two technical replicates (GC-MS runs/tree) for each hybrid will be analyzed (n=20). Volatile organic compounds and the phytohormonal profile of the hybrids will be analyzed using GC-MS. Leaf-polar metabolites, Starch and sucrose content and Callose deposition accumulation will analyzed using standard protocols.RNA sequencing will be conducted from total RNA extracted from greenhouse-grown CLas infected citrus trees and PCR markers will be developed based on the differentially expressed genes.Engage Stakeholders and Raise Awareness of the New Hybrids Being Developed: Information on the HLB tolerant Australian lime hybrids will be delivered to stakeholders, building on an existing web-based framework and citrus grower-focused outreach activities to facilitate grower adoption. We will invite stakeholders to visit the field trials being set up in CA and FL. We will also strive to make our technology understandable to non-scientists. Throughout the course of the project, we will 1) develop peer-reviewed extension documents published through UF EDIS (English and Spanish) to provide pertinent information for the cultivation of the Australian limes in the various citrus growing regions, 2) provide updates to growers through in person local and regional extension events hosted by our citrus extension agents, 3) provide timely articles for Citrus Industry Magazine and Citrograph, 4) share our research more broadly with colleagues through the Science for Citrus Health website, 5) participate in podcasts throughout the duration of this project to discuss Australian lime research with both scientific and grower audiences, and 6) produce a dedicated web page to provide timely information in a manner understandable for non-scientists.