Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
Huanglongbing (HLB) is a devastating disease threatening the US citrus industry. Growers in the United States have been seeking a solution to HLB for nearly 20 years. The current strategies for mitigating HLB are costly and unsustainable. The development and commercialization of HLB-tolerant scions proposed in this project is a possible solution for sustainable citriculture in the US. This project will investigate scions with demonstrated HLB tolerance and the potential to produce commercial-grade fruit despite testing positive for CLas, the agent believed to cause HLB. Research will be conducted on plants both infected in established groves and uninfected in a CUPS facility for a comparative analysis of uninfected material versus both tolerant and susceptible infected material.The long-term goals of this project are 1) to distribute HLB-resistant rootstocks to stakeholders; and 2) to understand the biological mechanisms behind HLB-tolerance for breeding HLB resistant scions and gene editing purposes. The objectives of this project are 1) verify HLB tolerance of preselected mature sweet orange and other scions; 2) transferring scion germplasm material with HLB tolerance to other citrus producing states; 3) delivery of data supporting HLB resistant scions to stakeholders; and 4) study underlying mechanisms of HLB Tolerance in sweet orange and other scions. This project will gather multi-omic data to gain knowledge of the multidimensional underpinnings of HLB tolerance in citrus scions. Multi-omic data includes data related to genetics, gene expression, physiology, and biochemistry, and these data will be compared to horticultural growth data and fruit and juice quality data. This project's objectives align with priority needs 1) development of commercial citrus varieties (scions) with genetic tolerance to HLB using traditional breeding techniques; and 4) A cure for HLB-infected trees and strategies for maintaining their productivity.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Goals / Objectives
Long Term GoalsThe long term goals of this project are 1) to commercialize and distribute HLB-tolerant scions to stakeholders in Florida, Texas, and California; and 2) to understand the biological mechanisms behind HLB-tolerance for breeding HLB tolerant and resistant scions and using this acquired knowledge to develop profitable selections for growers with biotechnology (gene editing) or conventional breeding (introgressing tolerant or resistant regions into sweet orange and fresh market selections). Objectives associated with Goal1 areObjective 2:Methodology for transferring scion germplasm material with HLB tolerance to other citrus producing states; and Objective 3:Delivery of data supporting HLB tolerant scions to stakeholders and project findings to the industry. Objectives associated with Goal 2 areObjective 1: Verify HLB tolerance of preselected mature sweet orange and other scions and determine their impacts on field performance and juice quality compared to conventional standards; and Objective 4:Study underlying mechanisms of HLB Tolerance in sweet orange and other scions and impacts of environment on scion performance, including fruit and juice quality. This project will use Citrus Under Protective Screen to compare with citrus in conventional groves to learn more abvout how tolerant accessions are able to grow better than susceptible industry standards. Although there is no transgenic nor gene edited citrus in this project, it is an intended goal that the knowledge gained from this project will assist scientists in the biotechnologyresearch space.
Project Methods
Efforts that will be used to create a Change in Knowledge include extension and outreach activities. These extension and outreach activities will take the form of workshops, field days and participation in the citrus industry's main events, including events such as Citrus Show, Citrus Expo, and Citrus Institute, which are heavily attended by growers. Evaluation will occur in several forms during the project period. For Objective 1, HLB tolerance will be verified at two locations (CUPS facilty and in the grove) bycollecting visual ratings to describe tree decline by HLB disease using a method that ranges from 1 to 4 score. Scores are indicative of the proportion of the tree with HLB symptoms from severe to healthy (Gottwald et al., 1989; Slinsk, 2016) and will be used along with existing spectral drone data. Digital SPAD quantitative data (unitless SPAD readings for leaf chlorophyll content) will also be collected to correlate with visual inspection and drone data (tree size, NVDI and NDRE). Plant growth will be monitored among the selections with conventional horticultural methods. These data will include plant growth, vegetative flush habits, flowering time, fruit set and fruit and juice data. For Objective 1 HLB testing, the testing will be carried out to verify disease status. Tree foliage will be divided into 4 sections and 4-5 leaves will be collected per tree. Leaf samples are a mix of symptomatic and asymptomatic samples. Fibrous root samples are collected by inserting a root auger (Cat No. 05-02) about 1 meter away from the citrus tree foot. Chopped midrib of leaves and fibrous roots samples are transferred into 2.0 mL polypropylene screw cap tubes pre-filled with 0.5 mm steel beads and stored at 4°C for DNA extraction.CLas detection via qPCR. To detect CLas in the selected citrus scion, DNA will be isolated from midrib of leaves and fibrous roots with a modified CTAB. DNA obtained from root tissues will be purified with Zymo Research Corporation OneStep™ PCR Inhibitor Removal Kit. DNA quality check will be carried out with the Nanodrop Lite to measure DNA concentrations and purity absorbance A260/A230 ratios. Real-time RT-qPCR will be performed on a StepOnePlus™ Real time PCR system. DNA samples extracted from leaves and root will be used for CLas detection with SYBR® Green qPCR with 16S rDNA primers LasLong.For verifying HLB tolerance of scion candidates and to determine impacts of tolerance on scion performance in CUPS versus the grove, the experimental design will include a multiple site design with at least six individuals (n ≥ 6) selected at random per site for horticultural, transcriptomic and metabolomic data collection. DNA and RNA will be sequenced for HLB tolerant scions to compare with the control selections. The sites will include one CUPS facility and two field sites with mature trees. At each site, a randomized selection of six trees will be tagged for and followed during the experiment. Trees along border or drive rows will not be selected to reduce the edge effect. Six biological replicates of HLB-tolerant advanced scion sweet orange and fresh fruit candidates and two conventional industry standard sweet orange scions (Valencia and Hamlin) will be evaluated for verification of HLB tolerance. At the CUPS site, conventional Hamlin and conventional Valencia will be analyzed as the clean, CLas-negative comparators for plant growth and juice quality. Horticultural data, including yield and juice quality data, will be gathered to compare with the field site trees. The trees will be evaluated over at least two seasons. Scion growth will be measured periodically over time. Fruit and juice data will be evaluated for horticultural traits. Verification of tolerance will be determined if conventional scions grow and produce poorer quality trees and fruit compared to the advanced selections by the end of the experiment. More importantly, the effects of tolerant scions on tree symptom development, and growth and fruit juice and yield data will be measured and collected to determine actual impact of HLB tolerance. This will include length of shoot during flush, number of leaves on shoots, number of shoots, leaf size in leaf area, SPAD instrument reading, and stem diameter, similar to Chater et al. (2017). An economist will be hired as a consultant to determine economic industry effects of HLB tolerance by comparing clean conventional scions (in CUPS) to the CLas infected susceptible and tolerant scions in the field.For Objective 2: Methodology for transferring scion germplasm material with HLB tolerance to other citrus producing states, briefly, scions that have been validated as HLB-tolerant will be shipped to California for distribution and conservation in the Citrus Clonal Protection Program and the National Clonal Germplasm Repository for Citrus and Dates. Scions with HLB tolerance of interest will be sent to the CCPP at the University of California, RiversideFor Objective 3: delivery of data supporting HLB tolerant scions and project findings to stakeholders, the objective will have a two phase extension program. The first phase of the extension program (Phase 1) will include grower visits to established trials and the CUPS versus field experiments, where the HLB tolerant scions can be viewed by the stakeholders (growers, processors, and nurseries). These visits will include surveys to collect baseline data for project metric evaluation mechanisms, including questions about knowledge of HLB plant tolerance and resistance, scion and rootstock availability and knowledge and perceptions of the project goals and activities. Phase 1 will include pre- and post-workshop questionnaires to determine the program's effectiveness in disseminating knowledge, especially knowledge gained from this project and during the extension activities but will also determine stakeholder engagement and opinion of project activities and progress. Phase 2 will use information gained from stakeholders from Phase 1 to understand grower and other stakeholder opinions on desirable scions and rootstocks, evaluate project success, and determine change in attitude over time during the project period.Objective 4 will include the acquisition of multi-omic data sets that will be analyzed separately and together to understand mechanisms of HLB tolerance in citrus. The multi-omic data that will be used in this project are genomic, transcriptomic, and metabolomic in scale along with plant and fruit and fruit juice phenotypic data. Differentially expressed genes (DEGs) will be identified in scions of control and infected trees in CUPS and in the field trials by short read RNAseq methods; likewise, differentially altered targeted and untargeted metabolites (DAMs) will be identified using the fruit. Whole genome shotgun sequencing (WGS) of these scion candidates, up to 60x coverage, will be conducted to support subsequent bioinformatic analyses to identify the potential genomic regions that are responsible for the detected DEGs and DAMs. Advanced machine learning regression methodologies, including the computationally efficient Best Linear Unbiased Prediction method (Wang et al., 2019) and commonly used least absolute shrinkage and selection operator approach (Tibshirani, 1996), will be leveraged to analyze these multi-dimensional data to identify the connections between genomic loci, differentially expressed gene transcripts, and altered relative metabolite abundances that are associated with CLas infection, sensitivity, tolerance, and fruit quality.