Progress 09/15/23 to 09/14/24

Outputs
Target Audience:The target audiences reached by my efforts during this reporting period include citrus growers, packers, processors, and research and extension faculty and staff at the University of Florida/IFAS. The reason for targeting these groups is because they are the intended stakeholders related to this project. This project intends to leverage the huanglongbing resistant varieties to determine if these rootstocks could help growers grow trees that end up being more profitable in their farming and cropping systems. This work, if successful, would aid other stakeholders downstream, such as the packers and processors, who rely on citrus fruit to run a profitable and sustainable business. If there are no fruit to pack for the fresh market or squeeze for the juice market, this could cause packing houses to close down and processing plants to close as well. The academic stakeholders were also reached during this reporting period, mainly during stakeholder workshops for growers and processors, and these academic stakeholders would likely benefit from this work as they learn about potentially disease resistant cultivars, their pedigrees and downstream there will be genes, gene transcripts and metabolites to target for research purposes included for breeding and gene editing purposes. The efforts utilized to reach these primary stakeholders include workshops, small and large grower meetings and scientific conferences. Changes/Problems:There have been twoproblems that are being addressed. 1. The inarching experiment trees were destroyed by the grove management crew with an unapproved herbcide mix and spray. This created a delay in the onset of this component of the project where we will need to plant the rootstocks after the threat of freeze is gone this planting seasonin 2025. We will follow the trees during the growing season and attempt to inarch them during planting with plant protection, which will likely be individual protective covers. We will likely ask for a no cost extension because of this problem. 2. The rootstock candidates that are believed to be HLB resistant have been growing off, but some have been growingslowly compared to the industry standard. We had to start the experiment due to the time constraints and we did not use the rootstocks thart were too small to conventionally graft. We instead ony selected the rootstocks that grew off well enough to graft. We plan to propagate additional individuals and restart the experiment with as many rootstocks as possible and ask for a no cost extension. What opportunities for training and professional development has the project provided?This project has provided a space for the graduate student on the project, and others, to be trained for various skillsets related to the project activities. The student is learning how to manage a greenhouse and irrigate citrus trees and is learning how to manage transcriptomic and horticultural data. In addition, the graduate student on the project is learning how to write proposals and manuscripts. The grad student is also learning how to collect, process and analyze horticultural data. The postdoc on the project in Co-PI Wang's lab is being trained on sampling citrus roots and leaves and how to handle and process the samples. The second postdoc is being trained and enhancing their skills related to gene expressions and analysis. This postdoc is being trained in transcriptomics to determine if thereare markers assocaietd with HLB tolerance or resistance. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Due to the extreme cold weather this season, the rootstocks of interest that will be used for inarching will be planted toward the end of winter or the beginning of spring. The second and third timepoints will be sampled this year and the stakekholder group will be updated on the progress of the project. The data from the preliminary experiment and the first, second and third timepoints will be processed and analyzed. I will work with the grove management crew to make sure that they do not destroy the plants planted for inarching. We have the conventional standards in the group and established and there will be inarching done in the early spring and the trees followed througout the year.

Impacts
What was accomplished under these goals? For the first goal, " to commercialize and distribute HLB-resistant rootstocks to stakeholders in Florida, Texas, and California", there has been not accomplishments because the project has not yet identified any material with validated HLB resistance, as this is something that will happen later in the project. For the second goal, "to understand the biological mechanisms behind HLB-tolerance and -resistance for breeding HLB resistant scions and gene editing purposes", there has been some key accomplishments. These key accomplishments include propagating the huanglongbing resistant accessions and the industry standards by means of hardwood cuttings and growing most of them off to a point where we can graft the infected "budsticks" or hardwood propagules on the candidates. Another key accomplishment is that we have successfully sequenced the genomes of most of the rootstock candidates in this project. Additionally, we have tested the rootstock candidates for CLas, the bacteria that is believed to be the cause of huanglongbing. Some of the candidate rootstocks were difficult to propagate with this method and failed the first round of propagation, but the second round of propagation was successful. We have also propagated the trees that will be used for the inarching experiment and have identified the grove in which this experiment will be executed. For the rootstock candidates that were successfully propagated during the first round of propagation, we have pruned the individuals back so that they can be easily grafted with the infected propagules. One selection was inarched successfullly into a prelimiary tree. Two rootstocks that were propagated from the HLB-affecte grove were found to have HLB. We are continuing to monitor these plants to0 see if they overcome the disease. For this reporting period, the trees for inarching had been planted at a site with four-year-old Valencia trees. The rootstocks that were ready for grafting had infected and non-infected budwood budded onto several of the candidate rootstocks. Samples were taken from these propagated Valencia scions for metabolomic and transcriptomic analysis. The transcriptomic analysis is underway with sequencing and the metabolomic analysis is in storage until the other time points are harvested. The rootstocks planted in the field by the four-year-old Valencia were destroyed with high dosage herbicide sprays and plants are being propagated to replant in a more controlled trial. There are three time points proposed for the greenhouse experiment. The first time point has been sampled and there remain two more time points until the project is completed.

Publications

Progress 09/15/22 to 09/14/23

Outputs
Target Audience:The target audiences reached by my efforts during this reporting period include citrus growers, packers, processors, and research and extension faculty and staff at the University of Florida/IFAS.The reason for targeting these groups is because they are the intended stakeholders related to this project. This project intends to leverage the huanglongbing resistant varieties to determine if these rootstocks could help growers grow trees that end up being more profitable in their farming and cropping systems. This work, if successful, would aid other stakeholders downstream, such as the packers and processors, who rely on citrus fruit to run a profitable and sustainable business. If there are no fruit to pack for the fresh market or squeeze for the juicemarket, this could cause packing houses to close down and processing plants to close as well. The academic stakeholders were also reached during this reporting period, mainly during stakeholder workshops for growers and processors, and these academic stakeholders would likely benefit from this work as they learn about potentially disease resistant cultivars, their pedigrees and downstream there will be genes, gene transcripts and metabolites to target for research purposes included for breeding and gene editing purposes.The efforts utilized to reach these primary stakeholders include workshops, small and large grower meetings and scientific conferences. Changes/Problems:So far, there are no major changes. It is likely that there will be a no-cost extension request to make sure we haveample time for data analysis and transfer of materials. This request will likley occur in March or April of 2024 if needed. What opportunities for training and professional development has the project provided?This project has provided a space for the graduate student on the project, and others, to be trained for various skillsets related to the project activities. The student is learning how to manage a greenhouse and irrigate citrus trees and is learninghow to manage transcriptomic and horticultural data. In addition, the graduate student on the project is learning how to write proposals and manuscripts. The grad student is also learning how to collect, process and analyze horticultural data. The postdoc on the project in Co-PI Wang's lab is beingtrained on sampling citrus roots and leaves. How have the results been disseminated to communities of interest?The results thus far have been disseminated to communities of interest via small and large group meetings. For the grower, packer, and processors stakeholder group, the results that we have so far (propagating and testing material for huanglongbing) has been disseminated at grower meetings. These grower meetings include annual grower meetings that host over 100 growers per year and smaller grower meetings, which host less than 100 growers per year. For the academic communities of interest, I have disseminated that results that we have thus far to a group of scientists at the USDA building that houses the Jefferson Auditorium in Washington D.C. All results have been disseminated via powerpoint presentation. In many cases, the dissemination of results thus far have included a few slides towards the end of presentations to demonstrate the status of this project. What do you plan to do during the next reporting period to accomplish the goals?The trees will be subjected to the preliminary experiment to test gene transcripts during peak physiological activity and during the pre-dawn or evening hours to determine diurnal effects of gene expression of these seemingly huanglongbing resistant rootstock candidates. After that, the trees will be grafted with huanglongbing-affected Valencia and the leavesand roots of these accessions will be subjected to metabolomic and transcriptomic profiling over two to three timepoints. Additionally, horticultural data will be gathered during the next project reporting period. These samples will be sent to the lab for processing and the resulting genomic, transcriptomic, and metabolomic data will be utilized in multi-omic bioinformatics modeling to determine differences in gene expression and metabolomic profiles as they relate to disease status and horticultural traits. We will meet with the stakeholder advisory board to report on progress. The first phase of the extension program will also occur during this reporting period. And finally, the inarching experiment will be planted during the next reporting period of this project.

Impacts
What was accomplished under these goals? For the first major goal, "to commercialize and distribute HLB-resistant rootstocks to stakeholders in Florida, Texas, and California", there has been not accomplishments because the project has not yet identified any material with validated HLB resistance, as this is something that will happen later in the project. For the second goal, "to understand the biological mechanisms behind HLB-tolerance and -resistance for breeding HLB resistant scions and gene editing purposes", there has been some key accomplishments. These key accomplishments include propagating the huanglongbing resistant accessions and the industry standards by means of hardwood cuttings and growing most of them off to a point where we can graft the infected "budsticks" or hardwood propagules on the candidates. Another key accomplishment is that we have successfully sequenced the genomes of most of the rootstock candidates in this project. Additionally, we have tested the rootstock candidates for CLas, the bacteria that is believed to be the cause of huanglongbing. Some of the candidate rootstocks were difficult to propagate with this method and failed the first round of propagation, but the second round of propagation was successful. We have also propagated the trees that will be used for the inarching experiment and have identified the grove in which this experiment will be executed. For the rootstock candidates that were successfully propagated during the first round of propagation, we have pruned the individuals back so that they can be easily grafted with the infected propagules.

Publications

• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Chater, J.M. (2023) "SP HLB-resistant rootstock candidates for the citrus industry: validating and understanding disease resistance" Washington D.C. November 3rd, 2023.