1. Please state project objectives and what work was done this quarter to address them:
The research objective is to determine if synthetic Plant Defense Inducers could provide additional benefits after a second year of applications for season long suppression of HLB following OTC injections or improve yield and quality of harvest in infected trees. These foliar applications could potentially protect trees during the months where OTC injections are not allowed and provide resistance management for OTC with a different mode of action. In addition to disease assessments, determination of citrus vigor, fruit drop, yield and fruit quality will be important to provide growers with tools for sustainable citrus production under Florida conditions.
– Research trial plot maintenance continues on a monthy basis.
– Individual tree leaf samples for PCR analysis was collected and sent to the Southern Gardens Diagnostic Laboratory in October to determine Liberibacter levels.
– PCR leaf data from the June sample date has been received from the HLB testing lab.
– Data analysis continues for the 2025 season to date.
– Fall applications have been made in October according to the trial protocol.
2. Please state what work is anticipated for next quarter:
– Data analysis and interim report writing will continue.
– In December leaf samples for PCR Liberibacter titer analysis will be collected. Also in December individual tree vigor and canopy volume (tree height and width) will be determined
3. Please state budget status (underspend or overspend, and why):
Quarterly budget of $20,875.00 according to funding schedule.
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Destroy declining plant material with no direct use to the citrus industry to make space for Stage I and Stage II trials (Years 1 and 2).
During this previous project period, more acreage has been removed at multiple locations on CREC property. The heavy equipment that is used to push trees at the Center was under repair, but it is now back up and running, now getting an approximate 10 acre space ready for two Stage II trials, one of which is the Coca Cola Company’s CRDF-sponsored trial and the other will be a trial with mostly sweet orange. The space is cleared for the consolidation block and this involved destroying additional plant improvement material that was in poor condition and seemed to be of no use to the citrus industry, as it was taking up space on CREC property and was not being used for crosses to the best of my knowledge. The irrigation has been installed in anticipation for the trees that were budded off from the healthiest in CREC Plant Improvement.
Objective 2: Conserve, move, consolidate, and propagate tolerant and resistant citrus trees to a centralized location. (Years 1 and 2).
Hundreds of trees have been propagated according to the nursery involved in the legal budding of these healthiest and most valuable accessions. I am currently waiting for a report from the nursery on what is ready and what needs to be budded again. Previously, I was informed of these hundreds of trees budded, nearly all have successfully been propagated and I have no reason to believe anything has changed. During this period, the tree spade was borrowed and damaged by USDA-ARS personnel. USDA-ARS personnel asked to use the spade for one day and then borrowed it for weeks, only to return it in a damaged, unusable state. I was required to contact the mechanic and get additional repairs. I used funding for this grant to repair those damages and it took several weeks to get the spade repaired and returned. This caused a major delay during a critical time of the project period. Based on the results of tree movement, it is likely that trees will need to be spaded, left in place until new a rootball is formed, and then moved to reduce stress. It is also likely that the largest, most promising trees may need a larger tree spade. Compost will be incorporated in the sandy soil to improve water retention and foliar fertilizers will be applied to help nurse trees that have been stressed. Several trees have survived movement and with the correct methods and equipment and personnel, I am confident that the healthiest trees can be moved to conserve the HLB tolerant specimens.
2. Please state what work is anticipated for next quarter:
Tree spading and developing less stressful methods of tree movement will continue as will removal of additional space. I intend to attend a CRDF meeting to report on the progress and to ask for guidance on moving forward with conserving the most HLB tolerant germplasm in the UF/IFAS citrus breeding program.
3. Please state budget status (underspend or overspend, and why):
Budget is underspent (81% time spent versus 64% funding expended) because we are waiting for the invoice from the nursery. We anticipate this to even out by the end of the project period.
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Screen breeding populations for new individuals with exceptional HLB tolerance and fruit quality as populations mature and begin to fruit.
We are currently overseeing five trials that have been planted, one trial was planted before the project period, four were recently planted during the project period. Two additional trials are ready to be planted as soon as the land is cleared and prepped, with a planting date in mid-September. Two additional trials are being propagated to replicate the Coca Cola Company trial in two additional locations. By the end of this reporting period, early mandarin cultivars with HLB tolerance will be evaluated for yield and fruit quality for fresh market. Drone flights have been flown to gather more information about HLB tolerance among accessions. There appear to be UF/IFAS and USDA selections with exceptional tolerance at Eagle Lake. Trees going into maturity are being followed and when the fruit are ready they will be evaluated for quality to determine value to the industry.
Objective 2: Plant new and manage ongoing Stage 2 semi-commercial trials of the selections most likely to succeed in the HLB environment versus commercial standard controls.
We will have a total of at least nine Stage 2 trials that will be planted during the course of this project, probably more. As mentioned above, we have one trial that was planted and established before the project period, a sweet orange trial that is now two years old. Some trees in that trial now have fruit which will be phenotyped this coming season. We have recently planted four trials that are in the ground now. One is a fresh market trial at CREC with some selections that are orange-like, another is a sweet orange trial at CREC and a third is a seedless lemon trial versus controls on CREC land. We have also planted a trial on private land in Eagle Lake, FL with sweet orange and other selections. All trials have standard controls and they are in randomized complete block designs. I will provide maps at an upcoming meeting among Rick Dantzler, Dr. Futch and Dr. Mattia. We are also leaving no stone unturned and propagating everything with potential value to the industry from the plant improvement team at CREC to evaluate for HLB tolerance and fruit quality. All trials have either control rootstocks or control combinations, depending on the trial. For the trial to be planted in mid-September, the trees are ready to plant and the nursery will deliver when the land is ready. We will incorporate compost into the soil to aid in increased cation exchange capacity, water holding capacity, nutrient delivery and oganic matter content for the sandy soil that is being used for this CREC trial site.
Objective 3: Utilize bioinformatic tools to identify genes or other variants involved in HLB tolerance and possible resistance for plant transformation and conventional breeding.
As mentioned previously, we have received the genomic, Hi-C, and transcriptomic data for assembly and annotation of the Hamlin 1-4-1 genome so that it can be compared to Hamlin N13-32 to look for genes and other variants associated with HLB tolerance. We have identified the many of the founders of the USDA and IFAS citrus breeding program and these founders will be deep sequenced to assemble, phase and annotate them so that these can be used to look for alleles that are associated with HLB tolerance among the 100 genotyped accessions. Several assembled, phased and annotated genomes will result from this work that should be useful to those at the Crop Transformation Center in Gainesville and others working on the HLB crisis. We have noticed that genomic data that we received from internal core services in Gainesville was not optimal compared to other labs we have worked with, so we are currently working on getting the highest quality data with the lowest possible cost and choosing a different lab accordingly. We are waiting on the most recent drone and phenotypic data from the Coca Cola Company to make decisions about the most tolerant accessions and how it relates to the drone data collected previously with the intention of choosing the most tolerant accessions for sequencing.
2. Please state what work is anticipated for next quarter:
We plan on deep sequencing, annotating, and phasing the founders and short read sequencing the 100 selections from USDA and IFAS breeding programs as soon as we find the best price for the highest quality data possible. We are actively prepping the land for the stage II trial with the Coca Cola Company at CREC and we plan on following ongoing trials to phenotype advanced selections to compare versus the standard controls. We plan to have meetings with the scientists at the Lawrence Berkeley National Laboratory to discuss data and research activities. The documentation with LBNL will be completed by the next report. We will phenotype all accessions that are earliest in the programs.
3. Please state budget status (underspend or overspend, and why):
We are underspent due to working on Lawrence Berkeley National Laboratory documentation and sequencing decisions. We will be more on track by next reporting period.
1. Please state project objectives and what work was done this quarter to address them:
The research objective is to determine if synthetic Plant Defense Inducers could provide additional benefits after a second year of applications for season long suppression of HLB following OTC injections or improve yield and quality of harvest in infected trees. These foliar applications could potentially protect trees during the months where OTC injections are not allowed and provide resistance management for OTC with a different mode of action. In addition to disease assessments, determination of citrus vigor, fruit drop, yield and fruit quality will be important to provide growers with tools for sustainable citrus production under Florida conditions.
– Individual tree assessment for health and vigor was conducted in June.
– Individual tree leaf samples for PCR analysis was collected and sent to the Southern Gardens Diagnostic Laboratory in June to determine Liberibacter levels.
– PCR leaf data has been received from the HLB testing lab.
– Data analysis has been initiated for the 2025 season to date.
– Summer foliar applications has been made according to the trial protocol.
2. Please state what work is anticipated for next quarter:
– Data analysis and interim report writing will continue.
– The next project milestones will be in October when leaf samples for PCR leaf analysis will be collected, individual tree vigor evaluated, and the fall foliar and soil treatment applications will be conducted according to the trial protocol.
3. Please state budget status (underspend or overspend, and why):
Quarterly budget of $20,875.00 according to funding schedule.
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Screen breeding populations for new individuals with exceptional HLB tolerance and fruit quality as populations mature and begin to fruit.
More data has been collected for the late season and data from the previous quarter has been analyzed, including fruit and juice blends derived from these materials. More tolerant material has been sampled. The late season oranges and orange-like accessions were sampled during the late season. Accessions that were treated with two years of OTC were compared versus those with one injection. New breeding material has been planted for long term evaluation and previously planted selections are being followed for fruiting and quality.
Objective 2: Plant new and manage ongoing Stage 2 semi-commercial trials of the selections most likely to succeed in the HLB environment versus commercial standard controls.
One Stage II with sweet orange is ongoing and this trial about 2 years old and is not fruiting yet, but the trees are growing off well and we anticipate the first fruit of that trial coming in next season. The two trials that were ready to plant have been planted. Data will be collected from these ongoing trials and they are being closely watched and well-maintained on CREC property. Also, we have reserved several acres for a large Stage II trial at CREC, which is a collaborative effort with the Coca Cola Company. We are planning to plant these trials are three locations (Citra, CREC and Ft. Pierce).
Objective 3: Utilize bioinformatic tools to identify genes or other variants involved in HLB tolerance and possible resistance for plant transformation and conventional breeding.
We have received the genomic, Hi-C, and transcriptomic data for assembly and annotation of the Hamlin 1-4-1 genome so that it can be compared to Hamlin N13-32 to look for genes and other variants associated with HLB tolerance. We have had our first official meeting with the Lawrence Berkeley National Lab scientist that will work with this group on identifying genes and other variants for HLB tolerance. We have identified the many of the founders of the USDA and IFAS citrus breeding program and these founders will be deep sequenced to assemble, phase and annotate them so that these can be used to look for alleles that are associated with HLB tolerance among the 100 genotyped accessions.
2. Please state what work is anticipated for next quarter:
We plan on deep sequencing, annotating, and phasing the founders and short read sequencing the 100 selections from USDA and IFAS breeding programs. We anticipate prepping the land for the three stage II trials with the Coca Cola Company and we plan on following ongoing trials to phenotype advanced selections to compare versus the standard controls. We plan to have meetings with the scientists at the Lawrence Berkeley National Laboratory to discuss data and research activities.
3. Please state budget status (underspend or overspend, and why):
We are underspent because we are waiting for the next round of paperwork from the Lawrence Berkeley National Laboratory. The paperwork that they sent was for only one year and we needed to make sure that the paperwork is correct before signing. We have requested a multiple year project agreement with an accurate scope of work.
1. Please state project objectives and what work was done this quarter to address them:
The research objective is to determine if synthetic Plant Defense Inducers could provide additional benefits after a second year of applications for season long suppression of HLB following OTC injections or improve yield and quality of harvest in infected trees. These foliar applications could potentially protect trees during the months where OTC injections are not allowed and provide resistance management for OTC with a different mode of action. In addition to disease assessments, determination of citrus vigor, fruit drop, yield and fruit quality will be important to provide growers with tools for sustainable citrus production under Florida conditions.
– Citrus harvest from the 2024 field trials was conducted at both locations in March 2025.
– Tree canopy height and width was measured to determine if Plant Defense Inducers is having an effect on tree growth.
– Spring spray applications (foliar and drench) has been conducted according to the trial protocol.
2. Please state what work is anticipated for next quarter:
– Individual tree assessment for vigor will be conducted in May
– Leaf samples will be collected for PCR analysis in May to determine Liberibacter titer levels.
– Summer spray applications will be conducted in June.
– Data analysis for an interim report will be conducted in June and July.
3. Please state budget status (underspend or overspend, and why):
Quarterly budget of $20,875.00 according to funding schedule.
1. Please state project objectives and what work was done this quarter to address them:
Objective 1: Screen breeding populations for new individuals with exceptional HLB tolerance and fruit quality as populations mature and begin to fruit.
Data has been collected on new material, including fruit and juice blends derived from these materials. Tolerant material has been sampled from material early in the harvest season. Drones have been flown over sites with new populations to select the healthiest trees for sampling. The late season oranges and orange-like accessions will be sampled during the late season. Accessions that were treated with OTC were compared versus the controls (non-injected).
Objective 2: Plant new and manage ongoing Stage 2 semi-commercial trials of the selections most likely to succeed in the HLB environment versus commercial standard controls.
One Stage II with sweet orange is ongoing trial is 1.5 years old and is not fruiting yet, but the trees are growing off well and we anticipate the first fruit of that trial coming in next season. Another two trials have trees that are ready to plant but we will wait until after the threat of freeze has passed before planting those. There are other trials that are ongoing and data is being or will be collected from them as the fruit become ready. Also, we have reserved several acres for Stage II trials.
Objective 3: Utilize bioinformatic tools to identify genes or other variants involved in HLB tolerance and possible resistance for plant transformation and conventional breeding.
The first Material has been sent to the ICBR at University of Florida for deep sequencing (1-4-1 Hamlin) to discover why, on a molecular level, the N-13-32 accession typically grows off better than Hamlin 1-4-1. We also recently discovered a Star Ruby grapefruit individual that may be a mutant with enhanced tolerance. We ran the SSR markers to determine that the budline was Star Ruby and the rootstock was US-941 and they were. The next step will be to see if there is mutagenesis that would confer HLB tolerance. We will also test the tree for HLB to see if it is resistant. The SSR work was done without significant expenditure because that work was done in-house with existing inputs.
2. Please state what work is anticipated for next quarter:
Next quarter, we will collect tree and fruit and juice quality data from the late season accessions in trial and in other plantings. We will propagate trees for more Stage II trials. We will reserve more space for Stage II trials. We will collect leaf material during late winter/early spring for the sequencing of the 100 accessions. We will also be in a position to receive and process the genomic data from 1-4-1 Hamlin and will collect transcriptomic data from this accession during flowering to annotate the 1-4-1 genome so that we can compare it to N13-32 Hamlin to look for differences in genome that could confer HLB tolerance in sweet orange and allow for identification of this tolerant budline. We are expecting to meet with the Laurence Berkeley National Laboratory group to discuss the coverage depth and platform of Illumina sequencing for the 100 accessions.
3. Please state budget status (underspend or overspend, and why):
We are 11% underspent (13% time spent vs 2% spent) due to the paperwork with the Laurence Berkeley National Laboratory and the careful planning of the sequencing effort with this group, who will be the ones focused on gene and variant discovery for CRISPR and transgenic work downstream at the Crop Transformation Center. We expect to be close to on track by the next reporting period. Additionally, we have had a change in federal administration which has come with some unanticipated outcomes that we are assessing.
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