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.
1. Project objectives: 1) Screen Bt toxins for activity against Diaprepes root weevil (DRW); 2) Identify the most effective dsRNA constructs against DRW; 3) Assess the combined action of dsRNA and Bt toxins; 4) Assess four Bt transgenic citrus lines for DRW resistance. Objective 1. Bacterial Pesticidal Proteins: During this first reporting period, 11 of some 15 bacteria-derived pesticidal proteins drawn from four different structural groups were expressed in Bonning’s lab by use of either E. coli- or Bacillus thuringiensis-based expression systems. Pesticidal proteins were harvested from E. coli or following Bt sporulation, purified and solubilized as required using standard procedures. Cry proteins were trypsin activated. These proteins are now ready for testing in initial bioassays against DRW. DRW Colony: Meanwhile, in Stelinski’s lab, a new colony of DRW has been established to generate insects for this project. The culture was initiated from adult weevils collected from citrus groves in central Florida in 2023. Larvae are reared on an artificial diet developed by Beavers (1982) using procedures described by Lapointe and Shapiro (1999).DRW Bioassay: To establish a reliable bioassay protocol for testing of bacterial pesticidal proteins against DRW larvae, soil-column, seedling, and meridic diet bioassay methods were compared using a proxy formulation of B. thuringiensis subsp. tenebrionis (Btt; CX-2330 85% [AI]). Bioassays were conducted to evaluate survival of DRW neonates and 5-week-old larvae after exposure to bacterial suspensions of Btt. While all bioassays indicated activity of the Btt treatment as compared with the control (particularly against neonate larvae), the meridic diet method produced the most consistent results with the least mortality observed in untreated control treatments. Moreover, this method was the easiest to establish, and the least expensive in material costs and time investment. While certain experiments may require use of other bioassay methods or variations thereof, these initial results indicate that the meridic diet bioassay will serve our needs for testing of bacterial pesticidal proteins. Objective 2. Gene Silencing RNAs: the gene silencing RNAs (dsRNAs) and primers have been designed for all DRW target genes by Killiny, and reagents necessary for dsRNA synthesis are on order. Citations: Beavers, J. B. 1982. Biology of Diaprepes abbreviatus (Coleoptera: Curculionidae) reared on an artificial diet. Florida Entomologist. 65: 263-269.Lapointe, S. L., & Shapiro, J. P. 1999. Effect of soil moisture on development of Diaprepes abbreviatus (Coleoptera: Curculionidae). Florida Entomologist, 82: 291-299. 2. Plans for the next quarter:Objective 1: Initiate screening of bacterial pesticidal proteins for toxicity against DRWObjective 4: Conduct bioassays to assess the survival of DRW on transgenic plants that express bacterial pesticidal proteins. 3. Budget status: Hiring of the postdoctoral researcher for work on objective 2 in Killiny’s lab has been delayed. Otherwise the project is on track.
Huanglongbing (HLB) is a systemic disease of citrus caused by the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) that limits citrus production worldwide. CLas is an obligate bacterial pathogen that multiplies in citrus trees and in the insect vector, the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. There is no cure for HLB currently and broad-spectrum antibiotics represent one possible therapeutic against disease symptoms. Single-stranded nucleic acid analogs, 2-deoxy-2-Fluoro-ß-D-arabinonucleic antisense oligonucleotides (FANA ASOs), can modulate gene expression by enzymatic degradation or steric blocking of an RNA target. FANA ASOs recognize and bind to specific RNA forms, including mRNA, miRNA, and long noncoding RNA, through complementary base pairing. Injection of oxytetracycline (OTC) into mature citrus trees with HLB ameliorated symptoms of disease, increased fruit yield, and quality of juice as compared with that produced by non-injected controls. Injection of trees with FANA ASOs also reduced CLas infection but did not improve fruit yield and quality above control levels at the injection dosage tested. Reduced pathogen titers following OTC or FANA ASO injection were coincident with lower CLas acquisition and inoculation by laboratory deployed and wild-type D. citri collected from the field, respectively.Trunk injection of OTC in rotation with antimicrobial technologies like FANA ASOs may be useful in management of HLB by reducing CLas infection in trees and disrupting transmission. Future investigations should prioritize optimizing FANA ASO dosage in trees and exploring the potential of multiplex FANA ASOs that simultaneously target multiple mRNAs to enhance efficacy against CLas infection.
1. Please state project objectives and what work was done this quarter to address them: Objectives:.1. Screen FANA antisense oligonucleotide targeting CLas for efficacy under field conditions. Our working hypothesis is that CLas-specific FANAs can be delivered using microinjection developed for RNAi-based technologies to reduce CLas in infected citrus trees.2. Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission. Our working hypothesis is that CLas will be inhibited explicitly in psyllids by using CLas-specific FANAs, resulting in reduced CLas acquisition and transmission by ACP under field conditions.Methods:Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy under field conditions. Field trials with laboratory-vetted FANAs were conducted in research groves at the UF Citrus Research and Education Center. Treatments were applied to 10-year-old, CLas-infected ‘Hamlin’ trees of a standard size and CLas titer. AUM LifeTech designed and synthesized FANA ASOs complementary to two essential CLas genes: the CLas NAD-dependent DNA Ligase gene (LigA) and the CLas DNA B-Helicase gene. As a negative control, a FANA ASO was designed as a scramble sequence with no complementarity with any citrus gene. Antibiotic application (Fireline – Oxytetracycline) and insecticide-only treatments were applied to trees as positive and negative control treatments, respectively. Each treatment was applied to 15 trees in 1-acre plots replicated three times in a randomized complete block design. Treatments were applied to both sides of the tree canopy using microinjection of dosages determined in our previous greenhouse assays. The first replicate of this experiment was conducted from spring and fall 2022 and spring 2023. It consisted of five treatments: untreated control (insecticide-only), oxytetracycline control (1.56 g of Fireline per tree), Scramble Control-FANA, CLas LigA-FANA, and CLas B Helicase-FANA. All FANAs dosages were 625 ppm per tree. Update: This report provides an update on the effect of FANA ASOs on CLas infection in trees, tree growth, and yield after two years of the study. In the fall of 2023, a fourth injection of treatments was performed. CLas infection remained significantly low in trees treated with oxytetracycline trees from day zero and up to 90 days following application. LigA-FANA-treated trees showed a significant reduction in CLas infection at day 7- and 30 days following application, and results were comparable with oxytetracycline-treated trees. However, at 45, 60, and 90 days following application, FANA-treated trees showed similar CLas infection levels to those in the insecticide-treated (negative contrl) trees. At the end of the experiment, ten dead trees were recorded: 2-3 trees in FANA-treated plots (13-20%, n = 15), three trees in insecticides-treated trees (20%, n = 15), and none in oxytetracycline-treated trees. Additionally, three health parameters (Canopy and CGU) were recorded at the end of the experiment to assess the cumulative effect of treatments on trees. These results are currently being analyzed and will be provided in the final report. 2023 Harvest season data:Fruit yield. In 2023, trees treated with oxytetracycline yielded 5 – 20 times more fruit than trees in FANA ASO and insecticide-treated plots, respectively. Among these treatments, trees treated with oxytetracycline yielded significantly more fruit per tree (40.4 fruit/tree) than the other treatments evaluated. The highest total fruit weight was produced by trees treated with oxytetracycline with a total of 124.27 lbs., followed by Helicase-B-FANA (9.24 lbs.), Control-FANA (8.1 lbs.), LigA-FANA (3.02 lbs.), and insecticides-only (1.47 lbs.) treated trees. The mean fruit weight per tree was significantly higher in oxytetracycline-treated trees, with 8.29 lbs. of fruit weight per tree compared with 0.3-1.32 lbs. of fruit weight produced per tree observed from the rest of the treatments.Fruit drop. Fruit drop was successfully recorded prior to the harvest of the plots. Results are currently being processed and analyzed and will be included in the final report. Juice yield and quality analyses. Oxytetracycline-treated trees produced the highest juice weight, with a total of 62.65 lbs. produced in 2023, followed by Helicase-B-FANA (3.97 lbs.), Control-FANA (3.7 lbs.), LigA-FANA (1.17 lbs.), and insecticides-only (0.65 lbs.) treated trees. The mean juice weight per tree was significantly higher in oxytetracycline-treated trees, with 4.18 lbs. of juice per tree compared to 0.13-0.57 lbs. of juice produced by the rest of the treatments. Moreover, oxytetracycline-treated trees also had a significantly higher Brixº/acid ratio, averaging 12.5 Brix/Acid in the juice compared to 9.72-10.62 Brix/Acid achieved by the rest of the treatments. Juice color parameters were improved in 2023 compared to 2022. The juice from oxytetracycline-treated trees had the highest color score of 31.87, followed by LigA-FANA (31.2 score), insecticides-only (31.2 score), Helicase-B-FANA (31.03 score), and Scramble-FANA (30.98 score) treated trees. However, no statistically significant differences among treatments were found in the juice color.Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission. Acquisition assay. Field assays with psyllids were conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP in the spring and fall of 2022 and spring and fall of 2023. Psyllid nymphs, which develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from FANA-treated infected citrus trees was compared with acquisition from untreated infected trees, using the treatments described in Obj.1. Seven days after treatments were applied, ten ACP (five males and five females) from uninfected laboratory cultures were caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated three times on individual trees. Following oviposition (seven days after), ACP adults (P1) were collected and preserved for CLas detection. Egg clutches were left on trees enclosed in mesh sleeves. After nymphs reached the adult stage (15 days after), psyllids (F1) and leaves from test plants were collected. The effect of FANA treatments on the acquisition of CLas was assessed by comparing the CLas titer in P1 and F1 ACPs caged on treated and untreated citrus trees.Overall, the fewest infected ACP adults were collected on oxytetracycline-treated trees, followed by LigA-FANA-treated trees. For emerging ACP nymphs, CLas infection was only statistically reduced on trees treated with oxytetracycline compared to the control. The last replication i scurrently finishing. Results are being processed and analyzed and will be added to the final report. To evaluate incoculation, a subsample of 10 ACP per treatment was collected from treated trees and transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP adults were enclosed on plants for inoculation feeding for seven days. The last replication of the experiment was performed in the first quarter of 2024. Results are currently being analyzed and will be added to the final report. 2. Please state what work is anticipated for next quarter:The remaining fruit and juice quality data will be compied analyzed. All acquisition and inoculation assays will be completed, final samples processd, and data will be analyzed in the next quarter. 3. Please state budget status (underspend or overspend, and why): The budget spending is on track as anticipated. 4. Please show all potential commercialization products resulting from this research, and the status of each: Not applicable at this time. THis project is evaluating registered and available products.
1. Please state project objectives and what work was done this quarter to address them: Objectives:.1. Screen FANA antisense oligonucleotide targeting CLas for efficacy under field conditions. Our working hypothesis is that CLas-specific FANAs can be delivered using microinjection developed for RNAi-based technologies to reduce CLas in infected citrus trees.2. Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission. Our working hypothesis is that CLas will be inhibited explicitly in psyllids by using CLas-specific FANAs, resulting in reduced CLas acquisition and transmission by ACP under field conditions.Methods:Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy under field conditions. Field trials with laboratory-vetted FANAs were conducted in research groves at the UF Citrus Research and Education Center. Treatments were applied to 10-year-old, CLas-infected ‘Hamlin’ trees of a standard size and CLas titer. AUM LifeTech designed and synthesized FANA ASOs complementary to two essential CLas genes: the CLas NAD-dependent DNA Ligase gene (LigA) and the CLas DNA B-Helicase gene. As a negative control, a FANA ASO was designed as a scramble sequence with no complementarity with any citrus gene. Antibiotic application (Fireline – Oxytetracycline) and insecticide-only treatments were applied to trees as positive and negative control treatments, respectively. Each treatment was applied to 15 trees in 1-acre plots replicated three times in a randomized complete block design. Treatments were applied to both sides of the tree canopy using microinjection of dosages determined in our previous greenhouse assays. The first replicate of this experiment was conducted from spring and fall 2022 and spring 2023. It consisted of five treatments: untreated control (insecticide-only), oxytetracycline control (1.56 g of Fireline per tree), Scramble Control-FANA, CLas LigA-FANA, and CLas B Helicase-FANA. All FANAs dosages were 625 ppm per tree. Update: This report provides an update on the effect of FANA ASOs on CLas infection in trees, tree growth, and yield after two years of the study. In the fall of 2023, a fourth injection of treatments was performed. CLas infection remained significantly low in trees treated with oxytetracycline trees from day zero and up to 90 days following application. LigA-FANA-treated trees showed a significant reduction in CLas infection at day 7- and 30 days following application, and results were comparable with oxytetracycline-treated trees. However, at 45, 60, and 90 days following application, FANA-treated trees showed similar CLas infection levels to those in the insecticide-treated (negative contrl) trees. At the end of the experiment, ten dead trees were recorded: 2-3 trees in FANA-treated plots (13-20%, n = 15), three trees in insecticides-treated trees (20%, n = 15), and none in oxytetracycline-treated trees. Additionally, three health parameters (Canopy and CGU) were recorded at the end of the experiment to assess the cumulative effect of treatments on trees. These results are currently being analyzed and will be provided in the final report. 2023 Harvest season data:Fruit yield. In 2023, trees treated with oxytetracycline yielded 5 – 20 times more fruit than trees in FANA ASO and insecticide-treated plots, respectively. Among these treatments, trees treated with oxytetracycline yielded significantly more fruit per tree (40.4 fruit/tree) than the other treatments evaluated. The highest total fruit weight was produced by trees treated with oxytetracycline with a total of 124.27 lbs., followed by Helicase-B-FANA (9.24 lbs.), Control-FANA (8.1 lbs.), LigA-FANA (3.02 lbs.), and insecticides-only (1.47 lbs.) treated trees. The mean fruit weight per tree was significantly higher in oxytetracycline-treated trees, with 8.29 lbs. of fruit weight per tree compared with 0.3-1.32 lbs. of fruit weight produced per tree observed from the rest of the treatments.Fruit drop. Fruit drop was successfully recorded prior to the harvest of the plots. Results are currently being processed and analyzed and will be included in the final report. Juice yield and quality analyses. Oxytetracycline-treated trees produced the highest juice weight, with a total of 62.65 lbs. produced in 2023, followed by Helicase-B-FANA (3.97 lbs.), Control-FANA (3.7 lbs.), LigA-FANA (1.17 lbs.), and insecticides-only (0.65 lbs.) treated trees. The mean juice weight per tree was significantly higher in oxytetracycline-treated trees, with 4.18 lbs. of juice per tree compared to 0.13-0.57 lbs. of juice produced by the rest of the treatments. Moreover, oxytetracycline-treated trees also had a significantly higher Brixº/acid ratio, averaging 12.5 Brix/Acid in the juice compared to 9.72-10.62 Brix/Acid achieved by the rest of the treatments. Juice color parameters were improved in 2023 compared to 2022. The juice from oxytetracycline-treated trees had the highest color score of 31.87, followed by LigA-FANA (31.2 score), insecticides-only (31.2 score), Helicase-B-FANA (31.03 score), and Scramble-FANA (30.98 score) treated trees. However, no statistically significant differences among treatments were found in the juice color.Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission. Acquisition assay. Field assays with psyllids were conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP in the spring and fall of 2022 and spring and fall of 2023. Psyllid nymphs, which develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from FANA-treated infected citrus trees was compared with acquisition from untreated infected trees, using the treatments described in Obj.1. Seven days after treatments were applied, ten ACP (five males and five females) from uninfected laboratory cultures were caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated three times on individual trees. Following oviposition (seven days after), ACP adults (P1) were collected and preserved for CLas detection. Egg clutches were left on trees enclosed in mesh sleeves. After nymphs reached the adult stage (15 days after), psyllids (F1) and leaves from test plants were collected. The effect of FANA treatments on the acquisition of CLas was assessed by comparing the CLas titer in P1 and F1 ACPs caged on treated and untreated citrus trees.Overall, the fewest infected ACP adults were collected on oxytetracycline-treated trees, followed by LigA-FANA-treated trees. For emerging ACP nymphs, CLas infection was only statistically reduced on trees treated with oxytetracycline compared to the control. The last replication i scurrently finishing. Results are being processed and analyzed and will be added to the final report. To evaluate incoculation, a subsample of 10 ACP per treatment was collected from treated trees and transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP adults were enclosed on plants for inoculation feeding for seven days. The last replication of the experiment was performed in the first quarter of 2024. Results are currently being analyzed and will be added to the final report. 2. Please state what work is anticipated for next quarter:The remaining fruit and juice quality data will be compied analyzed. All acquisition and inoculation assays will be completed, final samples processd, and data will be analyzed in the next quarter. 3. Please state budget status (underspend or overspend, and why): The budget spending is on track as anticipated. 4. Please show all potential commercialization products resulting from this research, and the status of each: Not applicable at this time. THis project is evaluating registered and available products.
1. Please state project objectives and what work was done this quarter to address them: Objectives:.1. Screen FANA antisense oligonucleotide targeting CLas for efficacy under field conditions. Our working hypothesis is that CLas-specific FANAs can be delivered using microinjection developed for RNAi-based technologies to reduce CLas in infected citrus trees.2. Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission. Our working hypothesis is that CLas will be inhibited explicitly in psyllids by using CLas-specific FANAs, resulting in reduced CLas acquisition and transmission by ACP under field conditions.Methods:Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy under field conditions. Field trials with laboratory-vetted FANAs were conducted in research groves at the UF Citrus Research and Education Center. Treatments were applied to 10-year-old, CLas-infected ‘Hamlin’ trees of a standard size and CLas titer. AUM LifeTech designed and synthesized FANA ASOs complementary to two essential CLas genes: the CLas NAD-dependent DNA Ligase gene (LigA) and the CLas DNA B-Helicase gene. As a negative control, a FANA ASO was designed as a scramble sequence with no complementarity with any citrus gene. Antibiotic application (Fireline – Oxytetracycline) and insecticide-only treatments were applied to trees as positive and negative control treatments, respectively. Each treatment was applied to 15 trees in 1-acre plots replicated three times in a randomized complete block design. Treatments were applied to both sides of the tree canopy using microinjection of dosages determined in our previous greenhouse assays. The first replicate of this experiment was conducted from spring and fall 2022 and spring 2023. It consisted of five treatments: untreated control (insecticide-only), oxytetracycline control (1.56 g of Fireline per tree), Scramble Control-FANA, CLas LigA-FANA, and CLas B Helicase-FANA. All FANAs dosages were 625 ppm per tree. Update: This report provides an update on the effect of FANA ASOs on CLas infection in trees, tree growth, and yield after two years of the study. In the fall of 2023, a fourth injection of treatments was performed. CLas infection remained significantly low in trees treated with oxytetracycline trees from day zero and up to 90 days following application. LigA-FANA-treated trees showed a significant reduction in CLas infection at day 7- and 30 days following application, and results were comparable with oxytetracycline-treated trees. However, at 45, 60, and 90 days following application, FANA-treated trees showed similar CLas infection levels to those in the insecticide-treated (negative contrl) trees. At the end of the experiment, ten dead trees were recorded: 2-3 trees in FANA-treated plots (13-20%, n = 15), three trees in insecticides-treated trees (20%, n = 15), and none in oxytetracycline-treated trees. Additionally, three health parameters (Canopy and CGU) were recorded at the end of the experiment to assess the cumulative effect of treatments on trees. These results are currently being analyzed and will be provided in the final report. 2023 Harvest season data:Fruit yield. In 2023, trees treated with oxytetracycline yielded 5 – 20 times more fruit than trees in FANA ASO and insecticide-treated plots, respectively. Among these treatments, trees treated with oxytetracycline yielded significantly more fruit per tree (40.4 fruit/tree) than the other treatments evaluated. The highest total fruit weight was produced by trees treated with oxytetracycline with a total of 124.27 lbs., followed by Helicase-B-FANA (9.24 lbs.), Control-FANA (8.1 lbs.), LigA-FANA (3.02 lbs.), and insecticides-only (1.47 lbs.) treated trees. The mean fruit weight per tree was significantly higher in oxytetracycline-treated trees, with 8.29 lbs. of fruit weight per tree compared with 0.3-1.32 lbs. of fruit weight produced per tree observed from the rest of the treatments.Fruit drop. Fruit drop was successfully recorded prior to the harvest of the plots. Results are currently being processed and analyzed and will be included in the final report. Juice yield and quality analyses. Oxytetracycline-treated trees produced the highest juice weight, with a total of 62.65 lbs. produced in 2023, followed by Helicase-B-FANA (3.97 lbs.), Control-FANA (3.7 lbs.), LigA-FANA (1.17 lbs.), and insecticides-only (0.65 lbs.) treated trees. The mean juice weight per tree was significantly higher in oxytetracycline-treated trees, with 4.18 lbs. of juice per tree compared to 0.13-0.57 lbs. of juice produced by the rest of the treatments. Moreover, oxytetracycline-treated trees also had a significantly higher Brixº/acid ratio, averaging 12.5 Brix/Acid in the juice compared to 9.72-10.62 Brix/Acid achieved by the rest of the treatments. Juice color parameters were improved in 2023 compared to 2022. The juice from oxytetracycline-treated trees had the highest color score of 31.87, followed by LigA-FANA (31.2 score), insecticides-only (31.2 score), Helicase-B-FANA (31.03 score), and Scramble-FANA (30.98 score) treated trees. However, no statistically significant differences among treatments were found in the juice color.Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission. Acquisition assay. Field assays with psyllids were conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP in the spring and fall of 2022 and spring and fall of 2023. Psyllid nymphs, which develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from FANA-treated infected citrus trees was compared with acquisition from untreated infected trees, using the treatments described in Obj.1. Seven days after treatments were applied, ten ACP (five males and five females) from uninfected laboratory cultures were caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated three times on individual trees. Following oviposition (seven days after), ACP adults (P1) were collected and preserved for CLas detection. Egg clutches were left on trees enclosed in mesh sleeves. After nymphs reached the adult stage (15 days after), psyllids (F1) and leaves from test plants were collected. The effect of FANA treatments on the acquisition of CLas was assessed by comparing the CLas titer in P1 and F1 ACPs caged on treated and untreated citrus trees.Overall, the fewest infected ACP adults were collected on oxytetracycline-treated trees, followed by LigA-FANA-treated trees. For emerging ACP nymphs, CLas infection was only statistically reduced on trees treated with oxytetracycline compared to the control. The last replication i scurrently finishing. Results are being processed and analyzed and will be added to the final report. To evaluate incoculation, a subsample of 10 ACP per treatment was collected from treated trees and transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP adults were enclosed on plants for inoculation feeding for seven days. The last replication of the experiment was performed in the first quarter of 2024. Results are currently being analyzed and will be added to the final report. 2. Please state what work is anticipated for next quarter:The remaining fruit and juice quality data will be compied analyzed. All acquisition and inoculation assays will be completed, final samples processd, and data will be analyzed in the next quarter. 3. Please state budget status (underspend or overspend, and why): The budget spending is on track as anticipated. 4. Please show all potential commercialization products resulting from this research, and the status of each: Not applicable at this time. THis project is evaluating registered and available products.
(863) 956-8817
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