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.
1. Please state project objectives and what work was done this quarter to address them:
1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. 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 in a field setting.
Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. 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 control treatments. 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.
Before treatment, four leaves were removed from each tree, two from each side of the tree’s apex and two from each side of the base of the canopy, for initial titer (T0) using quantitative real-time polymerase chain reaction (qPCR) assays. To monitor the effect of the FANA ASOs on the CLas titer of each tree, four leaf samples were removed from the same branches as the T0 samples after 2, 7, 30, 45, 60, and 90 days. The post-treatment CLas titer (TF) was calculated by qRT-PCR analysis each time. Leaf samples were run in duplicates, and the relative quantities of CLas in threes were calculated based on the comparative cycle threshold 2-..Ct method.
Update: In spring 2023, CLas infection declined significantly in antibiotic-treated trees from 0 to 60 days following application. Treatment and sampling time significantly affected CLas infection, although the interaction between treatment and sampling date was not statistically significant. CLas infection in FANA-treated trees was not statistically different from insecticide-treated trees.
In the fall of 2023, a fourth injection of treatments was performed, and all leaf samples were collected and processed for analysis of CLas infection.
Tree Health and Yield
Trees that received antibiotic treatments grew significantly wider canopies, followed by LigA-FANA-treated trees compared to the rest of the treatments. Similarly, monthly flushing patterns were affected by the interaction between treatments and sampling dates. Antibiotic and LigA-FANA treated trees had significantly more flush among treatments in June, July, and September of 2022; Similarly, in February and May of 2023. However, there was no statistical effect of antibiotic treatment and sampling date on tree canopy height and circumference at the graft union.
In November 2023, fruit were collected from all field plots and processed at the CREC Pilot Plant. Fruit yield, fruit drop, and juice quality will be reported in the next quarter.
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 of 2023. Psyllid nymphs, who 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.
Update:
In the spring and summer of 2023, a reduction in CLas acquisition by ACP adults feeding on Helicase-B-FANA-treated and antibiotics-treated trees was observed. Additionally, significantly fewer infected ACP were collected on antibiotic-treated trees compared to the rest of the treatments. Ants attacked ACPoffspring populations during the experiment; thus, insufficient replicates were collected. Therefore, comparisons of CLas infections between treatments could not be performed, and differences were not significant.
To date, fewer infected ACP adults were collected from antibiotic-treated trees, followed by LigA-FANA-treated trees compared to the rest of the treatments. CLas infection was lower among offspring that fed on antibiotic-treated plants. The final replication of the experiment was initiated in the fall of 2023. Samples will be collected during the next quarter and the results of the study reported in the the final report.
Inoculation assay.
A subsample of 10 ACP per treatment collected from treated trees was transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP adults were enclosed on plants for inoculation feeding for seven days. After that, ACPs were collected for subsequent CLas detection. After that, plants were sprayed with insecticides to eliminate any ACP progeny and were held for three months. Leaves were collected at 30, 60, and 90 days to assess the development of CLas infection following ACP exposure. For this assay, each treatment was replicated five times on individual trees.
Update: A final rplicate of the above assays was initiated in fall 2023. Samples will be collected during the next quarter and the results of the study reported in the the final report.
2. Please state what work is anticipated for next quarter:
Fruit and juice quality data will be analyzed following completion of fruit processing. Samples from the transmission and acquisision assays will be collected at the conclusion of the assays and processed to quantify CLas titers during 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:
The goal of this project is to protect citrus from Candidatus Liberibacter asiaticus by inducing the phloem to produce anchored, single-chain antibodies that will bind and immobilize the bacteria, allowing the plant to destroy them by natural defense mechanisms. Anchoring is accomplished by expressing the antibodies as extensions of native, phloem-specific sieve element occlusion (SEO) proteins.
On Oct. 16, 2023, 16 genetically transformed and 10 control Carrizo plants were shipped from Cornell to the Levy lab in Florida. The DNA constructs included the phloem-specific SEO promoter driving expression, in individual plasmids, of three different binding proteins (OmpA, CpaF, KpsF) as extensions of the native citrus SEO protein, for example, SEOp: OmpA-SEO. In controls the binding proteins were omitted. Plants were shipped with an APHIS epermit, according to USDA procedures. In Florida, the plants, which arrived in excellent condition, were retested for inserted sequences, repotted and placed in a secure greenhouse for additional growth.
Since Carrizo citrange plants, though readily transformed, can have low CLas titer (William Dawson, personal communication) it was decided to use the transformed Carrizo as rootstocks and untransformed Pineapple sweet orange (Citrus sinensis L. Osbeck) as scions. The grafted plants are now almost large enough to be tested for CLas mobility. The scions will be infected either by grafting infected tissue to them or by exposure to CLas-infected Diaphorina citri. CLas mobility will be tested by analyzing the Carrizo roots.
The Turgeon lab also synthesized SEO-anchored antibody constructs (the 3 different antibody genes above) using the strong phloem-specific rice tungro bacilliform virus (RTBV) promoter (Dutt et al., Tree Physiol. 32:83 2012) which should increase the number of binding proteins in the sieve elements. In addition, the Turgeon lab made constructs for RTBV driven dual-binding (bivalent) antibodies (OmpA-OmpA, CpaF-CpaF, KpsF-KpsF). Dual antibodies have been shown to have extremely strong binding potential (Bannas et al. Front. Immunol. 8:1603 2017). These constructs are now being used for Duncan grapefruit transformation.
2. Please state what work is anticipated for next quarter:
The Levy lab will soon begin testing the grafted trees by exposure to CLas and by grafting CLas-infected tissue to the scions. In the Turgeon lab, additional transformed trees are being produced, using Duncan grapefruit and Valencia sweet orange. The DNA constructs have been made, and transformation has begun, but the trees will not be ready for testing within the time frame of this grant. In order of priority the constructs are: 1) 35S promoter driving the three, anchored single-chain antibody constructs (these transgenics are being produced in the Levy lab); 2) RTBV promoter driving the three, anchored single-chain antibody constructs; 3) RTBV promoter driving the three, unanchored, dual-antibody constructs, and 4) 35S promoter driving the three unanchored, dual antibody constructs.
3. Please state budget status (underspend or overspend, and why):
The budget status is an anticipated with funds neither underspent nor overspent.
4. Potential commercialization products
No commercialization is expected during this grant period although we believe that if these constructs prove successful in arresting CLas, they will provide direction for the reconstruction of a CLas-free citrus industry.
1. Project objectives and work done this quarter: The goal of this project is to protect citrus from Candidatus Liberibacter asiaticus by inducing the phloem to produce anchored, single-chain antibodies that will bind and immobilize the bacteria, allowing the plant to destroy them by natural defense mechanisms. Anchoring is accomplished by expressing the antibodies as extensions of native, phloem-specific sieve element occlusion (SEO) proteins.
The Turgeon lab has sent transgenic plants to the Levy lab in Florida (with a required USDA permit) for testing. The Levy lab has further propagated these trees and now has a total of 79 rooted, transgenic plants of 5 phenotypes, as well as controls. Two strategies are being used to test the effectiveness of the constructs. First, CLas-infected sweet orange tissue is currently being side-grafted to the trees to determine whether CLas will move out of the graft region. Second, as a more natural infection approach, healthy, non-transgenic sweet orange stems are being grafted to the transformed plants and these stems will then be exposed to infected Diaphorina citri. We predict that CLas will travel downward in the sweet orange phloem to the transgenic portion of the stem but will be arrested there by the anchored antibodies.
2. Anticipated work for next quarter: In continuing work in the Turgeon lab, plasmids encoding SEO-anchored antibodies of three types (Omp, CpaF and KpsF) have been constructed using the 35S promoter and transformations will begin in the next two weeks. In another approach the Turgeon lab has made dual antibody constructs using the same antibody types as above, but in unanchored form, driven by 35S. Dual antibodies against surface antigens have been shown to be much more effective than single antibodies in arresting bacterial growth. (This work cannot be completed in the 1-year timeframe.)
3. Budget status: The budget status is as anticipated with funds neither underspent nor overspent.
4. Commercialization products: None were anticipated for this grant, although it may be possible to commercialize the transformed Carrizo citrus as rootstocks if they are sufficiently protected by our strategy.
Objectives:.
1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. 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 in a field setting.
Methods:
Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. 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 during spring and fall of 2022 and spring of 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 applied at 625 ppm per tree.
Before treatment, four leaves were removed from each tree, two from each side of the tree’s apex and two from each side of the base of the canopy, to determine initial titer (T0) using quantitative real-time polymerase chain reaction (qPCR) assays. To monitor the effect of the FANA ASOs on the CLas titer of each tree, four leaf samples were removed from the same branches according to the same procudures used to collect the T0 samples at 2, 7, 30, 45, 60, and 90 days post-treatment. The post-treatment CLas titer (TF) was calculated by qRT-PCR analysis during each interval. Leaf samples were run in duplicates, and the relative quantities of CLas in threes were calculated based on the comparative cycle threshold 2-..Ct method.
Update: The information regarding the effect of FANA ASOs in CLas infection in trees, tree growth, and yield is presented in this report.
In the spring of 2022, CLas infection decreased in trees 30 days after applying treatments. CLas infection was also lower in FANA ASOs and antibiotic-treated trees compared with trees treated with insecticide-only after 30 days. The lowest CLas infections were observed in LigA-FANA and antibiotic-treated trees 60 days after treatment. After 90 days, CLas infections were lowest among trees treated with Helicase-B-FANA or antibiotics.
In the fall of 2022, CLas infection declined in trees seven days after treatment with either LigA-FANA treated or antibiotics. CLas infection in was significantly reduced in trees treated with the FANA ASOs or antibiotic treatments 45 days after treatment as compared with control-FANA-treated trees. Similarly, at 90 days after treatments were applied, the lowest CLas infections were observed in LigA-FANA-treated or antibiotic-treated trees.
In spring 2023, CLas infection declined significantly in antibiotic-treated trees from 0 to 60 days following application. Treatment and sampling time significantly affected CLas infection, although the interaction between treatment and sampling date was not statistically significant. CLas infection in FANA-treated trees was not statistically different from insecticide-treated trees.
In the summer of 2023, a fourth injection of treatments was performed. Results will be added to the following report.
FANA ASOs Effect on Tree Health and Yield
Tree growth. Trees that received antibiotic treatments grew significantly wider canopies than control trees. Growth of trees treated with LigA-FANA was the second most increased as compared with the negative control. Similarly, monthly flushing patterns were affected by the interaction between treatments and sampling dates. Antibiotic or LigA-FANA treated trees had significantly more flush growth than the other treatments evaluated in June, July, and September of 2022. Similarly, flusing of trees treated with antibiotic or LigA-FANA also flushed more than trees from other treatments in February and May of 2023. However, there was no statistical effect of antibiotic treatment and sampling date on tree canopy height and circumference at the graft union.
Fruit yield. In 2022, fruit numbers from antibiotic-treated trees were 4.74 and 4.12.- times greater than from control or FANA ASOs treated trees. Among these treatments, trees treated with antibiotics had significantly higher mean fruit yield per tree (46 fruit/tree) when compared to the rest of the treatments, which averaged approximately 10-13 fruit/tree. Antibiotic-treated trees exhibited the largest fruit diameter, with an average of 19 cm per fruit, followed by LigA-FANA (17.53 cm/fruit), Helicase-B-FANA (17.35 cm/fruit), Control-FANA (16.99 cm/fruit), and insecticides-only (16.29 cm/fruit) treated trees; although these differences were slightly significant. The highest citrus fruit weight was produced by antibiotic-treated trees with a total of 156.07 lbs., followed by LigA-FANA (34.00 lbs.), insecticides-only (31.30 lbs.), Helicase-B-FANA (28.60 lbs.), and Control-FANA (28.30 lbs.) treated trees. The mean fruit weight per tree was significantly higher in antibiotic-treated trees, with 10.40 lbs. of fruit weight per tree compared with 1.88-2.43 lbs. of fruit weight produced per tree from the rest of the treatments.
Fruit drop. The month with the highest fruit drop was October 2022, when Hurricane Ian severely affected all trees. In November 2022, however, the fruit drop was minimal. Among the trees treated with FANA ASOs treatments, 254-287 fruits dropped. These treatments were the most severely affected by the hurricane and had the highest fruit drop in 2022. Trees treated with antibiotics had the lowest total fruit drop, followed by trees treated with insecticides only. Furthermore, antibiotic-treated trees had the lowest mean fruit drop per tree, only 11.26 fruit/tree. In comparison, FANA-treated trees had an average fruit drop ranging from 17-19 fruit/per tree. Trees treated with insecticides only had an average fruit drop per tree of 14 fruit/tree. However, these differences were only slightly significant. Antibiotic treatment also reduced the fruit drop percentage in treated trees, with the percentage of fruit dropped from the whole fruit harvested being significantly lower in antibiotic-treated trees (22% fruit drop) compared to the rest of the treatments (66-72% fruit drop).
Juice yield and quality analyses. Antibiotic-treated trees produced the highest juice weight, with a total of 78.47 lbs. produced in 2022, followed by LigA-FANA (16.27 lbs.), insecticide-only (15.19 lbs.), Helicase-B-FANA (13.65 lbs.), and Control-FANA (13.58 lbs.) treated trees. The mean juice weight per tree was significantly higher in antibiotic-treated trees, with 5.23 lbs. of juice per tree compared to 0.90-1.16 lbs. of juice produced by the rest of the treatments. Moreover, antibiotic-treated trees also had a significantly higher Brixº/acid ratio, averaging 15.79 Brix/Acid in the juice compared to 11.81-13.08 Brix/Acid achieved by the rest of the treatments. The juice from antibiotic-treated trees had the highest color score of 30.73, followed by Helicase-B-FANA (30.35 score), Scramble-FANA (30.34 score), LigA-FANA (30.20 score), and insecticides-only (30.13 score) treated trees. However, no significant differences were found in the juice color among treatments.
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 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 by psyllids was assessed by comparing the CLas titer in P1 (parental) and F1 (offspring) ACP caged on treated and untreated citrus trees.
Update: This report presents information regarding acquisition assays performed in the spring and fall of 2022 and 2023.
In the spring of 2022, we documented significant reduction in CLas acquisition by ACP adults feeding on FANA-treated trees, although it was not statistically different from that measured on trees treated with insecticides only. The lowest CLas aquisition by ACP occurred on antibiotic-treated trees. Similarly, in the spring of 2022, significantly fewer infected ACP adults were collected from trees treate with antibiotic than from other treatments evaluated. CLas acquisition by nymphs feeding on trees treated with FANA ASOs was significantly lower in the spring of 2023 than on control trees. Significantly lower CLas infection was observed in nymphs feeding on antibiotic-treated trees or those treated with FANA ASOs as compared with control trees. However, none of the treatments evaluated completely prevented CLas acquisition by psyllids.
In the fall of 2022, CLas acquisition by ACP adults was significantly reduced on trees treated with FANA ASOs or antibiotics as compared with controls. The lowest CLas infection was observed for ACP feeding on antibiotic-treated trees, followed by LigA-FANA, and Helicase-B-FANA. Additionally, significantly fewer infected ACP adults were collected on trees treated with antibiotic or LigA-FANA as compared with the other treatments evaluated. The trend observed in ACP offspring developing on trees was similar to that observed for adults. There was reduced CLas acquisition by nymphs feeding on trees treated with FANA ASOs. Significantly lower CLas titers were measured in ACP offspring feeding on trees treated with antibiotic or LigA-FANA as compared with trees treated with the FANA control. In addition, only 27% of all ACP offspring collected from trees treated with antibiotic were CLas infected as compared with 97 to 100% infection measured in the treatments.
In the spring of 2023, CLas acquisition was reduced for ACP adults feeding on trees treated with Helicase-B-FANA or antibiotics as compared with control trees. Additionally, significantly fewer infected ACP were collected on trees treated with antibiotic as compared compared with the rest of the treatments evaluated. Ants attacked ACP offspring populations during the experiment; thus, insufficient replicates were collected. Therefore, comparisons of CLas infections between treatments could not be performed, and differences were not significant.
Significantly fewer infected ACP adults were collected from trees treated with antibiotics or LigA-FANA as compared with the rest of the treatments evaluated. For ACP offspring, we recorded a reduciton in CLas infection in nymphs only on trees treated with antibiotic as compared with the control. In the summer of 2023, this experiment was replicated, and results will be added to the following report.
Inoculation assay.
A subsample of 10 ACP per treatment were collected from treated trees and then transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP adults were enclosed on plants for inoculation feeding for seven days. After that, ACP were collected for subsequent CLas detection. Thereafter, plants were sprayed with insecticides to eliminate any ACP progeny and were held for three months. Leaves were collected at 30, 60, and 90 days after inoculation to assess the development of CLas infection following ACP exposure. For this assay, each treatment was replicated five times on individual trees.
Update: This report presents information regarding the inoculation assay performed during the spring and fall of 2022 and the spring of 2023.
Thirty days after the inoculation access period, there were no differences in CLas infection rates among treatments. However, 60 days after incoulation, the lowest plant infection rates were observed in plants enclosed with ACP taken from antibiotic-treated plants, followed by plants enclosed with ACP taken from LigA-FANA-treated plants. A similar effect was observed 90 days after inoculation. However, the lowest plant infection rates were observed in plants enclosed with ACP removed from antibiotic-treated plants.
There was a statistically significant effect of treatment on the CLas inoculation rate for ACP collected from treated trees for the inoculation assay. ACP adults that fed on trees treated with LigA-FANA or Helicase-B-FANA infected other trees at statistically reduced rates (80 or 86%, respectively) than did psyllids that fed on insecticide-treated, control trees (98%). The lowest CLas inoculation rate was observed from ACP adults that fed on antibiotic-treated plants (64%).
In the summer of 2023, this experiment was replicated, and results will be added to the following report.
1. Please state project objectives and what work was done this quarter to address them: Objective 1: Using callose inhibitors to improve systemic uptake and reduce HLB symptoms.Objective 2: Maintaining water-saturated injection site: Sealed trunk injection ports that stay functional by avoiding woundresponses.Objective 3: Targeted root delivery.The field experiment initiated on sweet orange ~8 years old (Citrus x sinensis) trees. We selected and labeled treesaccording to 8 different treatments: (1) injected control (water), (2) Oxytetracycline (2 g per tree), (3) DDG (0.1 mM), (4)DDG (0.1mM) + Oxytetracycline (2 g per tree), (5) DDG (1mM), (6) 3AB (0.1mM), (7) 3AB (0.1mM) + Oxytetracycline (2 gper tree), (8) 3AB (1mM). Then we designed the appropriate map for the experiment. Before applying treatments,baseline stomatal conductance data and data related to trunk diameter, canopy volume and canopy density wascollected. Furthermore, six mature leaf samples per tree were collected to measure the CLas titer and quantify calloselevel of each tree. Then we employed the trunk injection (as described in (Vincent et al. 2022)). One week after applyingtreatments, we collected stomata conductance data to assess the treatment impact on leaf health. Leaf samples werecollected to measure the CLas titre and quantify the callose level of trees 1 and 2 weeks after treatments.We will evaluate the trees for callose level and Clas titre monthly. We will also measure the canopy volume and density,fruit drops via monthly data collection to determine the effect of injections on tree health. We will begin experiments for onobjectives 2 and 3. 2. Please state what work is anticipated for next quarter:We will evaluate the trees for callose level and Clas titre monthly. We will also measure the canopy volume and density,fruit drops via monthly data collection to determine the effect of injections on tree health. We will begin experiments foron objectives 2 and 3. 3. Please state budget status (underspend or overspend, and why):Underspend- since it took some time to initiate the work 4. Please show all potential commercialization products resulting from this research, and the status of each:We are still evaluating the effect of the callose inhibitor in the field trials
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