1. Please state project objectives and what work was done this quarter to address them:Objectives of this project are to determine the phytotoxicity of OTC and other potential partner antibiotics 1) by a citrus leaf assay in the lab, 2) by a greenhouse assay in seedling trees, and 3) to compare OTC with other antibiotics’ efficacy against CLas on mature trees in the grove. This quarter (Q1), we purchased most of the proposed antibiotics, except zhongshengmycin, which was not available from the manufacturers in China. Therefore, we replaced this antibiotic with another antibiotic, nalidixic acid, which was shown to be effective on various gram-negative bacteria. We have successfully completed tasks pertaining to Objective 1, where we screened all experimental antibiotics and imidacloprid, along with OTC to evaluate their phytotoxicity to citrus. In these lab experiments, we used a detached-leaf assay to assess ‘exaggerated’ concentrations quickly and to document any potential phytotoxicity. In repeated (at least three times) experiments, only oxolinic acid and nalidixic acid at 600 ppm caused light phytotoxicity, including petiole and midvein necrosis; whereas other antibiotics, namely OTC, kasugamycin, streptomycin, gentamicin and validamycin A, all at 600 ppm, and an insecticide imidacloprid at 800 ppm, did not show any phytotoxicity. Further evaluation of these two phytotoxic antibiotics revealed that the solvent used for dissolving them per manufacturer’s recommendations (NaOH 0.01M) was a potential culprit behind this phytotoxicity. Therefore, we concluded that none of the tested antibiotics and imidacloprid were phytotoxic to citrus leaves at these exaggerated concentrations.Next, we conducted similar phytotoxicity assays, this time on greenhouse-grown healthy Valencia seedlings in the greenhouse (Objective 2). Here, all the treatments we used in the leaf assays from above were used in 10X more exaggerated concentrations (i.e., 6000 ppm) per tree and additional 3000 ppm for gentamicin after seeing high phytotoxicity in that treatment in the first repetition. We used the drill-based trunk injection (Chemjet) technique to deliver these treatments from the seedling trunk. All treatments were taken up by the trees without any issues except for the imidacloprid (8000 ppm), which was in a formulation that was not recommended for trunk injection. In the subsequent trial, we replaced this formulation with the brand (Xytect) that was formulated specifically for the trunk injection of forest and ornamental trees. No uptake issue for this new formulation was observed. Regarding phytotoxicity in the greenhouse, at least one out of five trees per treatment in repeated trials showed some level of phytotoxicity–particularly in the drill site on the trunk–including on the trees that were injected with water. This suggested that drilling a hole in the trunk of young (1.5-2 years old) seedlings is very damaging; 10-70% of the drill sites showed signs of oozing, regardless of the treatment. Nevertheless, only a small number of trees showed some phytotoxicity on the leaves, including dieback, leaf discoloration, defoliation, and leaf curling. Among them, gentamicin was the most phytotoxic to citrus at 6000 ppm, as three out of five treated plants showed severe dieback, and eventually, those plants died. However, gentamicin at 3000 ppm was not as phytotoxic, and showed only leaf curling on 60% of the treated plants, with none of the treated plants dying. Much milder symptoms, such as leaf mottling, chlorosis, and vein yellowing, were observed in only a few plants treated with kasugamycin and streptomycin at 6000 ppm. None of the other antibiotics showed severe phytotoxicity, but only occasional leaf chlorosis or leaf curling, which did not affect the trees’ overall health. Additionally, Xytect–injectable imidacloprid–caused severe phytotoxicity, including oozing on the treatment sites (40%), dieback of the plants (40%), leaf discoloration (40%), defoliation (60%) and leaf curling (80%). 2. Please state what work is anticipated for next quarter:1) Greenhouse assays will be repeated one or two more times to have better insight into the phytotoxicity of these treatments. 2) We will finalize the phytotoxicity experiments, analyze data, and make decisions on what treatment and in what concentration they can be injected into citrus trees before moving onto field experiments. 3) The grove site with mature Valencia and Hamlin trees was identified at the SWFREC experimental grove. We have started taking measurements and disease ratings of trees (Time zero; T0), as well as labeled them to prepare for the upcoming trunk injection trials (Objective 3). Trees are flushing now, and we believe that at the mid-March or end of March, we will be injecting those trees. 3. Please state budget status (underspend or overspend, and why): The budget is spent as expected.
1. Please state project objectives and what work was done this quarter to address them: This is a new 3-year project. The goal is to evaluate new anti-CLas compounds in Florida singly and in combination with OTC to determine their effects on HLB tree health, yield, and injury. The study objective are:Objective 1: Pre-screening and quality control (purity, solubility, and bioactivity) of selected anti-CLas leads. (Initiated). Objective 2: Multi-year field evaluation of new chemicals, OTC, and combinations by trunk injections. (Initiated). We identified five new active ingredients for which synthesis and sourcing in sufficient amounts needed for the field trials were completed. Quality control experiments were initiated to ensure batch purity, solubility, and bioactivity, as determined using the CLas-citrus hairy root bioassays per our established methods (Irigoyen et al., 2020; Mandadi et al., 2020). Site and tree selection for the field trials in Florida have been initiated. The field trial will likely use 6-8-year-old HLB-affected sweet orange (Valencia) trees, either at the UF/SWFREC or a commercially operated citrus grove that has not already treated them with OTC in the prior year. 2. Please state what work is anticipated for next quarter: In the next quarter, we will complete all quality controls and initiate trunk injections in Florida. The plan is to inject the trees in the Spring (April or May 2024) after the new flush has hardened to ensure optimal delivery of the injected material. General grove management will follow practices determined by the grove operators. Baseline horticultural measurements (tree height, canopy spread, trunk diameter) will be conducted before injections, and then annually. 3. Please state budget status (underspend or overspend, and why): Ontrack with spending. 4. Please show all potential commercialization products resulting from this research, and the status of each: By the end of the study, we anticipate identifying one or more new active ingredients that improve the HLB-affected tree health and fruit yield. The new chemicals could be developed as commercial products singly or combined with OTC for HLB management.
1. Please state project objectives and what work was done this quarter to address them: This report is for the 3rd quarter of year 2 of Project 21-002C – Continued Support for the Southern Gardens Diagnostic Laboratory. This project provides HLB testing for researchers, growers and homeowners. A total of 9494 samples were run during the quarter. All samples were plant samples, mostly from research trials. To date, a total of 49,606 samples have been run for the 7 quarters that have been funded to date. This is slightly under the budgeted amount of 50,314 samples through 7 quarters. 2. Please state what work is anticipated for next quarter: Based on expected trends, it is expected that we will be slightly over the budgeted amount of 57,500 samples for the 2 years. 3. Please state budget status (underspend or overspend, and why): It is expected that the number of samples will slightly exceed the budgeted amount. If so, the budget will be adjusted to cover the cost of the overruns (expendibles only, labor will remain the same).
1. Please state project objectives and what work was done this quarter to address them: This report is for the 4th quarter of year 2 of Project 21-002C – Continued Support for the Southern Gardens Diagnostic Laboratory. This project provides HLB testing for researchers, growers and homeowners. A total of 10,058 samples were run during the quarter. All samples were plant samples, mostly from research trials. To date, a total of 59,664 samples have been run for the 8 quarters that have been funded to date. This is slightly overthe budgeted amount of 57,500 samples through 7 quarters. 2. Please state what work is anticipated for next quarter: This is the final report for the currently funded project. However, the lab will continue to operate on a year by year basis at the discretion of the CRDF board 3. Please state budget status (underspend or overspend, and why): A total of 59,664 samples were run for the two year period. This is 2,164 samples higher than the budgeted amount. As has been done in the past, we will bill CRDF for the cost of the expendibles for the overage. The labor and overhead will remain unchanged.
1. Please state project objectives and what work was done this quarter to address them: This project aims to test SAMP efficacy in the field and greenhouse on important citrus varieties in Florida and California. A. Foliar spray of SAMP before exposure to non choice feeding of ACP reduced the infection rate on sweet orange and Lisbon lemon trees in the greenhouse trials. After 10 months of infection, the SAMP-treated trees have a significantly lower infection rate (23%) compared to the mock treated plants (100%). Much less ACP eggs were hatched to nymphs after the SAMP treatment. Similar results were observed on Tango mandarin on Carrizo rootstock. We are at the end of this experiment, and are getting the last measurement for publication. This study suggests that SAMP also has negative impact on ACP transmission and development. B. We have established collaboration with Dr. Megan Dewdney in UF for all the field trials in FL. A large scale field test on 240 young Hamlin trees with 4 different treatments, 60 trees per treatment, was laid out in a completely randomized design. The treatments are 1. Untreated control, 2. Treatment in the nursery 1 week prior to planting and field applications (10 µM in 158 ml/tree) every 2 months, 3. No nursery treatment with field applications every 2 months starting 10 days post-planting, 4. The injection treatment with Invaio simple injection device every 6 months. 23 months after tree planting in the field, the trees are showing declining symptoms caused by HLB. In four treatments, the treatment 2 which is with nursery spray followed by foliar spray every two months has the lowest declining rate of trees. The pandemic also caused a few treatment delays which may reduce the significant of SAMP treatment. Overall, we observed that the treatment can reduce the declining rate. However, we also found that injection on young trees can cause damage sometimes which could also impact on growth of the trees. Recent Hurricane hit in Florida has caused significant damage on many trees, we have to extend this experiment for at least 6-9 months to allow the trees to recover.We will continue to monitor the tree growth and to measure the CLas titer during the next 12-18 months. C. A second field test on infected 4-year old ‘Hamlin’ sweet orange bearing trees was also initiated. We have located grove in the Lake Wales region. The trial involves a total of 125 bearing trees and was laid out as a randomized complete block design with five blocks and five replicates per block. The treatments in this trial were an untreated control and bimonthly foliar application. The foliar spray treatment volume one treatment is 25 gal of 10 µM (60 mg/L) SAMP solution. Three additional treatments from an additional funding source are to look at the peptide as various injection treatments. Due to the pandemic, Dr. Megan’s group didn’t manage to collect the fruits and measure the fruit yield before the farmers collected everything, so we had to wait for the measurement for this year. All the treatment and measurement is still on going. THe Hurricane also caused significant damage on these bearing trees. More variable results will be expected.2. Please state what work is anticipated for next quarter: Test I: Field Test I- SAMP efficacy test on 240 newly planted ‘Hamlin’ sweet orange treesWe will continue to monitor the tree growth, to measure the size, disease levels and CLas titers in trees and ACP to evaluate the effectiveness of SAMP and better delivery methods. Test II: Field Test II- SAMP efficacy test on 125 infected 4-year old ‘Hamlin’ sweet orange trees.The second field trial was set up to test if SAMP can remediate an HLB-affected young citrus grove. This experiment is ongoing in the Ridge region of Florida on the deep sandy soils. The trial began in August 2021, and we are making bi-monthly foliar applications and continuing to monitor tree growth, disease severity, and CLas titers in trees and ACP. Considering the hurricane impact last year, we expect to monitor the trial until mid 2024 to get enough data.3. Please state budget status (underspend or overspend, and why):This project is in combination with the greenhouse and field trials supported by other funding source USDA NIFA. The CRDF buget was fully spent on purchasing large quantity of synthetic SAMP. All the other cost, such as the personnel and consumable cost, were supported by USDA NIFA (which is also at the end of the grant). We are in urgent need of new funding support to continue this SAMP project.
1. Please state project objectives and what work was done this quarter to address them: Please state project objectives and what work was done this quarter to address them: This report is for the first quarter of year 2 of Project 21-002C – Continued Support for the Southern Gardens Diagnostic Laboratory. This project provides HLB testing for resarchers, growers and homeowners. A total of 6,016 samples were run during this 3 month-period. All samples were plant samples, mostly from large scale research trials. To date 35,232 samples have been run, which is under the budgeted amount of 35,938 for 5 quarters of work. 2. Please state what work is anticipated for next quarter:We appear to be on track or possibly slightly under what was budgeted. Whether or not we stay at this level most likely will depend on how many samples growers and researcher submit related to trials with OTC. 3. Please state budget status (underspend or overspend, and why): To date, the project is essentially on budget. A total of 35,232 samples were run versus a budget of 35,938. As has been done in previous years, the number of samples run at the end of year 2 will be reconciled against the budgeted amount and the last payment will be adjusted up or down as needed.
1. Please state project objectives and what work was done this quarter to address them: Please state project objectives and what work was done this quarter to address them: This report is for the second quarter of year 2 of Project 21-002C – Continued Support for the Southern Gardens Diagnostic Laboratory. This project provides HLB testing for resarchers, growers and homeowners. A total of 4,880 samples were run during this 3 month-period. All samples were plant samples, mostly from large scale research trials. To date 40,112 samples have been run, which is under the budgeted amount of 43,126 for 6 quarters of work. 2. Please state what work is anticipated for next quarter:We appear to be on track or possibly slightly under what was budgeted. Whether or not we stay at this level most likely will depend on how many samples growers and researcher submit related to trials with OTC. 3. Please state budget status (underspend or overspend, and why): To date, the project is essentially on budget. A total of 40,112 samples were run versus a budget of 43,126. As has been done in previous years, the number of samples run at the end of year 2 will be reconciled against the budgeted amount and the last payment will be adjusted up or down as needed.
1. Please state project objectives and what work was done this quarter to address them: Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Field trials 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. The following treatments were as trunk injetions in May 2022: FANA ASOs complementary to CLas NAD-dependent DNA ligase gene (LigA) and CLas DNA B- helicase gene, nontarget control FANA ASO, oxytetracycline (Fireline). Injected treatments were compared with an insecticide-only control treatments. Each treatment was applied 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. All FANAs dosages were 625 ppm per tree. To monitor the effect of treatments on trees CLas titers as assessed 0, 2, 7, 30, 60 and 90 days post-treatment by qRT-PCR analysis. A second replicate of the treatements in this experiment was applied in September 2022. Fruit were harvested to assess fruit quality and yield in November 2022. Results: CLas titers were initially similar across treatments, with all trees infected. CLas titers declined in FANA-treated and oxytetracycline -treated trees 30d after treatments were applied. CLas titers were lowest in trees treated with LigA-FANA or oxytetracycline at 30d and 60d post treatment. Trees treated with helicase-B-FANA also exhibited a reduction in CLas titer comparable to oxytetracycline treaments on 30d and 90d post-treatment. Overall, trees injected with ligA-FANA, helicase-B-FANA, or oxytetracycline had significantly lower CLas titers at 30, 60, and 90 days post treatment compared to the control treatments (insecticide or non-target FANA). Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Acquisition assay.Acquisition assays were replicated this quarter. Samples are currently being processed. Inoculation assay. A subsample of 10 ACP per treatment were collected from treated trees was transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP were enclosed on plants for inoculation feeding for seven days. Inoculated plants were sampled at 30, 60, and 90 days after inoculationsto assess the development of CLas infection following ACP exposure. For this assay, each treatment was replicated five times on individual trees. Results: Forty percent of trees exposed to ACP from LigA-FANA and Helicase-B FANA treatments were infected with CLas 30 days after inoculation, compared with 20% trees exposed to ACP from insecticide treatments. Sixty days after inoculation, 40-80% of trees were infected in the LigA-FANA, B-Helicase-FANA, scramble-FANA, and insecticide treatments Ninety days post-inoculation, a similar pattern was observed; however, plants exposed to ACP from insecticide-treated trees were 100% positive for CLas infection. Plants inoculated by ACP from oxytetracycline-treated trees were the only treatment to remain CLas-free after 30, 60, and 90 days post-inoculation.These initial results suggest that ACP collected from FANA-treated trees are less likely to inoculate healthy citrus. 2. Please state what work is anticipated for next quarter: Objective 1:Samples from the second round of treatment applications through day 60 will be processed during next quarter. Tree health and fruit quality/yield data will be reported next quarter. Objective 2: Acquisition assays were replicated and results will be reported next quarter. 3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments, which are the largest portion of the budget, have been purchased for the experiments. Remaining budget will be spent on field use charges and for payroll to complete sample analysis, ACP assays and colony maintenance. Microinjectors needing replacement will be purchased next quarter for injections in April.
. Please state project objectives and what work was done this quarter to address them: Objective 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. 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 to be complementary to two CLas essential genes. 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 May through August and consist of five treatments: untreated control (insecticide-only), oxytetracycline control (1.56 g of Fireline per tree), Scramble Control-FANA, CLas L-FANA, and CLas B H-FANA. All FANAs dosages were 625 ppm per tree. Prior to treatment, four leaves were removed from each tree, two from each side of the apex of the tree 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 leaves samples were removed from the same branches as the T0 samples after 2, 7, 30, 60 and 90 days. The post-treatment CLas titer (TF) were calculated by qRT-PCR analysis, at 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. Results: In this report, information regarding the first application of treatments is presented. At T0, CLas titters in trees were similar across treatments. Two and seven days after treatment were applied, CLas titers in trees fluctuated; and higher CLas titers were observed in FANA treated trees compared to antibiotic and insecticide treated trees. No differences were found in CLas titers among treatments 30 days after treatments were applied. However, CLas titers were lower in trees treated with L-FANA and titers were comparable with those trees treated with antibiotics 60 days after treatments were applied. Overall, trunk-injections of L-FANA and antibiotic treatments significantly reduced CLas titers in trees as compared with insecticide only or control FANA treatments. A second replicate of this experiment will be performed in September of 2022 to confirm our observations. Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Our working hypothesis is that CLas will be specifically inhibited in psyllids by using CLas-specific FANAs, resulting in a reduction in Las acquisition and transmission by ACP in a field setting. Acquisition assay. Field assays with psyllids were conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP in June 2022. Psyllid nymphs, who develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from FANA-treated infected citrus trees were 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 acquisition of CLas was assessed by comparing the CLas titer in P1 and F1 ACPs caged on treated and untreated citrus trees. Results: The results from a bioassay assessing acquisition of CLas following FANA treatments showed that ACP adults (P1) and offspring (F1) were able to acquire CLas across treatments 7 and 15 days following treatments, respectively. The lowest CLas titers were observed in P1 and F1 ACP feeding on antibiotic-treated trees; followed by L -FANA and H-FANA treatments. The highest CLas titers were observed on ACP adults and offspring feeding on Scramble-FANA and insecticides only treated trees. Overall, L -FANA, H-FANA, and antibiotic treatments reduced CLas acquisition by ACP as compared with scramble-FANA and insecticide treatments. A second replicate of this experiment will be performed in September 2022. 2. Please state what work is anticipated for next quarter: Objective 1:The second treatment application is planned for this period. Samples from the second round of treatment applications through day 30 will be processed during quarter #3. Tree health and fruit quality/yield will be assessed in quarter # 4. Objective 2: Acquisition assays will be replicated and partial results with P1 and F1 adults will be reported in quarter #4. Inoculation assays will also be completed in quarter #4, with samples collected for CLas detection. If rootstocks are used for inoculation, plants will be held for development of CLas infection and symptom development. 3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments, which are the largest portion of the budget, have been purchased for the experiments. Remaining budget will be spent for sample analysis, ACP assays and colony maintenance, research plots, and personnel. Microinjectors needing replacement will be purchased next quarter.
1. Please state project objectives and what work was done this quarter to address them: This report is for the third quarter of year 1 of Project 21-002C – Continued Support for the Southern Gardens Diagnostic Laboratory. This project provides HLB testing for resarchers, growers and homeowners. A total of 9,118 samples were run during this 3 month-period. All samples were plant samples, mostly from large scale research trials. To date 24,704 samples have been run. It is expected that we will be close to the budgeted amount of 28,750 samples for the end of year 1. During this quarter, the lab experienced some technical difficulties which were resolved by working with Dr. Ozgur Batuman’s lab by running comparison samples. The problem was traced to using EDTA in the elution buffer. Although the elution methodology has been the same for years, for some unknown reason it became an issue. Suppliers of the affected samples were notified and the problem was corrected. It is suspected that the supplier of the master mix changed their formulation which resulted in the inhibition by EDTA (which has been reported in the literature as a potential inhibitor of PCR reactions). The lab operations are back on track at this time. 2. Please state what work is anticipated for next quarter: It is expected that the number of samples will slow down for the last quarter of year 1 as this is the time of year when symptoms are not evident and the titer of the bacterium in the plant declines. 3. Please state budget status (underspend or overspend, and why): Based on current projections, it is expected that the lab will be on target for the budgeted amount of samples for year 1 (budget=28,750)
1. Please state project objectives and what work was done this quarter to address them: This report is for the fourth quarter of year 1 of Project 21-002C – Continued Support for the Southern Gardens Diagnostic Laboratory. This project provides HLB testing for resarchers, growers and homeowners. A total of 4,512 samples were run during this 3 month-period. All samples were plant samples, mostly from large scale research trials. To date 29,216 samples have been run, which is slightly over the budgeted amount of 28,750 samples for year 1. 2. Please state what work is anticipated for next quarter: It is expected that sample volume will be slightly higher in quarters 1 and 2 of year 2 due to CRAFT projects which are just starting to come on line. However this remains to be seen. 3. Please state budget status (underspend or overspend, and why): For year 1, the project is essentially on budget. A total of 29,216 samples were run versus a budget of 28,750. As has been done in previous years, the number of samples run at the end of year 2 will be reconciled against the budgeted amount and the last payment will be adjusted up or down as needed.
1. Please state project objectives and what work was done this quarter to address them: The overall objective in this proposal is to explore whether FANA oligonucleotides, which are synthetic RNA-based synthetic molecules that mimic DNA and inhibit bacterial gene expression can be used to mitigate CLas in infected trees.The first objective of this project is to s screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Treatments were applied to 10-year-old, CLas-infected Valencia trees of a standard size and CLas titer. FANA ASOs complementary to two CLas essential genes F(ANA-L and FANA-H), a scramble sequence control (FANA-SC), oxytetracycline (Fireline) and insecticide-only treatments are were applied to 15 trees in 1-acre plots, replicated five times in a randomized complete block design. Treatments are applied using microinjection of dosages determined in our previous greenhouse assays.Prior to treatment 1 (T1), four leaves were removed from each tree, two from each side of the apex of the tree and two from each side of th20%e base of the canopy, to determine initial CLas titer (T0). PCR of these leaves and leaves collected post treatment was conducted during Q2 of this project to monitor the effect of the FANA ASOs on the CLas titer of each tree. Pathogen titers in trees before treatment was determined using a quantitative real-time PCR (qPCR) assay. Trees were uniformly infected with mean Ct (qPCR cycle threshold) values ranging from 24 to 25. Oxytetracyline (Fireline) was initially the most effective in preventing increases in CLas titers, followed by FANA L. CLas titers increased 2 d following all other treatment applications. The percent increase in CLas Ct value was greatest in the FANA-H and insecticide-only treatments. Sample processing is still underway to assess the effect of treatments on tree infections7, 30, and 60 d after treatment applications. We expect that the treatments will take time to move throughout the trees and reduce CLas infection. The second objective is to evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Single females from uninfected laboratory cultures were caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated ten times on individual trees. Due insufficient survival of ACP in March 2022, this experiment was carried out in May-June2022 and is still underway. Adult psyllids have been collected from trees and are being process to assess acquisition. F1 adults will be collected in late June to assess acquisition during nymph development and to use in inoculation assays. 2. Please state what work is anticipated for next quarter: Objective 1:Samples from first round of treatment applications through day 60 will be processed during quarter 3. A second round of treatments will be applied at the end of quarter 3. Objective 2: Results of acquisition assay #1 with P1 and F1 adults will be next quarter. Inoculation assays will also be completed in Q3, with samples collected for CLas detection. If rootstocks are used for inoculation, plants will be held for development of CLas infection and symptom development. 3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments, which are the largest portion of the budget, have been purchased for the experiments. Micorinjectors and research plot charges have also been spent. Remaining budget will be spent to process samples, ACP assays, and on personnel.
The previous report noted that trees on Swingle citrumelo and Carrizo rootstock were located and ordered for delivery in January when they would be used to repeat the HLB-Tylenchulus semipenetrans trial. Also that Kuharske plants were added to seedlings for a rootstock trial to evaluate tolerance to Radopholus similis in traditional rootstocks and newer releases, with inoculation to occur also in January. However, our greenhouse was disassembled without notice during the first week of January. The walls were open to the outside and a job that was to be finished in few days was not completed for more than three months. We moved all the citrus material to our soil processing laboratory to prevent infestation by Asian citrus psyllid. Eventually, we were forced to move our nematode cultures as well, due to high ambient temperature when the cooling system was removed. As a consequence, inoculation of these experiments was not possible.
The commercial and potential rootstock seedlings to be screened for tolerance to burrowing nematode were moved from the laboratory to the rebuilt greenhouse in early April and inoculated with Radopholus similis in late May. A new screenhouse was constructed and the Swingle citrumelo and Carrizo rootstock Valencia trees were moved from the laboratory, pruned to initiate new root and shoot growth. They will be inoculated with citrus nematode and graft-inoculated with CLas in the first week of June. A trial comparing the effects of burrowing nematode and CLas on rootstocks susceptible and resistant to burrowing nematode was terminated. Neither pathogen significantly affected the shoot weight of either rootstock; however burrowing nematode increased the root weights (P=0.001) and the effects differed for the two rootstocks. On the susceptible Carrizo citrange the nematode increased root weight by 12% (17.2g vs 19.2g) and on the resistant Kuharske Carrizo by 24% (54.1g vs 66.8g). Unlike the adverse effect of CLas on citrus nematode populations (July 2021 report), nearly twice as many burrowing nematodes per gram of root occurred on Carrizo seedlings infected by CLas than on seedlings only infected with burrowing nematode (65.9 vs 34.3), but the effect was not significant. There were no burrowing nematodes recovered from the resistant rootstock Kuharske. The repeat experiment is ongoing. The project ended on 30 April. Covid-related delays related in the NCE request and the unannounced 3-month closure of our greenhouse have delayed the completion of several experiments that include a repeat of the trials evaluating interactions between CLas and burrowing nematode described above and citrus nematode in which the nematodes increased the virulence of Las in citrus plants, whereas Las-infected plants were less suited to nematode population growth than non-infected plants. These and the trial screening rootstock resistance to the burrowing nematode are ongoing and will be completed and reported in a supplemental report in 2022.
1. Please state project objectives and what work was done this quarter to address them:The overall objective in this proposal is to explore whether FANA oligonucleotides, which are synthetic RNA-based synthetic molecules that mimic DNA and inhibit bacterial gene expression can be used to mitigate CLas in infected trees.The first objective of this project is to s 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. We have initiated field trials with laboratory-vetted FANAs in a research grove at the UF Citrus Research and Education Center in March 2022. Treatments are applied to 10-year-old, CLas-infected Valencia trees of a standard size and CLas titer. FANA ASOs complementary to two CLas essential genes and a scramble sequence control are synthesized by AUM LifeTech, initiated in December 2021. Antibiotic application (Fireline) and insecticide-only treatments are applied to trees as control treatments. Each treatment is applied to 15 trees in 1-acre plots, replicated five times in a randomized complete block design. Treatments are applied using microinjection of dosages determined in our previous greenhouse assays.Prior to treatment 1 (T1), four leaves were removed from each tree, two from each side of the apex of the tree and two from each side of the base of the canopy, for initial titer (T0) using quantitative real-time polymerase chain reaction (qPCR) assays. PSamples are removed from the same branches as the T0 samples after 2, 7, 30, 60 and 90 days. The second objective is to evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Field assays with psyllids are conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP. Single females from uninfected laboratory cultures are caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment is replicated ten times on individual trees. This experiment was initiated in March 2021, as outlined in our proposal timeline. The effect of FANA treatments on acquisition of CLas will be assessed by comparing the CLas titer in ACP caged on citrus trees before and after treatments and across time. 2. Please state what work is anticipated for next quarter: Objective 1: PCR of these leaves and leaves collected post treatment will be conducted during Q2 of this project to monitor the effect of the FANA ASOs on the CLas titer of each tree. Objective 2: Initial results after 1 treatment will be reported next quarter. 3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments, which are the largest portion of the budget, have been purchased for the experiments. Personnel spending has been low to date, as December 21- January 22 were primarily spent designing and synthesizng FANA molecules for the experiment.
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