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 (completed), 2) by a greenhouse assay in seedling trees (completed), and 3) to compare OTC with other antibiotics’ efficacy against CLas on mature trees in the grove (in progress). This quarter (Q3), we completed all injections on mature trees in the field, which corresponds to objective 3. All phenotypic tree measurements were taken, such as trunk diameter, canopy area, and height of each tree. We also recorded the visual parameters of citrus diseases that were present, which included HLB ratings, canker ratings, canopy color, and canopy density. Flower and new shoot formation percentages per tree were tracked to determine later if the treatment was potentially influencing/altering these parameters.We tracked all the points of injections (injection hole), one by one, and found no phytotoxicity (in terms of oozing or cracking) as of now. In general, no phytotoxicity was found on the last visit to the trial (dated 8.13.2024). Leaf samples were taken (one month after injection) and will be processed for CLas titers to compare with time zero. We will continue to sample other time points.Greenhouse phytotoxicity data (objective 2), collected during Q1 and Q2, were presented at the American Phytopathological Society’s annual Plant Health conference in Memphis, Tennessee, in July 2024. Per CRDF’s advisory request, we included additional treatment in our ongoing field trial. On August 12, we injected treatment consisting of kasugamycin +OTC (1/2 dose each; 5500 ppm) mixed together in the same FlexInjec injector (2 FlexInject per tree; 50 ml per FlexInject), as well as kasugamycin alone (1/2 dose; 5500 ppm), and OTC alone (1/2 dose; 5500 ppm) into mature Hamlin trees at the SWFREC trial site. 2. Please state what work is anticipated for next quarter:We will continue to sample leaves for CLas titers and process them to determine if a particular treatment significantly reduces CLas (using qPCR). Phytotoxicity will continue to be tracked every week. Visual and disease ratings, as well as canker incidence, will be tracked on a monthly basis. Flowers and shoots will also be counted together, along with the visual ratings. 3. Please state budget status (underspend or overspend, and why): We are paying the salary of one of the students who is working 60% on this project. The project is underspending as of now, but we anticipate being back on track soon. The reason that we are currently under budget is that a few of the most expensive antibiotics involved in the trial did not need to be purchased initially, as we had them on our hands to start. In the next couple of months, we will buy more of these antibiotics and use these funds.
1. Please state project objectives and what work was done this quarter to address them: This is a first year, third quarter report. 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 objectives 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). Based on CLas-citrus hairy root effiacy assays (Irigoyen et al., 2020), we shortlisted five new active ingredients for which synthesis and sourcing in sufficient amounts needed for the field trials were completed. All quality control assays (solubility, bioactivity, dose determination) were completed. We aimed to dissolve all compounds in water to avoid using any other solvents on the trees. Three out of five compounds were fully soluble in water. Two were partially soluble. All the molecules were shipped to Dr. Albrecht lab. Trunk injections were conducted on ~7-year-old HLB-affected sweet orange (Valencia/US-897) trees at the UF/SWFREC, as per the experimental design. 2. Please state what work is anticipated for next quarter: In the next quarter, trees will be monitored for potential unintended injury or toxicity issues. General grove care and management activities will continue. Any changes in horticultural traits will be also monitored. 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 year 3 (July 1, 2023 – May 30, 2024) 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 11,030 samples were run during the year. All samples were plant samples, mostly from research trials. To date, a total of 70,600 samples have been run for the 3 years that have been funded to date. The number of samples for year 3 represents a substantial reduction from the number of samples received in the past (averaging about 25,000/yr). This could be due to the state of the industry or to the perception that PCR analysis does not provide the resolution necessary to discriminate between treatments. As an attempt to provide more applicable data to determine if HLB mitigation treatments were working, SGDL is in the process of adapting and validating an assay developed by Dr. Ping Duan (USDA) to indirectly measure activity of the CLas bacterium. This assay, if validated, will be offered in the future as a means of evalutating treatments (e.g. OTC injection) to see if CLas metabolism and growth is being affected.2. Please state what work is anticipated for next quarter:SGDL will continue the testing and validation of the assay developed by Dr. Duan to determine it’s potential for use as a method to evaluate the effectiveness of HLB mitigation strategies. If it proves to be an effective method, this assay as well as the convential qPCR and qPCR with copy number determination will be offered to growers and researchers going forward.3. Please state budget status (underspend or overspend, and why):Due to the reduction in the number of samples and cofunding by one of the private companies that is utilizing the laboratory, the spending was well under the not to exceed cap that CRDF placed on the spending. Although the cost per sample went up (due to the fixed cost of the technicians being spread out over a smaller number of samples), the amount funded by CRDF was approximately 62% of the not to exceed cap.
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.
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