1. Please state project objectives and what work was done this quarter to address them: Objective. To determine the influence of compost during the first three years of tree establishment on growth, productivity, and root and soil health of citrus trees on rootstocks with different vigor-inducing capacity. Annother round of compost was applied in May at the usual rate.Leaf flush ratings were conducted in May, June, and July.Soil moisture was measured in May, June, and July.Fibrous root respiration was measured in June.Fibrous root length and weight were determined to calculate the specific root length. We continue to organize and analyze the data. 2. Please state what work is anticipated for next quarter:We will collect soil samples for macro and micro nutrient analysis and determination of other soil traits (organic matter, CEC, pH, etc.). We will collect leaves for micro and macro nutrient analysis.We will continue with flush ratings and soil moisture determination.We will continue with data analysis. 3. Please state budget status (underspend or overspend, and why): Approximately 45% of funds have been spent, which is mostly in accordance with the timeline.
HLB is known to make citrus roots more susceptible to Phytophthora root rot. It also reduces the efficacy of chemical management of Phytophthora root rot, creating a difficult management scenario. Current Phytophthora management recommendations are based on pre-HLB work done in the 1980s. These three conditions raise the question of whether yield improvement from Phytophthora management is enough to pay for the management costs themselves. The goal of this project is to develop new soil propagule density managment thresholds and recommendations for chemical management of Phytophthora root rot based on ecomonic analysis of yield responses in different soil conditions. Objective 1) Determine if labelled Phytophthora management maintains efficacy in the field on HLB-affected trees for reducing fibrous root loss and improving yield.The spring, early summer and late summer treatments have been put out in the trial plots. Summer Phytophthora soil samples and propagule counts have been taken.In the next quarter we expect to put the last treatment for the year on the fall root flush and take a fall Phytophthora propagule count from all plots. As an expansion of the original scope, we will also rate the blocks for brown rot. This rating will be both a field assessment and a lab based one by taking a weighed subsample of fruit for assessment of brown rot development under optimal conditions in the lab. The weight of fruit sampled for brown rot will be recorded and considered when determining total yield at harvest. Depending on fruit maturity we may start harvesting Hamlins, but this will likely happen in the following quarter. Objective 2) Determine benefit-cost thresholds for Phytophthora treatment on HLB-affected treesAs planned in the proposal, this objective awaits this years yield results to begin calculating benefit-cost based on a combination of change in yield from the previous year and comparison among treatments within blocks.
Progress report for the third quarter of the 2020/2021 project year
The purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruits. The objectives of the project are to:
1. Determine optimal nutrient concentrations in roots and leaves for multiple grapefruit and orange varieties.
2. Compare and contrast fertigation, soil, and foliar fertilization to identify best application method for uptake of nutrients into both underground and aboveground components.
3. Investigate the relationship between root and leaf nutrient contents to tree health, yield, and fruit quality as well as bacteria titer.
4. Generate updated and new guidelines for optimal nutrient contents for roots and leaves for HLB-affected trees.
Progress to date:
The project is being conducted at three sites: Citrus Research and Education Center (CREC), Southern Gardens Citrus near Clewiston, FL and Indian River Research and Education Center (IRREC). Data collection continued during this quarter on root scanning, canopy size determinations and soil sampling on the central Ridge and southwest Flatwoods along with fertilizer treatment applications. All sampling for the third quarter of the third year of the project are complete.
The aim of the study at the UF/IFAS IRREC in Fort Pierce, FL was to relate nutrient concentrations in grapefruit leaves and roots to indicators of tree health and root growth. The research was conducted on flatwoods soils in a randomized complete block design field study on Ruby Red grapefruit. Micronutrients (B, Fe, Mn and Zn) were applied using three different concentrations (1x, 2x, and 4x current UF/IFAS guidelines) in the form of either dry granular water-soluble fertilizer, controlled-release fertilizer, or liquid fertilizer. A total of 600 trees divided in 40 experimental units were employed. We collected leaf and root nutrient concentrations, canopy volume and tree height twice a year. Mini-rhizotrons were installed at the beginning of the experiment and root images were taken four times a year. Results showed increased micronutrient concentrations in the leaves among all treatments. There were no significant differences in tree height, canopy volume, root length, and root diameter. Yield and fruit quality data were collected in February and are being analyzed.
Graduate student Lukas Hallman completed his MS program under the supervision of Dr. Rossi. Portions of his work will be published in peer reviewed journals. PI Kadyampakeni and graduate student Tanyaradzwa Chinyukwi published a review article in the Journal of Plant Nutrition.
Plans for Next Quarter
The team will continue with fertilizer treatments and data collection including HLB rating assessments, canopy size and root growth measurements and reporting on the progress of the project. Former graduate student Tanyaradzwa Chinyukwi and graduate student Lukas Hallman will present their works at the Florida State Horticultural Society Meeting in Daytona Beach, FL in September 2021. Lukas will also present his work at the American Society of Horticultural Science in Denver, CO in August 2021.
Dr. Ferrarezi departed from IRREC this quarter and his tasks will be completed by co-PI Dr. Alan Wright to make sure all project milestones are completed as planned.
Objective 1. To illustrate whether application of bactericides via trunk injection could efficiently manage citrus HLB and how bactericides via trunk injection affects Las and HLB diseased trees. 1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against LasThis has been completed for both streptomycin and oxytetracycline against Las. A manuscript entitled: “Residue dynamics of streptomycin in citrus delivered by foliar spray and trunk injection and effect on Candidatus Liberibacter asiaticus titer” was accepted for publication by Phytopathology.1.2. Effect of bactericides via trunk injection on citrus HLB disease progression, tree health, yield and fruit quality in different aged trees with a different disease severityThe field experiments were performed at four different groves on different aged trees with a different disease severity. They are one located in Avon Park, FL, 3-year old Valencia trees; one in Bartow, FL, 2-year old W. Murrcot trees; and one in Auburndale, FL, 7-year old Hamlin trees (planted in 02/2012). The last one is in CREC-, Lake Alfred, FL, 20-year old Hamlin trees. The HLB disease severity and tree size (canopy volume and trunk diameter) in the four groves were estimated immediately prior to treatment application. For the field tests, the experiment design is a randomized complete block design (RCBD) for 9 treatments, including 6 injection treatments (3 different doses for OTC or STR), 2 spray treatments (OTC or STR spraying), and one No treatment as a negative control. Each injection treatment consisted of 9 or 15 trees divided into 3 blocks of 3 or 5 trees each. Each spray treatment consisted of 30 trees divided into 3 blocks of 10 trees each. For all the four field trials, the injection treatment applications were completed by the end of April 2019. The 1st application of spray treatments were completed during spring flushing in February or March 2019, the 2nd applications were conducted in late June to early July 2019, and the 3rd applications were conducted in early to middle October 2019. Leaf samples have been collected from the treated trees at the following time points: 0 (pre- injection), 7, 14, 28 days, 2, 4, 6, 8, 10 and 12 months after treatment (MPT). The estimation of Las titers in these leaf samples are ongoing with qPCR assays. The first estimation of HLB disease severity and growth performance (height, trunk diameter, and canopy volume) of immature trees after treatment were performed in May 2019 (three months after the injection) and continued in a 3-months interval. Fruit yield and quality data were collected for the Bartow trial (W. Murrcot), Auburndale trial (Hamlin), and CREC trial (Hamlin) in January 2021. We investigated the effect of 10 adjuvants on oxytetracycline absorption via foliar spray. Four adjuvants including Flame slightly increased the antimicrobial effect of OTC on Las.Objective 2. To examine the dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system of trees and characterize the degradation metabolites of bactericides in citrus. Leaf and root samples have been collected from OTC or STR treated trees in the Avon Park grove at the following time points:0 (pre- injection), 2, 4, 7, 14, 28 days, 2, 4, 6, 8, 10, and 12 months after injection. The samples have been processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. Fruit samples were collected for the Bartow trial (W. Murrcot), Auburndale trial (Hamlin), and CREC trial (Hamlin) during harvest in January 2020, and for the Avon Park trial (Valencia) in April 2020. The samples were processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. We have collected data for 60 and 360 days post treatment. We have analyzed the residues of OTC and STR for fruit samples harvested in January 2021. Objective 3. To determine whether trunk injection of bactericides could decrease Las acquisition by Asian citrus psyllids (ACP)Twenty 1.5-year old citrus (Valencia sweet orange) plants were graft-inoculated by Las carrying buds in February 2020. These plants are being tested for Las infection and 4 plants were confirmed with Las infection (Ct values are between 34.0 and 35.0) at 4 months after grafting. They will be subjected to OTC or STR treatment by trunk injection and ACP acquisition access for 7 to 14 days. We have determined the time points to test OTC and STR treatment on ACP acquisition of Las. We have conducted the test regarding how trunk injection of OTC affect ACP acquisition of Las. OTC signficantly reduces ACP acquisition of Las. Objective 4. To monitor resistance development in Las against bactericides and evaluate potential side effects of trunk injection of bactericides Monitoring resistance development in Las against bactericides. Leaf samples for this test have been collected from 5 trees injected with OTC and 5 trees injected with STR at the highest doses in each of the three groves at 6 and 9 months after the injection, respectively. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared with the reported sequence. We are further confirming the results. Evaluation of potential side effects of trunk injection of bactericides have been completed. We have collected another set of samples to monitor Las resistance against OTC and STR. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared to the reported sequence, indicating no major changes in bacterial resistance against antimicrobials. Leaf samples were collected from OTC or STR injected trees in the Avon Park grove at two and four months after treatment for the analysis of the degradation metabolites of the bactericides. The extraction of the degradation metabolites were completed and will be subjected to HPLC assays with the three OTC metabolites: 4-epi-oxytetracycline, a-apo-oxytetracycline, and ß-apo-oxytetracycline as standards.
The purpose of this project is to assess nitrogen (N) fertilization rates for high-density plantings of different scion and rootstock combinations in four commercial citrus growers. The overall approach is to 1) determine the appropriate N rates needed to support tree growth and productivity, 2) compare data obtained with existing UF/IFAS N recommendations, and 3) revise best management practices (BMPs) for N application in young trees planted in high-density plantings affected by Huanglongbing (HLB). The Ph.D. student and Research Assistant dedicated to the project were hired. We contacted the grower collaborators participating in the project: IMG Citrus (Brian Randolph), Peace River Citrus (Larry Black Jr.), Agromillora Florida/ Lost Lake Groves (Clay Pedersen), Graves Brothers (David Howard), and scheduled/performed site visits to all locations. The planting densities and variety/scion combinations were chosen, field maps generated, experimental units identified with permanent signs, and the current information regarding N fertilization requested to proceed with the treatment applications. Dr. Ferrarezi contacted Yara (Bill Easterwood) and received fertilizer donations for the first year (20 tons or 40k lbs). Unfortunately, the delivery was delayed due to logistics finding a local distributor, blending the four different formulations, and delivering in Fort Pierce. We were able to receive both calcium nitrate and ammonium nitrate despite the challenges of finding this fertilizer. That was only possible because of the partnership with Yara. Treatments are in the process of being applied in all locations; we should complete the 2nd fertilization for the season on 07/16/2021. The first tree size and leaf area index data collection was performed as expected. We contacted AgerPoint to collect tree size data using Lidar sensors potentially, but the cost was prohibitive. We partnered up with AeroBotics to analyze images collected from drones. The project is on schedule.
1. Please state project objectives and what work was done this quarter to address them: The overall goal of the proposed research is to test the effectiveness of Frateuria defendens (Frd) in reducing HLB damage in FL citrus. The original proposal was revised for a 1-year feasibility study. Objective 1. Determine the optimal application method of Frd to citrus trees.We continued with the spray experiment that was initiated in Dec 2020 and data (horticultural and plant health assesment; CLas and Frd titer determination) are being collected bimonthly. The last dataset collected (April) suggested a positive but weak effect of weekly foliar sprays of Frd on the number of leaves and internodes and a slight reduction of CLas titers compared to the plants sprayed biweekly and non-sprayed controls. We are continuuing our experimentation regarding viability/survival of Frd in citrus tissues. Our studies indicate that an organisms or compound that is associated with CLas (but not CLas itself) inhibits Frd growth in CLas infected leaves. Because of the growth promoting effects of Frd observed in the first experiment, a follow-up experiment was initiated to test the hypothesis that Frd influences the hormonal balance in citrus plants. For this experiment Frd is applied by leaf infiltration. Plant growth is evaluated on a bimonthly basis. We have also initiated an experiment to assess Frd efficacy against CLas when delivered by stem injection. For this experiment Frd is compared against oxytetracycline. Objective 2. Determine if Frd pre-treatment can prevent trees from getting infected with Candidatus Liberibacter asiaticus (CLas).So far, efficacy of Frd applications (foliar or injected) in restoring health of HLB affected plants was not found to be strong enough to justify initiation of this experiment. 2. Please state what work is anticipated for next quarter: We will continue with the foliar spray, leaf infiltration, and stem injection experiments, collection of horticultural information, and CLas and Frd titer determination. 3. Please state budget status (underspend or overspend, and why): Approximately 85% of funds have been spent which, as expected, is slightly overspent because funds were less than what is needed to fund a postdoc for one full year.
This project is an continuation of an objective of existing CRDF funded project (# 00124558 ; ended in March 2019, final report submited to CRDF) with some added treatments to be evaluated in comparison to control (dry conventional fertilizer with foliar micronutrients). Objective 1 which is the continuation of # 00124558 included 10 treatments.Altogether currently there are 25 treatments of citrus nutrition that are being compared to control. Within this quarter we have successfully harvested the trial. The Mn and B treatment is still the best performing treatment across all the treatments in Fort Meade. Where treatment with CRF and tiger micronutrients (6-6-3-1) is consitent good performer at both the sites. Altogether our data so far suggests that use CRF and soil applied micronutirents with slightly acidic pH is beneficial for the trees. No significant differences have been oberserved in new added treatments, it is speculated that two two years is not long enough duration to statitiscally impact the yield. We anticipate that we whould be able to see effect of treatments in upcoming year.Unfortunately, due to Covid-19 related restriction we could not perform sensory analysis, however we collected juice for flavor analysis in order to asses the effect of nutritional treatments on sensory aspect of juice. Currently, we are in process of data organization and analysis of field data and flavor analysis of juice samples.Within this quater the we successfully collected data on tree health, leaf and soil nutrition. We continue to analyze the data.
1. Please state project objectives and what work was done this quarter to address them:Project objectives:Objective 1: Determine the effect of systemic acquired resistance (SAR) in enhanced tolerance to HLB Objective 2: Monitor tree health and HLB levels Objective 3: Conduct Juice analysis and evaluate quality parameters from selected Parson Brown trees. We have identified and collected leaf samples in the spring from 8 Parson Brown groves. Groves are spread all around the major citrus growing regions. We have evaluated the samples collected from the first quarter. All trees are HLB positive with Parson Brown Ct values comparable to the Hamlins. Parson Brown trees in most samples had elevated PR1 and PR2 . DNA and RNA analysis in this quarter will allow us to see if the trend continues through the summer. 2. Please state what work is anticipated for next quarter:In the next quarter, we will again sample the same groves and collect similar leaf samples. HLB levels will be evaluated using qPCR and we will evaluate the genetic differences between the samples. We will also collect data on some physiological parameters. 3. Please state budget status (underspend or overspend, and why):We are on target with the spending for this project.
This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants plus the addition of a new strategy using gene editing. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. We also are in the process of testing citrus that has been transformed to modify the bs5 gene to enhance resistance to the citrus canker bacterium. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. We are in the process of characterizing if different TAL effector variants activate the transgene in order to determine if variants of TAL effectors are effectively targeted. In order to do this TAL variants we are in the process of inoculating into leaves of the transgenic plant, JJ5, and 5′-RACE will be used to identify transcription starts. We will be able to determine if several unique TAL effectors are able to activate transcription. As for developing a different transgenic with ProBs314EBE:avrGf2, we have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contain an additional selectable marker that allowed for identifying putative transgenics with a higher success rate. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. However, there were issues with the construct. Therefore a new construct was made. This construct was recently sent to Vladimir Orbovic, who is in the process of creating additional transformants. Objective 2. To determine if EFR-generated transgenic grapefruit plants are resistant to the citrus canker bacterium in field experiments in Fort Pierce. We have grafted our two most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and planted them in the field at Fort Pierce in collaboration Dr. Ed Stover. We will rate these in the next few weeks. We will be conducting futher greenhouse assays on ROS activity to determine if other EFR transgenics may have more potential for resistance to citrus canker than the two currently in the field. Objective 3. To determine if bs5-generated transgenic Carrizo plants are resistant to X. citri and to generate transgenic grapefruit carrying the pepper bs5. We have recently received budwood from UC Berkeley. The budwood was from two transgenic events and a third was from a tree that was run through the transformation process, but that was negative for the gene. The latter was to serve as the negative control as it had undergone the transformation process. We have grafted the buds and several have developed into branches. We are currently growing these. In the past few months neither of the branches that were created by crispr were actively growing. Recently both of the branches for the transgenic trees have begun growing and we plan on conducting further population experiments in order to confirm previous results on the the level of resistance.
Nematicide treatments for the final spring season were applied. Sting nematode populations were monitored monthly in row middle treatments of cover crop or native vegetation. Results to date were reported at the Florida Citrus Show in May and in the June issue of Citrus Industry.
Nematicide treatments for the final spring season were applied. Sting nematode populations were monitored monthly in row middle treatments of cover crop or native vegetation. Results to date were reported at the Florida Citrus Show in May and in the June issue of Citrus Industry. The primary information conveyed to both outlets is that 1) during two years, perennial peanut reduced the number of sting nematodes in row middles by more than 90%, which may be beneficial as the trees grow and root systems expand, 2) to the extent that the different nematicides reduced sting nematode numbers, the trees responded with increased vegetative (fibrous roots and trunks) growth, 3) during the first two years of production, tree growth rate was inversely related to number of fruit, 4) preliminary observation of tree growth in groves heavily damaged by sting nematode indicates that tolerance of sting nematode herbivory is likely conferred by blocking HLB occurrance via use of individual protective covers.
Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars.During the 2nd quarter of the third year of our grant, the Stover lab has completed production of the transgenics needed for this project. Many lines have been sent to the Rogers lab and the last few are growing well in the greenhouse and will be large enough to sent soon. The Rogers lab has continued small-scale no-choice psyllid inoculation experiments. ARS facilities are still at a maximum of 25% occupancy due to the COVID-19 pandemic; we are teleworking the remaining time. This continues to slow down progress on grant milestones. We are very much hoping to be allowed to move to the next phases of reopening soon, which will allow for much more rapid progress. In addition, the grant-funded post-doc, Dr. Tamara Collum has accepted a permanent position; her last day will be July 2, 2021. We wish her well and are glad she isn’t going far and will be available to answer questions while we hire another post-doc to continue most of the day-to-day work of this project.
In the last quarter, CLas inoculations of experimental trees occured and 2 and 7 week samples have been taken for RNAseq and metabolomics by the postdoc in Dr. Johnson’s and Dr. Wang’s labs respectively. Soil samples were also collected from around the sampled roots for metabolomic analysis of root leachate. These samples are currently being stored at -80 degrees celsius. The second sampling was taken at 7 weeks instead of 6 weeks to allow enough root growth for sampling as a root flush was actively occuring. Biweekly samples have been taken to assess CLas infection of the roots. Once positive trees are confirmed metabolomic and RNAseq analysis of the appropriate collected samples will begin.
HLB is known to make citrus roots more susceptible to Phytophthora root rot. It also reduces the efficacy of chemical management of Phytophthora root rot, creating a difficult management scenario. Current Phytophthora management recommendations are based on pre-HLB work done in the 1980s. These three conditions raise the question of whether yield improvement from Phytophthora management is enough to pay for the management costs themselves. The goal of this project is to develop new soil propagule density managment thresholds and recommendations for chemical management of Phytophthora root rot based on ecomonic analysis of yield responses in different soil conditions. Yield was collected on the Valencia plots in the last quarter. Irrigations systems for chemigation treatments has been installed and the spring treatment has been put out. In all but one treatment the first treatment of the year is phosphite. We have begun HLB inoculations of Phytophthora postive rootstocks for a greenhouse experiment to determine if the newly labeled chemistries have the same limitation on HLB-affected plants as fosetyl-Al and mefanoxam have shown. Many of the new chemistries are directly effective against Phytophthora in the soil rather than acting after uptake by the roots, so this is likely to reduce the limitations of Phytophthora management in HLB-affected groves and test the hypothesis.
Objective 1. To illustrate whether application of bactericides via trunk injection could efficiently manage citrus HLB and how bactericides via trunk injection affects Las and HLB diseased trees. 1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against LasThis has been completed for both streptomycin and oxytetracycline against Las. A manuscript entitled: “Residue dynamics of streptomycin in citrus delivered by foliar spray and trunk injection and effect on Candidatus Liberibacter asiaticus titer” was accepted for publication by Phytopathology.1.2. Effect of bactericides via trunk injection on citrus HLB disease progression, tree health, yield and fruit quality in different aged trees with a different disease severityThe field experiments were performed at four different groves on different aged trees with a different disease severity. They are one located in Avon Park, FL, 3-year old Valencia trees; one in Bartow, FL, 2-year old W. Murrcot trees; and one in Auburndale, FL, 7-year old Hamlin trees (planted in 02/2012). The last one is in CREC-, Lake Alfred, FL, 20-year old Hamlin trees. The HLB disease severity and tree size (canopy volume and trunk diameter) in the four groves were estimated immediately prior to treatment application. For the field tests, the experiment design is a randomized complete block design (RCBD) for 9 treatments, including 6 injection treatments (3 different doses for OTC or STR), 2 spray treatments (OTC or STR spraying), and one No treatment as a negative control. Each injection treatment consisted of 9 or 15 trees divided into 3 blocks of 3 or 5 trees each. Each spray treatment consisted of 30 trees divided into 3 blocks of 10 trees each. For all the four field trials, the injection treatment applications were completed by the end of April 2019. The 1st application of spray treatments were completed during spring flushing in February or March 2019, the 2nd applications were conducted in late June to early July 2019, and the 3rd applications were conducted in early to middle October 2019. Leaf samples have been collected from the treated trees at the following time points: 0 (pre- injection), 7, 14, 28 days, 2, 4, 6, 8, 10 and 12 months after treatment (MPT). The estimation of Las titers in these leaf samples are ongoing with qPCR assays. The first estimation of HLB disease severity and growth performance (height, trunk diameter, and canopy volume) of immature trees after treatment were performed in May 2019 (three months after the injection) and continued in a 3-months interval. Fruit yield and quality data were collected for the Bartow trial (W. Murrcot), Auburndale trial (Hamlin), and CREC trial (Hamlin) in January 2021. We investigated the effect of10 adjuvants on oxytetracycline absorption via foliar spray. Four adjuvants including Flame slightly increased the antimicrobial effect of OTC on Las.Objective 2. To examine the dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system of trees and characterize the degradation metabolites of bactericides in citrus. Leaf and root samples have been collected from OTC or STR treated trees in the Avon Park grove at the following time points:0 (pre- injection), 2, 4, 7, 14, 28 days, 2, 4, 6, 8, 10, and 12 months after injection. The samples have been processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. Fruit samples were collected for the Bartow trial (W. Murrcot), Auburndale trial (Hamlin), and CREC trial (Hamlin) during harvest in January 2020, and for the Avon Park trial (Valencia) in April 2020. The samples were processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. We have collected data for 60 and 360 days post treatment. We have analyzed the residues of OTC and STR for fruit samples harvested in January 2021. Objective 3. To determine whether trunk injection of bactericides could decrease Las acquisition by Asian citrus psyllids (ACP)Twenty 1.5-year old citrus (Valencia sweet orange) plants were graft-inoculated by Las carrying buds in February 2020. These plants are being tested for Las infection and 4 plants were confirmed with Las infection (Ct values are between 34.0 and 35.0) at 4 months after grafting. They will be subjected to OTC or STR treatment by trunk injection and ACP acquisition access for 7 to 14 days. We have determined the time points to test OTC and STR treatment on ACP acquisition of Las. Objective 4. To monitor resistance development in Las against bactericides and evaluate potential side effects of trunk injection of bactericides Monitoring resistance development in Las against bactericides. Leaf samples for this test have been collected from 5 trees injected with OTC and 5 trees injected with STR at the highest doses in each of the three groves at 6 and 9 months after the injection, respectively. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared with the reported sequence. We are further confirming the results. Evaluation of potential side effects of trunk injection of bactericides have been completed. We have collected another set of samples to monitor Las resistance against OTC and STR. The samples were sent out for sequencing to monitor the mutations of OTC and STR resistance related genes.
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