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. Finding and getting to sites during the research ramp-up has proven more difficult than predicted. We are still looking for suitable grower sites with Phytophthora pressure. Meanwhile we are planning an additional greenhouse Phytophthora and HLB experiment to determine if the newly labeled chemistries have the same limitation on HLB-affected plants as fosetyl-Al and mefanoxam have shown.
The objectives of this study are to identify optimal pH range for root function and minimize root turnover on HLB-affected rootstocks and how uneven pH levels in the root zone (e.g. irrigated vs. row middle portions of root system) affect the overall health of the tree. This is being done in a split root system in the greenhouse where pH of different parts of the root system can be controlled an maintained. The following progress reported is based on a July 31st report date. The breakdown of the first experiment has been completed and preparation for setup of the 2nd experiment is underway. Initial findings from the first experiment will be presented virtually as a poster at the American Phytopathological Society annual meeting the 2nd week of August. Final dry mass, gene expression, and CLas population data is still being collected on samples from the first breakdown. Initial observations from breakdown suggest that roots suffer minimal damage at some soil pH environments pH 7.5 for US942 and pH 5.5 for Swingle. Swingle roots were most damaged by HLB at pH 7.5 and US942 roots were most damaged at pH 5.5. More information will be provided when the quantitative data has been analyzed from the first experiment.
Progress report for the second quarter of the 2019/2020 project yearThe purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruit. 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. While it is early to determine some trends, it appears soil and root nutrient levels, for example, follow the pattern of treatments e.g. 4x>2x>1x, for soil applied treatments. Similarly, for foliar applied treatments, the pattern in leaf nutrient concentration follows 1x<2x<4x. We are following through with correlations in roots, leaves and soil with canopy size, trunk size, yield, bacteria titers and so forth. Root growth tends to be more pronounced with soil applied nutrients compared with leaf applied nutrients and this will be confirmed and validated further in the following year of measurements. At the southeast Flatwoods, all grove caretaking activities were performed on time and the treatments have been applied as expected along with data collection on tree size, soil/leaf/root nutrient levels, and HLB ratings. All sampling for the third quarter is complete and data analysis is underway and will be reported in the next quarter. Graduate students Tanyaradzwa Chinyukwi and Lucas Hallman presented portions of their research at the American Society of Horticultural Science virtual Annual Meeting between August 10 to 13, 2020. Plans for Next QuarterThe 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. Graduate students working on the project will present at the Florida State Horticultural Society. One graduate student working on the project will be submitting her MS thesis to the UF graduate school this fall and will prepare articles for refereed journal publications.
Objective 1 – Determine the effect of the ratio and concentration of Fe2+ + organic acid on hydroxyl radical production and stability. Objective complete. Objective 2 – Determine the phytotoxic levels of Fe2+ + organic acid solutions on citrus. Objective complete. Objective 3 – Determine the effect of Fe2+ + organic acid solutions on HLB titer using a rapid greenhouse, HLB-infected citron, rooted shoot bud assay. Continuing work to develop a rapid greenhouse screening system. Current systems are not yet ready for screening methods to cure or manage HLB. Experimentally robust screening systems are probably the single most important research tool that are needed by the HLB research community. Objective 4 – Requires screening system – see objective 3 discussion.Objective 5 – Requires screening system – see objective 3 discussion. Objectives 6 and 8 (Note: there is no 7) – These are the field tests for the various ferrous iron (Fe2+) and citric acid treatments on HLB status and horticultural measures for both mature (HLB symptomatic) and nonbearing (non-symptomatic) trees. The seven treatments have been applied according to the schedule specified in the project protocol to both bearing and nonbearing age class trees. The nonbearing trees as a class continue to exhibit vigorous growth and are maturing with a good canopy density. There were no significant differences in growth between treatments based on trunk calipers measured on 06/05/2020. Interestingly, there were significant differences in the incidence of HLB symptomatic leaves between treatments (Table 1). Three treatments (Aqua F11-C, Tracite Iron and chelated iron) exhibited fewer incidences of HLB leaf patterns than the untreated control (Table 1).———————————————————————————-Table 1. Number of trees with no HLB symptoms. Each treatment is composed of 12 trees. Treatment # of Trees w/ no HLB Symptoms F11-C (30 ppm) 7 F11-C (60 ppm) 6 Tracite 5% Fe 7 FRC 249 4 Chelated Fe 10 Ferrous sulfate + citric acid 6 Fe Solution 2 Control 5 ———————————————————————————- Aerial images were taken of the young trees and images processed by machine learning to extract various size and shape measures, including canopy area (2D) and canopy density. These data were analyzed and no significant differences were detected between the treatments and the untreated control trees. The bearing grapefruit trees as a class were abandoned on 03/01/2020 due to severe decline caused by irreversible root damage, primarily due to the Phytophthora/Diaprepes complex exacerbated by HLB. Aerial images have been taken of the mature (bearing age) plot immediately prior to tree removal. Images are ready for processing, quantification, and analysis. Also, tree condition assessments, crop yield, and fruit drop counts were made and documented in early January 2020 and revealed no significant differences between treatments. Throughout this quarter as well as the project timeline, conventional pesticide spray applications were applied to all the treatments in the trial and were based on scouting and were in accordance with IFAS guidelines. Similarly, irrigation events were made based on tree and field conditions as determined by soil feel and appearance, tensiometer readings, water table observation well measurements and visual assessment of tree canopy. Fertilizer applications were made via fertigation and were `spoon fed’ with frequent small applications bi-weekly. No dry fertilizer was used.
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. Horticultural measurements, scheduled for the last quarter but not completed due to the Covid-19 related halt of all research activities, were completed. These include tree height, canopy size, and trunk diameters.Fibrous root samples were collected for assessment of root traits. Rhizosphere samples were collected for microbial community analysis. Root trait analysis and rhizosphere DNA extractions are in progress. Soil sample were collected for nutrient and physical attributes analysis. 2. Please state what work is anticipated for next quarter: We will complete the leaf sample collection for nutrient analysis and continue with processing of samples in the laboratory and statistical data analyses. 3. Please state budget status (underspend or overspend, and why): Approximately 28% of funds have been spent, which includes funds for the initial tree establishment. Spending is in accordance with the timeline.
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. 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 2020.Fruit yield was estimated for the Avon Park trial (Valencia) in April 2020.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. 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.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. Evaluation of potential side effects of trunk injection of bactericides have been completed.
The contract executed between CRAFT and CRDF was effective November 6, 2019. Project development and approval continued through the period ending June 30, 2020 and invoices for payment from CRAFT to CRDF continued to be sent to CRDF accounting for the total 2,002 contracted acres. PROJECT SELECTION & DEVELOPMENT:Throughout this Quarter, CRAFT Technical Working Group and staff continued to work with applicants to finalize experimental designs and contracts related to all proprosed projects. A total of 45 projects were established and sucessfully enrolled in the CRAFT program at the end of this Quarter, amounting to a total of 2,002 program acres.Pre-audits of each project are used to confirm the planting of trees and compliance with contract to date. The contract and experimental designs for each project are available upon request. As of June 30, 2020 all 45 projects were either fully planted or underway and extended pursuant to CRDF contract as amended. The 16 projects extended based on widespread delays in the delivery of contracted trees from citrus nurseries will continue to be planted as trees are received from the nurseries. At this time the initial data for the first and second quarterly report has been collected for the completed CRAFT participant contracts/projects on the USDA portal. Growers have each been assigned their own portal survey access and dashboard. The data from growers is expected to be entered in real time or at least quarterly. The data portal will include a separate entry point for data/ measurements from third party partners (such as FDACS/DPI) and has not been finalized. Data entered by participants and third parties for each project will be visible initially by the growers for their individual projects. In addition to execution of contracts and commencement of projects, CRAFT staff conducted numerous industry information sessions and communications outreach events. Communications include articles in multiple industry and general publications; public meetings of the Technical Working Group and Board of Directors; public workshops including a two-day Zoom workshop focusing on Cycle Two of the program; and more. A full list of communications efforts is available upon request. One hundred percent of objectives completed for the period of November 6, 2019 – June 30, 2020 based on full enrollment of program acres in demonstration projects, data collection and outreach activities.
his 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. The added treatments from objective 21. CRF + Tiger Micronutrients+ Mn 50%2. CRF + Tiger Micronutrients+ Zn 50%3. CRF + Tiger Micronutrients+ Fe 50%4. CRF + Tiger Micronutrients+ B 50%5. CRF + Tiger Micronutrients+ Mn +Zn 20%6. CRF + Tiger Micronutrients+ Mn +Fe 20%7. CRF + Tiger Micronutrients+ Zn +Fe 20%8. CRF + Tiger Micronutrients+ Zn +B 20%9. CRF + Tiger Micronutrients+ Fe + B 20%10. CRF + Tiger Micronutrients+ Mn +Zn 50%11. CRF + Tiger Micronutrients+ Mn +Fe 50%12. CRF + Tiger Micronutrients+ Zn +Fe 50%13. CRF + Tiger Micronutrients+ Zn +B 50%14. CRF + Tiger Micronutrients+ Fe + B 50% The treatment for objective 3: 1.CRF + Foliar Micronutrients + Tiger 90; 2.CRF + Tiger MicronutrientsAltogether currently there are 25 treatments of citrus nutrition that are being compared to control. Within this quater the we succefully collected harvest data on all 25 treatments. In addition, we were able to collect fruit quality and size data on the harvested fruit. This data is currently being analyzed statistically.Unfortunately, we were not able to do sensory analysis due to Covid-19 related slowdown of UF activities. In order to compensate, we have made an effort to collect juice from all the treatments and freeze-store. We plan to conduct a flavor compound and sugar analysis on these samples in order to gain some insight. We plan to conduct a sensory analysis next year with freshly harvested fruit and run a flavor and sugar analysis on those to get two years replicated data on flavor of juice.
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 second year of our grant, the Stover lab continues producing transgenic plants and the Rogers lab continues to propagate the transgenic lines. However, currently ARS employees have been ordered to maximize telework due to the COVID-19 pandemic. This has slowed down progress on the grant milestones. Still, there are 16 additional Hamlin transgenic lines, 1 Poncirus line, and 5 Carrizo lines ready to be shipped to the Rogers lab once we are able to reopen. The Rogers lab is increasing our Asian citrus psyllid colony in preparation for exposing the transgenic lines. The post-doctoral researcher, Tami Collum, has finished optimizing citrus nucleic acid extraction and immunoprecipitation protocols for the extraction of high quality translatome RNA. We hope conditions will allow shipments and starting CLas infections of transgenic lines during the 3rd quarter.
The objectives of this study are to identify optimal pH range for root function and minimize root turnover on HLB-affected rootstocks and how uneven pH levels in the root zone (e.g. irrigated vs. row middle portions of root system) affect the overall health of the tree. This is being done in a split root system in the greenhouse where pH of different parts of the root system can be controlled an maintained. The following progress reported is based on a April 30th report date. Modified pH irrigation and image collection and analysis continued through the COVID-19 research pause, but further sampling and testing was delayed as well as the setup of the second experiment. This pause did allow for root tracing of a backlog of images that was created by the longer than expected time for CLas infection of the inoculated trees. As research resumes we will initiate the second experiment and perform final sampling and breakdown of the first experiment.
Sugarbelle appears to be one of the most HLB tolerant scion varieties and UFR-4 is a rootstock that does not suffer from HLB-induced root loss. Valencia and Swingle are susceptible to HLB damage. This project has two main goals based on different graft combinations of these two scion and two rootstock varieties. First is to determine if there is a benefit to only half of a grafted tree being tolerant or if both scion and rootstock need to be tolerant to ameliorate some of the physiological effects of HLB. Second is to identify the mechanism of UFR-4 resistance to HLB-induced root loss and to identify the systemic signal of HLB-induced root loss with the hope of finding CRISPR targets to move this resistance into other rootstock varieties. These experiment will use a previously developed split root rhizotron system to maintain CLas infected and uninfected root systems on the same tree Rootstock seedlings (Swingle) and cuttings (UFR-4) have been selected from our greenhouse for grafting of SugarBelle and Valencia. The first round of grafting had an unexpectedly low success rate, so a new source of budwood was found and trees were regrafted. They are now growing in preparation for the first rep of the experiment. Aluminum framed rhizotrons continue to be constructed to prevent the loss of experimental trees that occured in previous wood framed rhizotrons as they aged. Much of the assembly has been possible at employees homes, reducing the delay due to COVID restrictions
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. While potential field sites have been identified, Phytophthora count testing to confirm the usefulness of the locations was delayed by COVID response, delaying plot design and treatment. Testing and plot layout will likely happen in June allowing for first year treatments to be applied during the most significant seasons for Phytophthora, especially with the dry spring that did not favor Phytophthora development.
As mentioned in the earlier report, we conducted an experiment to determine the effect of various factors on the ground penetrating radar (GPR) depth measurement. In the sixth quarter of this project, we analyzed the data of this experiment and produced the results for the comparison of the new calibration system with the previously available calibration methods. After the analysis, we found that: (a) the GPR measured root depth becomes smaller as the dielectric constant increases, (b) the error of GPR measured root depth becomes bigger with the increase of depth, and (c) the GPR frequency and root diameter seems to have little to no effect on the GPR measured depth. Due to the COVID-19 pandemic, the SWFREC has been closed since March 24th which has disabled us from working on the physical development of the automated GPR system. As of this time, we remain closed. Currently, we are all working from home and we have used this time to prepare a draft of a paper titled: A multi-point layered calibration method for citrus root depth measurement using Ground Penetrating Radar. We are currently going through the process of revision and editing of this draft; we will submit this paper to a scientific journal soon.We expect to return to work in the coming weeks, and we will be resuming work on the physical development and testing of the automated GPR system.The abstract of the paper (draft) can be found below:Abstract: This paper proposes and evaluates a new multi-layered point calibration method for citrus root depth measurement using ground penetrating radar (GPR). The effects of several factors such as (i) roots diameter, (ii) dielectric constant, and (iii) root depth on the GPR depth measurement have been investigated. Several multi-factor experiments were conducted using two different GPR antennas (900 MHz and 1600 MHz), different dielectric constants (2.5, 3.5, 5, 9 and 13) and at different depths (10, 20, 30, 40, 50 and 60 cm) to evaluate the influence of above-mentioned factors using five citrus branches and a stainless-steel bar in a citrus grove located at SWFREC. It has been found that the GPR measured root depth becomes smaller as the dielectric constant increases, the error of GPR measured root depth becomes bigger with the increase of depth and GPR frequency and root diameter seems to have little to no effect on the GPR measured depth.
Progress report for the second quarter of the 2019/2020 project yearThe purpose of the project is to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruit. 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 in the second quarter of 2019/2020, focused particularly on soil, root and leaf nutrient concentrations, HLB disease ratings, tree physiological characteristics, and root growth and longevity. We also collected second fruit yield and juice quality data on the research site near CREC, and first year fruit yield and juice quality data at the IRREC. Data collection continues, and analyses will be done as more data are collected. Root measurements, soil characterization, and canopy size determinations are completed every 6 months. Data analysis and comparison of early trends continues. We will provide further updates on the outlook of results for the orange and grapefruit trees in the next quarter. We will continue to monitor the trends over the coming months and report any observations accordingly. Root image tracing is almost up to date and preliminary quantification should be completed soon. One set-back during this quarter affected one deliverable on the project. The project could not collect harvest and fruit quality data for the Clewiston site due to COVID-19. Nevertheless, the yield and fruit quality data collected near Lake Alfred and at IRREC will provide the updates for this year. Plans for Next QuarterThe 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. Preliminary root longevity analysis will be done on the traced root images from Lake Alfred and Southern Gardens sites comparing lifespans of root flush cohorts in response to fertilizer treatment.
The purpose is to evaluate the control effect of bactericides via trunk injection.Objective 1. 1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against Las We developed a new method for evaluating the effects of oxytetracycline (OTC) treatment on Las titers in planta, and determined the relationship between OTC residue levels and control levels achieved for Las using mathematical modeling in greenhouse and field experiments. In greenhouse, OTC injection at 0.05 g/tree decreased Las titers to an undetectable level (Ct value = 36.0) from 7 to 30 DPA, and produced a residue level of OTC at 0.68-0.73 µg/g fresh tissue over this period. In the field, OTC injection at 0.50 g/tree resulted in the decline of Las titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27-0.33 µg/g fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for Las achieved. The results suggested that the minimum concentration of OTC required to suppress Las populations in planta to below the detection limit is 0.68 and 0.86 µg/g, and the minimum concentration of OTC required for initial inhibition of Las growth in planta is approximately 0.17 and 0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target Las in planta for effective control of citrus HLB. This study has been published by Phytopathology in a manuscript entitled: The in planta effective concentration of oxytetracycline against Candidatus Liberibacter asiaticus for suppression of citrus Huanglongbing. In addition, we evaluated the inhibitory activity of streptomycin (STR) against Las in a greenhouse experiment. Citrus trees were trunk-injected with STR, and leaves were inspected for Las populations and STR residues using qPCR and HPLC assays respectively, at various times after STR injection. Assays for Las titers and STR concentration in leaf samples from field trials are also ongoing. We are summarizing the data for publication and presenting the information to citrus growers. 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 2020.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. Examination of dynamics and residues of bactericide injected into citrus and systemic movement within the vascular system. 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), 7, 14, 28 days, 2, 4, 6 and 8 or 9 months after injection. The samples are being processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples are being determined by HPLC assays. Determination of the residue contents of bactericides in fruit and juice in each harvest. 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 are being processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples will be determined by HPLC assays. Analysis of degradation metabolites of bactericides injected into citrus trees. 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.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. Once Las presence is confirmed in these plants, they will be subjected to OTC or STR treatment by trunk injection and ACP acquisition access for 7 to 14 days.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. Evaluation of potential side effects of trunk injection of bactericides. We evaluated possible phytotoxity caused by OTC or STR in immature trees (3-year old Valencia) in the Avon Park grove from one week to one month after injection. The trees were be examined for the following symptoms: fruitlet drop, fruit drop, quantity of leaf drop, non-insect related leaf rolling, and leaf discoloration. There was no significant difference in fruitlet drop, fruit drop, quantity of leaf drop, or non-insect related leaf rolling between OTC or STR treatment and untreated control. About 20% (3 out of 15) trees injected with OTC or STR at the highest dose (2.0 g/tree) showed leaf discoloration (yellowing) on some young shoots. These phytotoxicity-like symptoms disappeared at 6 months post injection.
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