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.
Objective 1 – Ongoing. Objective 2 – Determine the phytotoxic levels of Fe + organic acid solutions on citrus. Preliminary range experiments were started using F11 to determine the concentration where leaf burn occurs. F11 is the Japanese Fe + citric acid product developed from the patent. In discussions with the Japanese inventors (who visited Florida to see the project), we just learned that another product, F11-C, is the product that was developed from this patent specifically for citrus. Therefore, we have modified all experiments, greenhouse and field, to use F11-C rather than F11. Aichi Steel, the manufacturer of F11 and F11-C, have provided us with all the product required for the project. Objective 3 – Determine the effect of Fe2+ + organic acid solutions on HLB titer using a rapid greenhouse, HLB-infected citron, rooted shoot bud assay. Being modified. Originally, we planned on using HLB-infected citron bud cuttings because they show clear HLB symptoms, but are still able to grow. This is unlike HLB-infected grapefruit that can barely be kept alive. However, in our experiments to characterize the citron system we have been able to detect very little growth differences between HLB-infected vs healthy citron plants. This means that treatment effects on HLB-infected plants cannot be determined, only the general effects on plant growth. We are working on another system based on very young sweet orange plants and psyllid inoculation where the preliminary experiment looked very promising. The HLB effect was very strong, the requirement for a good system to test treatments. The validation experiment is now being rerun at a larger scale. If it validates, then we will use this system on the objective 3 (and objectives 4 and 5) experiments where it should work quite well. Objective 4 – See objective 3 discussion.Objective 5 – See objective 3 discussion. Objectives 6 and 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. As reported in the previous progress reports, both tree classes have been measured and recorded for baseline ground data. Likewise both classes have been flown with a camera equipped UAV and aerial data has been documented and analyzed. Tree dimension data for both ground and aerial data have been correlated and display a significant degree of accuracy. In this quarter, 8 treatment applications have been performed. After the initial application, phytotoxic assessments were made for each treatment at 5 DAT and 10 DAT. Virtually all the ferrous iron (Fe2+) products with citric acid exhibited minor leaf burn whereas the chelated iron products did not cause burn. The nonbearing trees as a class are exhibiting vigorous growth and excellent health in that they do not reveal any HLB symptoms whereas the mature trees continue to exhibit moderate decline due to HLB. Neither tree classes reveal any visually discernable treatment effects.
This project was undertaken to assess the impacts of red-dyed and undyed particle films to repel the Asian citrus psyllid (Diaphorina citri; ACP) in the field in Florida. Previous work has indicated that under controlled conditions white kaolin particle films reduced arrival of ACP on citrus plants. Additionally, the same test showed a significant further reduction when the kaolin particle film was dyed bright red. We set out to test these results over time under field conditions. The overall conclusions of this project support the initial studies: white particle films reduce ACP and delay infection, and red-dyed film further reduces ACP and infection rate. Furthermore, these two products enhance growth, with the red-dyed film enhancing growth to a greater degree than the white film. They both increase canopy growth, trunk growth, and yield when compared to insecticide controls and to a treatment with no ACP control. This growth enhancement continued even after HLB infection. Infection reduced the plant growth rate, but the red-kaolin treated HLB-infected plants maintained a higher growth rate than infected untreated plants and a similar growth rate to HLB-free plants without particle films. Studies with potted plants indicate that red film enhances citrus growth relative, in some cases, to treatments with particle films of other colors. We followed up on these studies to assess how the red film produces this effect and concluded that it reduces water use, avoiding leaf water deficits. Overall, we can recommend both red and white particle films to enhance growth and repel ACP, delaying infection.
The contract executed between CRAFT and CRDF was effective November 6, 2019. The first invoices for payment from CRAFT to CRDF were sent to CRDF after March 31st, which was the end of the first full quarter since execution. Therefore, there was no Quarterly report for the Quarter October 1-December 30th, however deliverables during that period will be reported herein. CRAFT PARTICIPATION APPLICATION PROCESS: The CRAFT Technical Working Group, USDA/ARS and CRAFT staff were all involved with preparing the CRAFT application content for the online application experience. The application period was open to Florida citrus growers from September 20-October 15, 2019. CRAFT received 43 applications representing 2,046 acres, including 1618 acres for solid set plantings and 428 acres for reset plantings. The geographic distribution represented 16 counties and all citrus-producing regions in Florida. PROJECT SELECTION & DEVELOPMENT:After review of all projects submitted and the coincidence of application numbers correlating to the goal of 2,000 program acres, with good distribution by variety and project size, it was determined by the Technical Working Group that all of the projects could be accepted, dependent upon satisfactory development of experimental design, and be within the budget allocation by CRDF to CRAFT of $2 Million.Based upon the projects presented, grower participants were organized within subgroups of similar projects (for purposes of replication) and assigned a group leader to help develop the project design. The groups are as follows:1. Rootstock/Scion2. Soil/Tree Fertility3. Pest Management4. Biostimulants5. ResetsAll of the applications approved for grower contract finalization during the reporting period are available upon request. The contracts finalized and included in this report for completion of the deliverables are as follows: 1. 19_001R: Polk County, 40 Acres, Resets Group, Planted December 2019 Factor Tested: Individual Plant Covers on orange in combination with other practices 2. 19_003R: Polk County, 40 Acres, Resets Group, Planted January 2020 Factor Tested: Individual Plant Covers on orange in combination with other practices 3. 19_004R: Indian River County, 40 Acres, Resets Group, Planted January 2020 Factor tested: Individual Plant Covers on Red Grapefruit in combination with other practices 4. 19_015S: Hardee County, 20 Acres, Soil/Tree Fertility Group, Planted November-December 2019 Factor Tested – High and Low rates of specified inputs compared to standard practice 5. 19_022S: DeSoto County, 100 Acres, Rootstock/Scion Group, Planted January 2020 Factor tested Four selections ( UFR 4, US896, US1281 and UFR 6 budded on same Valencia clone) Pre-audits of each project are used to confirm the planting of trees and compliance with contract to date. The project designs for each contract are also available upon request. As of March 31, 2020, 29 applications representing 1,493 acres have been approved by the CRAFT Board of Directors and are finalizing contracts and planting. At this time the initial data for the first quarterly report has been collected for the five CRAFT participant contracts/projects above on the USDA portal, which is still under construction. The portion of the USDA portal that is completed is that for the grower supplied data. 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. Data with actual production inputs by growers is expected for the first five projects prior to the next quarterly report.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; the design of the CRAFT website (craftfdn.org); public meetings of the Technical Working Group and Board of Directors; public workshops; and more. A full list of communications efforts is available upon request.
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. 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 Micronutrients Altogether currently there are 25 treatments of citrus nutrition that are being compared to control. This quater (December, January, and February) the fertilizer was applied for spring 2020. The data collection on leaf and soil nutrient analysis, canopy volume and density has been completed.The results of this trial were presented in citrus industry article, March issue, ‘No one size fits all”. The article has resulted in lot of interest among the grower community. Currently, we are getting prepared for the harvest as well as data collection on fruit quality and size.This a fertilizer evaluation trial and the progress on it is timely and as per expectations.
This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. 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. This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. 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. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium or to alternate target susceptibility genes in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. As stated in a previous report, the transgenic Duncan grapefruit containing the Bs3-executor transgene, designated JJ5, shows a high level of resistance to an array of strains representing a worldwide collection. Furthermore, using real time PCR, we have validated that the gene is activated by one or more TAL effectors and that there is minimal activation without these genes. Buds from the original transgenic tree were grafted onto two rootstocks (812 and Sour Orange) and planted in late March in the field at Fort Pierce in collaboration Dr. Ed Stover. Citrus canker has developed on plants in the field and the trees were rated for disease in December and there was considerable disease on all susceptible Duncan trees, but no evidence on the transgenic, JJ5. Statistical analysis revealed that there was a significant difference in disease both at the second and third ratings. We have also identified two other possible transgenics from plants received from Dr. Vladimir Orbovic. Both, and an additional one which has since been identified, responded to infiltration with a high concentration of bacterial cells by exhibiting a hypersensitive reaction within 4 days of infiltration. One of the transgenics appeared to have a growth defect, but recently has developed normal shoots. All three transgenic trees contain the avrGf2 gene (based on PCR for detection of avrGf2). These transgenics were grafted onto rootstock and are in various stages (i.e, some of buds have broken and the shoots are developing while others are still dormant). During the past three months 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 that contained the targeted construct. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. The construct was sent to Vladimir Orbovic, who developed 45 putative grapefruit and sweet orange transformants. We have screened these via inoculations and so far the ones we have received are susceptibe. More will be available in the future. 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. They were planted in the field in late March. There was some citrus canker on the trees, although they were not uniformly infected. In September the trees were rated for disease severity and the transgenics carrying EFR had considerably more disease than the susceptible wild-type Duncan grapefruit. We then rated the trees in early December. Disease ratings for the trees for the two EFR events were not significantly different from the Duncan grapefruit control trees. We have identified additional transgenics from plants received from Dr. Vladimir Orbovic that that have been grafted onto rootstocks. The these will be tested for ROS activity and for EFR gene expression one the plants are in the right state once we are able to carry out research. 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, serves as budwood that had undergone the transformation process but that was negative for the transgene. This will serve as a negative control. We have grafted the buds and several have developed into branches. We are currently growing these. Unfortunately the control grafts have not developed any branches as of now. Once they are of an appropriate size we will send DNA to Berkeley. Of course this will only occur once we are allowed to conduct research.
No work other than weeding and maintenance of irrigation equipment in the perennial peanut plots was done in the two trials at Frostproof.A site near Ft. Pierce was sampled twice to characterize sting nematode population patterns. The site had previously been a young grove that was removed due to drainage issues and extensive damage by sting nematode. We abandoned the trial when the grower decided to plant trees on beds covered by landscape fabric mulch (for water conservation and faster tree growth). Fabric would preclude sampling to document the effects of nematicides on the nematode populations. Sampling is ongoing in neighboring groves of young trees to establish a trial in the flatwoods.
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 January 31st report date. Trees have been inoculated for 7 months and we have had a lower and less synchronized infection rate than expected from previous experiments. This led to a modification of the experimental setup to allow for blocking based on time of initial infection. However, we have decided to proceed with the treatments at this time. Irrigation in the first rep of the experiment has been initiated and we are currently collecting and testing leachate for shifts in pH. Physiological measures of canopy and root health have begun to track tree health under the differing irrigation pH environments. Trees for the second experiment have been inoculated and were just sampled to identify how many were successfully inoculated on both sides of the split root system.
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. Grafting was completed and we are monitoring bud survival and hoping to push the scion growth soon. Aluminum framed rhizotrons are being constructed to prevent the loss of experimental trees that occured in previous wood framed rhizotrons as they aged.
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. We have begun looking for field sites and preparing materials and application equipment for the trials. With low soil temperatures during much of this time we were not able to sample prospective groves for soil propagule counts.
Objective 1: Investigate the efficacy of bactericides treatments for preventing new infections for young citrus trees protection.Bactericidal treatments were applied from May 2019 through March 2020. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of the CLas-infection rate in citrus leaves from May to September are described. Currently, citrus leaves tissue samples from October through March are being processed to analyze the CLas-infection rate. Trees and ACP were considered CLas-infected (positives) when CT values were below 35. 1. Bactericides (monthly rotation): Before the bactericide application, the overall CT mean of the treatment was 34.5. After bactericide applications from May to September, the CLas bacterial titers have fluctuated without showing constant patterns. In June, the overall CT mean of the treatment increased to 35.1. In July, the overall CT mean of the treatment decreased to 33.6. In August, the CT mean increased to 36.38. In September, the CT mean of the treatment decreased to 33.14 representing a higher rate of CLas infection on the trees sampled. 2. Bactericides (quarterly rotation): Before the bactericide application, the overall CT mean of the treatment was 39.18. After bactericide applications from May to September, the CLas bacterial titers have also fluctuated without showing constant patterns. In June, the overall CT mean of the treatment decreased to 33.99. In July, the overall CT mean of the treatment increased to 34.07. Similarly, in August, the CT mean increased to 36.38. However, In September, the CT mean of the treatment decreased to 34.28 representing a low rate of CLas Infection on the trees sampled. 3. Negative Control (insecticide + Tree defender exclusion netting): Before bactericide application, the overall CT means of the treatment was 38.61. After bactericide applications from May to September, the CLas bacterial titers showed similar patterns from August to September In June, the overall CT mean of the treatment decreased to 34.65. In July, the overall CT mean of the treatment decreased to 33.69. In August and September, the CT mean were 37.69 and 37.45, respectively, meaning low rates of CLas infection on this treatment. 4. Positive Control (insecticide only): Before the bactericide application, the overall CT mean of the treatment was 39.08. After bactericide applications from May to September, the CLas bacterial titers showed similar patters from August to September. In June, the overall CT mean of the treatment decreased to 36.11. In July, the overall CT mean of the treatment decreased to 34.48. In August and September, the CT mean were 37.69 and 37.45, respectively, meaning low rates of CLas infection on this treatment. ACP adults were counted bi-weekly from May 2019 through March 2020. Preliminary results showed a low ACP population in citrus locations due to the active vector management performed by the farm manager. Therefore, no ACP adults were collected to analyze the CLas-infection rate. Objective 2. Determine the effect of bactericides application frequency on Las infection of citrus.We applied bactericide treatments from May 2019 through March 2020. CLas titer was monitored in leaf tissue and the Asian Citrus Psyllid (ACP) in response to antibiotic treatments using quantitative real-time PCR analysis. In each treatment, the CLas infection rate was analyzed on five females and five males. In this report, the results of the CLas-infection rate in citrus leaves and ACP from May through September 2019 are described. Currently, citrus leaves tissue and psyllid samples from October through March are being processed to analyze the CLas-infection rate. 1. Bactericides (monthly rotation): Before bactericide application, the overall CT mean of the treatment was 28.09. After bactericide applications from May to September, the CLas bacterial titers fluctuated from May to June. However, the CT mean showed similar infection rates from July to September. In June, the overall CT mean of the treatment increased to 29.24. In July, the overall CT mean of the treatment increased to 31.12. In August and September, the CT mean decreased to 29.47 and 29.76. Bi-weekly collected ACP from the treatment from May and July showed CT means of 34.5 and 37, respectively. 2. Bactericides (quarterly rotation): Before bactericide application, the overall CT mean of the treatment was 27.89. After bactericide applications from May to September, the CLas bacterial titers fluctuated from May to June. However, the CT mean showed similar infection rates from July to September. In June, the overall CT mean of the treatment increased to 29.90. In July, the overall CT mean of the treatment increased to 30.73. In August and September, the CT mean decreased to 29.49 and 29.15. Bi-weekly collected ACP from the treatment from May and July showed CT means of 35 and 38, respectively. 3. Positive Control (insecticide only): Before the bactericide application, the overall CT mean of the treatment was 28.09. After bactericide applications from May to September, the CLas bacterial titers shown a constant pattern in the CT means from June to September ranging from 29.5-30 approximately. Bi-weekly collected ACP from the treatment from May and July showed CT means of 36 and 37, respectively.
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