Project Objectives:
The purpose of the project was to develop new guidelines for restoring root health and improving overall tree nutrition for Florida oranges and grapefruits. The objectives of the project were 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.
Summary Description of the Project:
The project was 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 on root, leaf and soil nutrient evaluations, root scanning, canopy size determinations and soil sampling on the central Ridge and southwest Flatwoods along with fertilizer treatment applications were done throughout the project. Mini-rhizotrons were installed at the beginning of the experiment and root images were taken monthly and root density data were measured half-yearly. Fruit yield and juice quality and bacteria titer data were measured yearly. We compared standard fertilization with elevated (1x and 2x of current recommendations) macronutrients (potassium, magnesium and calcium) along with elevated (1x, 2x and 4x of current recommendations) micronutrient blends (iron, zinc, boron and manganese). At the UF/IFAS IRREC in Fort Pierce, FL 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.
Major accomplishments of the project:
One key finding in HLB-affected oranges is that it is not beneficial to double macronutrients such as Ca, Mg and K, but rather increasing micronutrients such as Fe, Mn, B and Zn by 1x to 4x because fruit yield, root health and canopy volume were optimized where micronutrients were elevated compared to current guidelines. For HLB-affected grapefruit, the results of this study may show evidence that rehabilitation of HLB-affected trees may be limited by the age of the tree and number of years it has been HLB-affected and should start early at the establishment of a grove. Two graduate students completed graduate degrees in Soil and Water Sciences and Horticultural Sciences. We made 8 grower presentations, published two MS theses, 5 citrus industry articles and 3 refereed journal articles. Next steps Additional 5 articles will be published in refereed journals in the next few months upon completion of data analysis. This will also be followed by extension bulletins based on our current results and follow-up studies to follow on the inconsistent results on yield, canopy size, root growth and root density patterns and bacteria titers. In the next phase of the research, we plan to use other tools we have learned from growers and other projects such as using real-time artificial intelligence to measure nutrient deficiency, soil amendments and using variable rate fertilization tools, besides fertigation and controlled release fertilizers.
Please state budget status (underspend or overspend, and why):
We completed the project milestones and spent about 99.5% of the budget. We are grateful to the Florida Citrus Growers and CRDF for their financial support.
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. We are in the process of analyzing data related to how adjuvants affect bactericide delivery via foliar spray, bactericide residues in plants, and effect of bactericides on HLB positive citrus tress of different ages.
The purpose of this project is to assess nitrogen (N) fertilization rates for high-density plantings of different scion androotstock combinations in four commercial citrus growers. The overall approach is to 1) determine the appropriate N ratesneeded to support tree growth and productivity, 2) compare data obtained with existing UF/IFAS N recommendations, and3) revise best management practices (BMPs) for N application in young trees planted in high-density plantings affected byHuanglongbing (HLB). The Ph.D. student and Research Assistant dedicated to the project worked with the three growercollaborators participating in the project: IMG Citrus (Brian Randolph), Peace River Citrus (Larry Black Jr.), AgromilloraFlorida/ Lost Lake Groves (Clay Pedersen), Graves Brothers (David Howard), and performed the first applications of thefertilizers. Fertilizers were donated by Yara (Bill Easterwood) for the first year (20 tons or 40k lbs). Treatments were applied in all locations and the 2nd fertilization for the season was completed on 07/16/2021. The first tree size and leaf area index data collection was performed and analyzed as expected. After being informed by CRDF that the project will be prematurely concluded in December 2021, cooperating growers were informed that we will not proceed with further applications and data collections.
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. In late March, 2019, in the field at Fort Pierce in collaboration Dr. Ed Stover, the transgenic material was planted. Citrus canker has developed on plants in the field and the trees were rated for disease in November 20, 2021and there was moderate disease on Duncan grapefruit trees but none on JJ5. We rated the plots again on July 28 (not June as mentioned in May report) 2021 and there were similar trends as in November 2020 although disease was lower given significant defoliation in the plots. We have also continued analyzing JJ5 for response to strains from Nian Wang to determine if those strains could overcome the resistance. Interestingly we noted unique phenotypes in plants inoculated with these strains although they were not typical disease reactions but more of a watersoaked appearance. We also used a 5′ Race kit to determine the transcription start in our JJ5 construct and observed that all strains tested activated trasncription of our 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. We have created a second construct for Vladimir Orbovic given the first attempt was not successful.The construct is with Vladimir Orbovic, who is in the process of creating additional transformants. He currently has identified one putative transgenic that has been potted and has several others that will be potted in the near 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 and were recently rated in late July. The trees were rated for disease in November, 2020 and there was considerable disease on all EFR plants with disease being more severe than on susceptible Duncan control. We rated the plots again on July 28 (not June as mentioned in May report) 2021 and there were similar trends as in November 2020 although disease was lower given significant defoliation in the plots. 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 have cut back the material and will analyze for susceptibility and bacterial growth once the leaves grow out again.
Frateuria defendens (Frd) is a recently discovered endophytic bacterium belonging to the family of Rhodanobacteraceae. The application of Frd by foliar spraying and soil drenching was documented to reduce the symptoms associated with phytoplasmas and liberibacters in various crops, including grapevine and carrots. It is thought that Frd has multiple modes of action including secretion of antimicrobials and priming of host defenses. This project was part of a collaborative effort between US (UF/IFAS SWFREC) and Israel (ARO) to develop an environmentally safe, affordable, and effective solution for the management of citrus HLB. It aimed to test whether Frd can effectively reduce CLas titers and HLB disease symptoms in citrus trees and can be developed as a commercial biocontrol agent. The project was originally intended as a 3-year study but was revised to conduct a 1-year feasability study. The overall goal of the project was to test the efficacy of Frd in reducing HLB disease effects in Florida citrus. The specific objectives were 1) to test the curative effects of Frd against HLB under greenhouse conditions, and 2) to optimize delivery of Frd and determine suitability for large-scale control of HLB in field conditions. Objectives had been modified slightly from the original proposal to make the study feasible within the reduced time frame of the funding period.Our studies showed that, under the conditions of our study, Frd had difficulties establishing in citrus leaves. This effect may have been associated with Frd-unfavorable biochemicals in the leaves and seemed to have been exacerbated by CLas infection.There was no clear evidence that Frd interacted with CLas directly (i.e., Frd applications did not reduce CLas titers) and/or had a curative effect against HLB disease. However, Frd applications had positive effects on plant growth and seemed to be able to mitigate some of the damage caused by HLB or to retard disease progression of severly affected plants under greenhouse conditions.The direct delivery of Frd via leaf infiltration or trunk injection was more effective than foliar sprays.
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. In this quarter we recorded a downregulation in the expression of CsPR1 and CsPR2 when compared with the first sampling in May in most of the experimental sites Parson Brown trees from two locations in Polk county had enhanced expression of CsPR1. There was no significant difference between Hamlin and Parson Brown performance from trees in the Immokalee site during July sampling. In addition to gene expression, samples have been collected for the following physiological parameter evaluation: Total chlorophyll content, starch content, alpha Amylase content, beta Amylase content, Carbohydrate content, Antioxidant content, Total phenolic compound content, Ascorbate content. A magazine article has been published in the Citrus Industry magazine to enhance public awareness on the parson brown oranges. 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 and evalute them. HLB levels will be evaluated using qPCR. Juice quality from all sites will be evaluated through the CREC pilot plant. 3. Please state budget status (underspend or overspend, and why):We are on target with the spending for this project.
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). Altogether currently there are 25 treatments of citrus nutrition that are being compared to control. Within this quarter we have applied second round of fertilizer. Within this quater the we successfully collected data on tree health, leaf and soil nutrition. We continue to analyze the data.Altogether, 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. Our data so far suggests that use CRF and soil applied micronutirents with slightly acidic pH is beneficial for the trees. In spring 2020 we saw a signifcant decrease in soil pH with extended use of Tiger micronutirents and therefore we stopped the use of Tiger product and applied non-sulphur micronutrients to the soil. The withdrawal of Tiger micronutirens for about 1.5 years has helped in bringing to soil pH to optimal ranges (5.8-6.5). No deleterious effects are observed on trees with discontinuation in use of tiger micronutirents. This suggests that the tiger micronutirent application can be altered with non-sulphur prill micronutrients to balance soil pH. We continue to closely monitor the soil pH and tree growth patterns. We are plan to apply fertilizer in first week of october and final harvest will be in Aril 2022.
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 an(he 3nd quarter of the third year of our grant, the Stover lab continues to grow the last few transgenic citrus in preparation for shipping them to the Rogers lab, which should happen early in the 4th quarter. The Rogers lab has continued small-scale no-choice psyllid inoculation experiments on the many lines they have received from the Stover lab and has begun RNA extractions. 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, Dr. Rogers is advertising for a new post-doc to work on finishing this project.
The contract executed between CRAFT and CRDF was effective March 18, 2021, however work began on Cycle II projects beginning in July 2020 and deliverables executed 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 Cycle II online application experience. The application period was open to Florida citrus growers from July 20-September 4, 2020. CRAFT received 86 applications representing 4,411 acres, including 3,014 acres for solid set plantings and 1,397 acres for reset plantings. The geographic distribution represented 11 counties and all citrus-producing regions in Florida. PROJECT SELECTION & DEVELOPMENT:After review of all projects submitted, it was determined by the Technical Working Group based on fulfillment of minimum requirements and scientific merit that 63 of the projects would be recommended for further development of experimental designs while the remaining 23 would be placed on a waitlist for future consideration dependent on funding availability. Following final consideration and review by the Board of Directors, a total of 59 projects were approved. 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 approved grower contracts for inclusion in Cycle II are included in Attachment 1. The project designs for each contract are also available upon request. As of June 30, 2021, 59 applications representing 2,636 acres have been approved by the CRAFT Board of Directors, finalized contracts and are pending planting. Following completion of planting, pre-audits of each project are used to confirm the planting of trees and compliance with contract to date. Growers are expected to report production data in real time or at least quarterly in to the USDA-CRAFT Data Portal. The data portal will include a separate entry point for data/ measurements from third party partners such as FDACS/DPI and Aerobotics. 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 continued communications and outreach efforts including, articles in multiple industry and general publications; updating of the CRAFT website (craftfdn.org); public meetings of the Technical Working Group and Board of Directors; and more. A full list of communications efforts is available upon request.
1. Please state project objectives and what work was done this quarter to address them: To determine how many leaf nutrient sampling per year are required to effectively capture the tree nutritional statusand adjust fertilizer accordingly.2. To establish the relationship of leaf nutrient concentration with yield, fruit drop, and canopy density3. To determine how the leaf nutrient (all 14 nutrient) levels change in the tree throughout the year.4. To evaluate how the leaf age affects the leaf nutrient status.TThe project was initiated in January 2021. In this quarter we were able to analyze the pretretament tree health and nutritional data. We have also initiated bimonthly leaf sampling for nutrient analysis. Based on the leaf nutrient analysis were were able to apply first round of fertilizer according to the leaf nutrient analysis. The preliminary findings were presented at annual meeting of ASHS, Denver, Colardo. 2. Please state what work is anticipated for next quarter: 1. Data analysis and interpretation2. Processing samples for starch3. Tagging summer flush4. Collecting samples for nutrient analysis5. Applying fertilizer treatments based on leaf results 3. Please state budget status (underspend or overspend, and why): Currently, the project is in early phase.The budget is being spent as per the plan where major funds have been used for nutrient anlaysis. We have found dedicated personnels to work on this project starting this quarter.
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.
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