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.
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 1st quarter of the second year of our grant, the Stover lab continues producing transgenic plants and shipping the ones that are ready to Ft. Detrick. CTV-infected plants were shipped to Ft. Detrick in February and are expressing nicely. This has allowed the post-doctoral researcher, Tami Collum, to finish optimizing citrus nucleic acid extraction protocols and perform immunoprecipitation and extraction of high quality translatome RNA from p35S::HF-RPL18 and pSUL::HF-RPL18. Another shipment of transgenic plants is anticipated in the 2nd quarter.
During this 5th quarter of the Project our work continued without problems. Objective 1: We continued monitoring parameters including tree trunk diameter (rootstock and scion) and canopy areas. Some differences are starting to emerge in trunk diameter, although not statistically significant yet: Covered trees are starting to show bigger diameters in both rootstock and scion. All IPC-covered trees are still HLB-negative. We are continuing documenting canopy area expansion by photography and also by leaf are index measurements after replacing the old 4-ft IPCs with new 8-ft covers, donated by The Tree Defender, Inc. Objective 2. We have finished planting the 700 trees of SugarBelle, Tango and Early Pride mandarins, and we started to monitor regularly tree parameters (trunk diameter, and leaf sampling, for CLas, chlorophyll and sugar analysis). Objectives 3 and 4. We set up blooming and fruit development experiments this season inside the IPCs and in our CUPS facility, which is now fully operative. We have performed deficit irrigation to induce blooming in both IPC and CUPS this season. We are currently collecting data on these experiments. Outreach, Professional Presentations and Extension Activities for this quarter : – A CUPS Day.CUPS, mini-CUPS and other strategies to manage HLB. Talk on “Individual Protective Covers” . SWFREC, was delivered on Dec 17. 45 people registered, 44 attended. Impact: 100% of the attendants found the presentation useful and manifested a gain in knowledge. Half of the attendants will improve their management practices, which, in this case, may mean to adopt IPCs. In addition, 32 CEUs and 10 CCAs were certified. -As a follow up of the International invited seminar at IVIA, Valencia, Spain entitled “Living with HLB. The new reality of Florida Citriculture” that was presented by Fernando Alferez last December 4, 2019, the spanish professional magazine Phytoma published an article showcasing, among others, our IPC work. It can be seen here:https://www.phytoma.com/noticias/noticias-de-actualidad/florida-conviviendo-con-el-hlb-y-los-huracanes -Citrus Extension Agents presentation on “The Citrus Horticulture Program at SWFREC”. Immokalee, January 28, 2020. As a result of this presentation, I have been invited to deliver these presentations in the next months: -“Individual Protective Covers”. O.J Break Series, Highlands Co. Extension Office. March 20, 2020. -“Individual Protective Covers” In Service Training invited speaker. CREC, Lake Alfred, April 8-9, 2020.
In the fifth quarter of this project, we started developing a new calibration system to get a better estimation of the depth of the roots when compared to the existing calibration methods. The existing calibration methods use dielectric constant measured using soil type and soil moisture level to get the depth of the roots. This new method combines dielectric constant calibration in the field with the post-processed data that is extracted in the lab. This method uses two different dielectric constant values at different depths, one at a deep level and another at a shallow level. These both values will be used to correct the GPR estimated depth of the root and increase the accuracy of root depth estimation. For evaluating this calibration method, we conducted field experiments at SWFREC citrus grove to determine: (i) effect of roots diameter on GPR depth measurement, (ii) effect of dielectric constant on GPR depth measurement and (iii) effect of root depth on GPR depth measurement. Upon performing these experiments, it is determined that the accuracy of depth of shallow roots was better than the depth of roots present at a deeper level and also, the new calibration method can be used to improve the accuracy of depth estimation for roots present at a level closer to the ground. We have found that this is due to the dielectric constant value being constant at shallow levels, because there is almost no change in the soil moisture content. However, as the depth increases, the soil moisture content rises and the dielectric constant increases, leading to the use of average value of all the changing dielectric constant values at deeper levels. All the results and discussion of these experiments are being prepared into a manuscript and will be reviewed further.We are also working on the development of an adjustable arm connection for the GPR to citrus trees. Several 3D modelled arms have been designed and are being tested to improve the functionality of the arm. The important functionality points we are considering implementing in the arm development is that (i) the arm fits all different tree sizes varying from young to old trees. (ii) the arm contains a measurement scale that can adjusted and fixed so that the 360 degree rotation around the tree remains constant at all time. In the coming quarters, we will:1) Develop further the new calibration method to improve the accuracy of depth estimation.2) Review the above mentioned manuscript containing results and discussion of the new calibration method and submit it for publication.3) Continue the development and testing of the automated GPR system to increase the efficiency of the system.
We have set up all of the experiments in the greenhouse and field needed to accomplish those objectives. We have also set up experiments in paralell with the field experiments. Given that citrate is the preferred carbon source for Liberibacter and that plants load citrate into phloem in order to acid mine insoluble phosphate from the soil, we expect foliar phosphate fertilization to reduce citrate in phloem so drastically, that it starves CLas.
We now know that foliarly-applied potassium phosphate will decrease phloem citrate levels in half after just one month of treatment compared to citrus saplings that were fed calcium phosphate to the roots. We are now analyzing samples to see whether citrate levels continue to decline after three months. We will keep this experiment going for the length of the project and will sample phloem citrate levels every three months.
The first greenhouse experiments on needed phosphate levels taught us the appropriate levels of potassium phosphate to spray on citrus trees in the field. In April 2019, a field trial commenced in a grove o 20-year-old infected trees near Lake Hamilton in Polk County. There were 10 replicates and four treatments in a randomized complete block design. We spray the trees with 0, 1x, 3x, and 9x the optimal level observed in the greenhouse every two months. Even at 1x, the plants are receiving enough P for flushing and fruit development. The plants are sprayed six times a year including after each flush. We sample these trees for phloem CLas levels every three months. The samples from the first nine months are now being analyzed. We expect to see CLas titer declines in the foliar phosphate-treated plants after one year of treatment.
A second field trial was established in August 2019 in the Immokalee area. That trial is the same design as that in Polk County. The idea was to have a trial on the ridge (Polk County) and flatwoods (Collier County) regions of citrus production. In both trials, the outcomes being measured are CLas titer in leaf midribs and leaf area index. Baseline samples were taken prior to the first spray and subsequent sampling will be done at 6, 12, and 18 months after the first spray. We have now sampled these trees for the first six months after treatment.
As we wait for field results, we are now testing the effect of a foliar potassium phosphate spray (compared to root-applied calcium phosphate) on CLas titer in graft-infected trees in the greenhouse. We are assaying those plants for CLas titer now and expect to have results in a week. This experiment will tell us whether foliar phosphate treatments can prevent CLas infection.
Given the speed with which foliar potassium phosphate can reduce organic hose plants acids levels in citrus phloem, we expect to see positive results in the field in reducing CLas titer in the first quarter of 2020. I am pleased to report that this project is working as planned so far. Foliar potassium phosphate does reduce citrate levels and the levels of other organic acids in phloem. As organic acids, particularly citrate, are the preferred carbon source for Liberibacter crescens, we expect this treatment to starve the pathogen.
We expect to observe CLas titer declines in these experiments over the next two months. If we do, a manuscript will be prepared very quickly in order to disseminate our results as quickly as possible. We will also ask CRDF for help in identifying more field locations for this work.
An extension article will follow shortly thereafter.
Our team (Triplett, Vincent, Killiny, and Wang) are working very well together and meet to discuss the project every two weeks.
In this quarter, we received permission to continue much our work on this project during the pandemic shutdown. All experiments in the greenhouse and field continue to be maintained.
We also obtained exciting results from our greenhouse experiment where Nabil Killiny graft-inoculated citrus saplings with CLas. We then applied the nutritional treatments to this plants to test the notion that leaf fertilization of potassium phosphate would reduce CLas titer in the leaf midribs compared to root fertilization with calcium phosphate.
Attached with this report are the data. With potassium phosphate fertilization of leaves, the propotion of unfected plants after 3 and 6 months is 80% and 86.7%, respectively. Plants fed calcium phosphate to the roots (analogous to field plants receiving no phosphate fertilizer) had only 40% and 38.5% of unifected plants at 3 and 6 months, respectively. Control plants given neither fertilizer were 26.7% and 38.5% uninfected at 3 and 6 months.
In contrast, after 6 months, 23.1%, 13.3%, and 0% of the highly infected plants were given leaf, root, or no fertilizaiton. Highly infected plants did improve over time with the foliar potassium phosphate fertilization.
So the greenhouse experiments have confirmed our hypothesis, foliar phosphate fertilization prevents and alleviates CLas infection. Treating plants with the equivalent of rock phosphate that is found in Florida citrus groves (calcium phosphate) encourages infection and it gets worse over time.
Now we need the field confirmation of these results. We were in the process of doing qPCR measurements of CLas leaf titer from our two field experiments, when we were ordered to stop data collection and reduce staff in the lab. This happened about the same time as the state-wide stay-at-home orders.
We expect to be able to get back to the qPCR measurements by mid-May.
We are also continuing with our greenhouse experiment on the effects of foliar phosphate treatments on citrate levels in phloem. Again, our efforts on this were curtailed on this with the shutdown. But we will be back at it soon.
All field and greenhouse experiments are on-going. Nothing was lost during the shutdown.
I submitted a preproposal to the USDA to expand our phosphate trials to 20 field experiments over the current two. That preproposal also includes a new disease model. I am eager to share those results with CRDF when the experiments are done.
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