Objective 1: Investigate the efficacy of bactericides treatments for preventing new infections for young citrus trees protection. Hypothesis: Bactericidal treatment will protect young trees from CLas colonization.Initial leaf samples were collected before treatments to evaluate CLas titers in the uninfected trees. Antibiotic treatments were applied from May 2019 through September of 2020. From early June through September, 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 and ACP from May 2019 through June 2020 are described. Currently, citrus leaves tissue samples from July through September 2020are being processed to analyze the CLas-infection rate. Results: CLas titers were highest during the past quarter compared to previous quarters across all treatments. The average tree CT value prior to the beginning og the study was 37.5-39.5. Trees are considered CLas-infected (positives) when CT values are below 35. Trees receiving monthly or quarterly applications of Firewall/Fireline tested CLas-positive beginning January 2020 and December 2019, respectively. Trees covered by Tree Defender bags or treated with insecticide only tested CLas-positive beginning December 2019 and February 2020, respectively.During May-June 2020, CLas values reached the higest level across all treatments since the study was initiated. CLas titers were lowest in the insecticide only treatment during this period, followed by monthly applications of Firewall/Fireline rotation. Trees receiving quarterly applications of Firewall/Fireline had the second highest titer of CLas, compared to other treatments. The highest titers of CLas during May-June 2020 were observed in trees covered with Tree Defender bags. Objective 2. Determine the effect of bactericides application frequency on Las infection of citrus. Hypothesis: Bactericidal treatment will reduce CLas infection in mature trees. Antibiotic treatments were applied from May 2019 through September of 2020. From early May through June, CLas titer was monitored in leaf tissue and Asian citrus psyllid adults in response to antibiotic treatments. Currently, citrus leaf samples from July through September are being processed to analyze the CLas-infection rate. Results: The average CT value of citrus trees at the onset of the study was 28. From May 2019-June 2020, trees receiving monthly applications of Firewall/Fireline had the lowest CT values, as compared with trees receiving quarterly antibiotic applications or insecticide only, with the exception of January 2020 when mean CLas titers across all treatments were not significantly different.
The purpose of this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Achieving this outcome will require progress in the following three tasks: (1) to compare the delivery efficacy of bactericides with three application methods (foliar spraying, truck injection, and root administration) based on the uptake and dynamic movement/distribution of the bactericide within the tree; (2) to clarify the systemic movement and transportation mechanisms of bactericides within the phloem of tree; and (3) to investigate the effects of citrus variety and age on the delivery efficacy of bactericides. This project requires a combination of greenhouse studies and field trials, and a reliable and accurate method for quantifying bactericides (oxytetracycline and streptomycin) in citrus tissues. This project officially started on December 1, 2018. This is the 3rd quarterly progress report covering 6/01 to 08/31, 2019. During this period we have started and/or completed the following work/research tasks:
1) The extraction method for oxytetracycline and streptomycin in citrus samples was further improved. Now we have a simpler and faster extraction method with a small SPE cartridge (Waters Oasis HLB 1 cc, 30 mg) to replace the previously used big one (Waters Oasis HLB 6 cc, 500 mg). Besides, the newly developed method was validated in terms of specificity, linearity, precision, accuracy and recovery with reference to the guideline established by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use Validation of analytical procedures.
2) We finished the planned citrus foliar spraying experiment. Plant samples (leaves, branches, and roots) were collected from the treated citrus trees at 0 (control), 7, 15 and 30 days after treatment. The samples were extracted and analyzed for bactericides using the above newly developed method. Preliminary results showed that both oxytetracycline and streptomycin could be detected in all the plant samples even at day 30 after spraying except for streptomycin in root. More biological replicates will be analyzed to verify the results.
3) For the trunk injection experiment, work was performed to develop some specific apparatus for trunk injection, and some trial tests were conducted to optimize flow speed of bactericide solution and ensure steady absorption of antibiotics into citrus trees under field conditions.
The work planned for the next quarter:
The major goals of research for the next three months are to obtain the final results of foliar spraying experiment and evaluate the delivery efficacy of bactericides within citrus trees based on the three different application methods (foliage spraying, trunk injection, and root administration). The following research tasks will be conducted in the 4th quarter: 1) More biological replicates of plant samples from the foliar spraying experiment will be analyzed to verify the present result; 2) The planned trunk injection experiment will be conducted; 3) Prepare for root administration of bactericide experiment.
Objective 1: Investigate the efficacy of bactericides treatments for preventing new infections for young citrus trees protection.
Hypothesis: Bactericidal treatment will protect young trees from CLas colonization.
Initial leaf samples were collected prior to treatments to evaluate CLas titers in the uninfected trees.
We applied bactericide treatments from May through September. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of CLas-infection rate in citrus leaves from May and June is described. Currently, citrus leaves tissue samples from July through September are being processed to analyze the CLas-infection rate.
*Trees were considered CLas-infected (positives) when CT values were below 35.
1. Antibiotics (monthly rotation): Prior to bactericide application (May), 15% of trees (20 trees/treatment) were CLas positive (Ct<35). After the bactericide application (June), 35% of trees were CLas positive (Ct<35).
2. Antibiotics (quarterly rotation): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After bactericide application (June), 40% trees were CLas positive (Ct<35).
3. Negative Control (insecticide + Tree defender exclusion netting): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After the bactericide application (June), 45% trees were CLas positive (Ct<35).
4. Positive Control (insecticide only): Prior to bactericide application (May), 100% of trees were CLas negative (Ct>35). After the bactericide application (June), 5% trees were CLas positive (Ct<35).
Counting of ACP adults using taps was conducted bi-weekly from May through September, presence of other life stages such as eggs and nymphs were scouted visually. Preliminary results showed a low ACP population in citrus locations due to the active vector management performed by farm manager. As consequence, no ACP adults were collected to analyze the CLas-infection rate using quantitative real-time PCR analysis. The overall number of eggs and nymphs were low or undetectable in citrus trees from May to September. Also, to determine the effect of citrus vegetative growth (flush-like structures) in CLas-infection rate, 1 ft.3 was used to count the number of flush-like structures per tree. Results showed that the presence of flush-like structures incremented from May to July and decreased in September.
Objective 2. Determine the effect of bactericides application frequency on Las infection of citrus.
Hypothesis: Bactericidal treatment will reduce CLas infection in mature trees.
We applied bactericide treatments from May through September. CLas titer was monitored in leaf tissue in response to antibiotic treatments using quantitative real-time PCR analysis. In this report, the results of CLas-infection rate in citrus leaves from May and June is described. Currently, citrus leaves tissue samples from July through September are being processed to analyze the CLas-infection rate.
*Trees were considered CLas-infected (positives) when CT values were below 35.
1. Antibiotics (monthly rotation): Prior to bactericide application (May), 100% of trees (20 trees/treatment) were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35). Although positive, bacterial titers declined in trees receiving antimicrobial treatments.
2. Antibiotics (quarterly rotation): Prior to bactericide application (May), 100% of trees were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35).
3. Positive Control (insecticide only): Prior to bactericide application (May), 100% of trees were CLas positive (Ct<35). After the bactericide application (June), 100% of trees were CLas positive (Ct<35).
Counting of ACP adults using taps was conducted bi-weekly from May through September, presence of other life stages such as eggs and nymphs were scouted visually. Preliminary results showed high ACP populations in treatments from May to August, excepting for June. The number of eggs and nymphs were not collected during May and first collection of June. However, populations increased from late June to August and reached high population levels. Currently, ACP adults that were collected bi-weekly are being processed to analyze the CLas-infection rate using quantitative real-time PCR analysis. Also, to determine the effect of citrus vegetative growth (flush-like structures) in CLas-infection rate, 1 ft3 was used to count the number of flush-like structures per tree. Flush was not collected during May and June. However, results showed that the presence of flush was high in July and August.
August 31, 2019 – In this quarter, we have continued to work on objectives outlined in our chronogram.
Objective 1. We have completed assessment of trees planted in our pilot study (planted 22 months ago) for CLas infection and HLB symptoms. All the non-covered trees are PCR-positive for CLas whereas all trees covered with IPC have tested negative. We are continuing with quantification of leaf drop and comparing leaf drop in both treatments; 6-month cumulative data show no significant differences in leaf drop in IPC-covered trees compared with non-covered trees. Interestingly, when counted seasonally, in spring leaf drop was significantly higher in non-covered trees as compared to IPC trees, whereas in summer, it was slightly higher inside IPCs. This fact points out a seasonal component that we will investigate as the project progresses.
In August, we have replaced the old 4-ft IPCs with new 8-ft covers, donated by The Tree Defender, Inc, because the trees had filled the volume of the cover completely. This also has opened the possibility of studying the dynamics of branch unfolding, which we are doing visually (photography documentation) and by measuring canopy growth and leaf area index. We have also assessed other pest and disease incidences inside the IPCs. We have found less incidences of canker inside IPCs and approximately equal incidences of greasy spot. However, greasy spot severity is higher inside the IPCs. We have found more incidence of other pests such as mites, armyworms, and leafrollers inside the IPCs, and a total absence of predators (beneficials). This suggests that relying only on IPC for insect control is not sufficient, and insect management must still be conducted. No psyillid have been found inside the IPCs.
Objective 2. To study the edge effect in different IPC layouts, we are now preparing to plant 700 trees of SugarBelle, Tango and Early Pride mandarins and using 3 different arrangements (targeted, alternated and patterned, as described in the proposal) of IPC. We have performed initial measurements of the tree parameters (trunk diameter, and leaf sampling, for CLas, cholorophyll and sugar analysis).
Objectives 3 and 4. We are continuing to measure fruit set and development inside the IPCs and comparing this with our CUPS planting. We are taking fruitlet and fruit samples regularly for biochemical analysis.
Outreach, Professional Presentations and Extension Activities for this quarter :
-Grower Presentation: “Growing Young Citrus Trees Under Individual Protective Covers (IPCs): What We Know After 18 Months” Citrus Expo 2019, August 15, Fort Myers, Fl.
-Industry Magazine Article: “Individual Protective Covers for Psyllid Exclusion and HLB Disease Prevention in Young Trees”. Article submitted to Citrus Industry Magazine in July to be published in October issue.
-Our Project was also noted in the September’s issue of Citrus Industry Mag’s UF/IFAS. The Citrus State Opinion Column by Jack Payne highlighted this work as an example of collaboration between growers, extension agents, and scientists in Florida. The column was entitled “Collaboration breeds solutions”.
August 2019 The objectives for this proposal are 1) Conduct ground and aerial applications of fungicides to determine the efficacy and economics of fungicide treatments; 2) Determine if Luna Sensation has enough systemic activity to protect flowers from before they fully develop and open; 3) Determine if the period flowering of trees affected by huanglongbing can be narrowed to eliminate the offseason bloom that contributes to the PFD inoculum increase in groves. Field trials were conducted each year to test new compounds and try new programs with existing compounds. Unfortunately, in all years but 2016, there was minimal PFD which did not allow for great confidence in the trial results We tried three different methods to investigate the question of Luna Sensation and did not come to a satisfactory answer. The first year, the experimental approach was flawed and the remaining years, there was insufficient disease to be able to generate an answer. We tried to induce flowering in the greenhouse but failed. Three years of PGR field trials were undertaken and it was found that GA does compress the flowering period but did not reduce yeild when applied once a month from September to December. It also appears to increase the amount of force needed to remove fruit from trees and reduce fruit drop. The Citrus Advisory Scheduler was developed based on a Brazilian model. It was modified and optimized for the Floridian situation. It brings data in from the FAWN system and will allow growers to monitor their risk for PFD. It also has a log in so growers can get alerts on their phones or e-mail systems. We did 4 years of validation trials for the Citrus Advisory Scheduler. We determined that the system is very reliable when indicating there is low risk for a PFD infection event. Unfortunately, there was limited disease during this program and we were unable to evaluate how well it worked when predicting moderate or high risk of PFD infection.
The grant covered continued funding of the SGCDL from July 1, 2017 through June 30, 2019. The lab has been under continuous operation since mid-2006 and has run over 350,000 samples since its inception. Sample results have been kept confidential with reports only being released to the sample submitter. Over the two-year grant period, 49,362 samples were run (down from 63,486 for the previous funding period). Of the samples run, 95% were plant samples (mostly leaves but some roots) and 5% were psyllid samples. Approximately 58% of the samples were run during year 1 of the funding (28,478) and 42% were run during year 2 of the funding (20,488). The great majority of the samples were from research trials. The fact that most of the samples were from research trials is similar to the previous funding cycle but represents a change from the early years where the samples were primarily diagnostic in nature. Since the samples were from trials, sample submissions tended to come in based on the timing required in the trial (i.e. twice a year, quarterly, monthly, etc.). Also depending on the trials, the samples tended to come in as large groups as opposed to individual small lots of samples. At times, samples arrived in groups of a 500 or more. This made scheduling a little difficult as the lab never knew when and how many samples were going to arrive. Thus sample turnaround varied from less than a week to up to 4-6 weeks depending on the sample load.During the previous funding period and continuing this funding period, and at the request of the customers, the SGCDL began quantifying the amount of DNA in the sample and running a standard curve in order to provide copy number in addition to the raw CT values. As time progressed, more and more of the samples were reported in this matter. For the current funding period, copy number was reported on approximately 50% of the outside samples (does not include internal Southern Gardens samples). The reduction in sample during the funding period is in part due to the effects of Hurricane Irma (which changed the planned sampling for many trials) and to a decline in the sampling of some of the CRDF trials, which traditionally has been the largest customer of the laboratory.
In the third quarter of this project, we summarized the results of our previous experiments that evaluated the influence of several limiting factors on the performance of a ground penetrating radar (GPR) to accurately detect HLB-affected citrus roots and determine their main structural characteristics. We prepared a manuscript that includes most of our experiments/results, and submitted it for peer-review to the Agronomy Journal, Special Issue of Precision Agriculture. The manuscript was accepted and published:Zhang X., Derival M., Albrecht U., and Ampatzidis Y., 2019. Evaluation of a Ground Penetrating Radar to Map Root Architecture of HLB-infected Citrus Trees. Agronomy (Special Issue: Precision Agr.), 9(7), 354. https://doi.org/10.3390/agronomy9070354. Received: 3 May 2019 / Revised: 23 June 2019 / Accepted: 1 July 2019 / Published: 3 July 2019.We have acknowledged CRDF:Funding: This research was funded by the University of Florida Citrus Initiative and the Citrus Research and Development Foundation. As explained in the previous reports (and based on our preliminary experiments), in order to collect accurate data from the GPR, a layout of the scans has to be prepared. This layout involves three concentric circles with a distance of 1 foot from each other with the trunk as the center. These concentric circles are drawn manually on the ground after measuring the distance from the trunk using a measuring tape and making marks. Once the circles are drawn around the tree, the scanner is moved along the circles manually by an operator moving under the trees. This process can be sometimes challenging as there may not be enough space to perform the scanning. As this process is done manually, the circles may not be perfect and there is a chance for errors to show up in the results due to irregular marking and drawing. In order to eliminate these problems and irregularities and also as an attempt to ease the scanning process we plan to automate the whole marking and scanning process.The idea is to put the scanner in an enclosed unit that is capable of being controlled remotely using the user. This enclosure is connected to the tree using an adjustable length bar to allow radial movement of the scanner. The adjustable length bar can have lengths varying from 1 to 3 feet. To achieve this, we built an aluminum chassis with wheels in a way that the scanner is touching the ground at all the time. However, with this design, we noticed some issues, especially with the wheels when rotating was stuck in the soil. This could be solved by increasing the size of the wheels but, since the scanner has to be in contact with the ground all the time, we did not proceed with this approach. Therefore, our next idea was to build an agile tracked chassis which can have a much better movement in soil and rough terrains. The track is connected to two drive wheels on either side of the chassis, which are in turn connected to motors controlled by a microcontroller. The motors are connected to a Cytron SmartDriveDuo Smart Dual Channel 10A Motor Driver, which allow the user to control the motors remotely using a remote controller. A RadioLink T8FB 2.4GHz 8CH Transmitter w/ R8EH 8CH Receiver was used for this purpose. Both the motors are connected to Motor Driver circuit and each of them can be controlled independently by the remote controller, which allows the chassis to make turns and perform radial movements. With this chassis, the scanner moves well on the ground even when there are irregularities present. In the next quarter, we will attach the adjustable length bars to the aluminum chassis and recognize the best way to attach them to the tree. The challenging aspect here is the varying size of the trunks, so the tree holder has to have a capability to adjust itself to hold on to varying trunk sizes and also at the same time allow the chassis to rotate around itself. We are currently working on this issue and have found certain methods to solve this problem to develop an automated system.
Our main goal was to find the reasons for inconsistent responses of HLB-affected citrus to Enhanced Nutrition (EN) programs and to develop feasible and economical remedies that can consistently replicate successful HLB mitigation with ENs in Florida groves. The two research objectives are summarized below.Research Objective 1: Establish nutrient sufficiency guidelines for leaf tissues of HLB-affected trees Using a survey data collection method of quarterly sampling in multiple groves of three Florida citrus production regions (Ridge, Indian River and Southwest), we assembled a large database of leaf, soil, and tree characteristics from a wide variety of HLB-symptomatic trees. Trees were classified into 5 HLB-severity classes and also grouped into “responding” or “non-responding” classes. Data were analyzed with ANOVA, DRIS, segmented regression, Cate-Nelson partitioning, and artificial neural networks in order to evaluate every possible dimension and interaction to elucidate linkages between measurements and tree performance or HLB severity. Using both segmented regression and Cate-Nelson methods with DRIS allowed us to identify new critical threshold concentrations (CTCs) for leaf nutrients of HLB-affected trees that can immediately be used to make tentative recommendations that supercede the somewhat aged CTCs published in UF/IFAS SL253 and elsewhere. There is definitely a need to update the IFAS guidelines for interpretation of sweet orange tree leaf nutrient analysis and we now have proof. When making decisions pertaining to the nutrient needs of HLB infected trees, it is important to realize that healthy trees and diseased trees behave differently. The evidence gathered in this objective shows a real need to modify these guidelines to reflect the increased need for all 11 nutrients, specifically potassium, magnesium, boron, zinc, manganese, iron and copper. With a larger database of responding and non-responding tree values these values could be improved. The results of this research were presented at grower events: Citrus Nutrition Day, Bartow; Citrus School, Arcadia; Citrus Institute, Avon Park, and published in the April 2019 Citrus Industry magazine. Research Objective 2: Determine soil conditions that favor root hair and VAM proliferation in HLB-affected treesUsing solid-phase tricalcium phosphate (TCP) as the sole source of phosphorus in a liquid culture experiment with Carrizo trees, we were able to limit the amount of available phosphorus in solution (< 1 ppm) and thereby stimulate the development of abundant root hairs by a factor of nearly two compared to a standard control with about 10 ppm soluble phosphorus. After exposing the experiment to HLB-carrying psyllids for two months, the TCP-treated trees had the lowest CLas titer, and we continued to see and measure healthy root hair development (now a 32x difference in root hair abundance, post-HLB) on the TCP treated tank solution plants compared to the control. The control tree's roots were nearly devoid of root hairs after trees became HLB positive, while the TCP-treated trees only suffered a minor reduction in root hair density. We conclude that these differences in root hair abundance could significantly improve tree health if the results could be replicated with soil-applied and incorporated TCP in the field. A paper on this research was presented at the FSHS conference in June.
The purpose is to evaluate the control effect of bactericides via trunk injection. Objective 1. To illustrate whether application of bactericides via trunk injection could efficiently manage citrus HLB and how bactericides via trunk injection affects CLas and HLB diseased trees.Three field trials have begun to investigate how the application of bactericides via trunk injection affects citrus growth, production, HLB symptom development, and CLas population in different aged trees at different levels of HLB disease severity. We developed a new method for evaluating the effects of oxytetracycline (OTC) treatment on CLas titers in planta, and determined the relationship between OTC residue levels and control levels achieved for CLas using mathematical modeling in greenhouse and field experiments. In either greenhouse or field, OTC spray did not reduce the titers of CLas and produced undetectable or mild levels of OTC residue in leaves within 7 days post application (DPA). In greenhouse, OTC injection at 0.05 g/tree decreased CLas 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 CLas 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 CLas achieved. The results suggested that the minimum concentration of OTC required to suppress CLas 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 CLas 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 CLas in planta for effective control of citrus HLB. Trunk injection is a promising delivery method for bactericides to control HLB. A manuscript entitled: The in planta effective concentration of oxytetracycline against Candidatus Liberibacter asiaticus for suppression of citrus Huanglongbing has been accepted for publication by Phytopathology.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. A field trial has begun to determine the concentrations of bactericides in leaf, stem, root, flower, and fruit using HPLC at the following time points: 2, 7, 14, 28 days, 2, 4, 6, 8, 10, 12 months after injection at different doses. The leaf samples are being collected at different time points for testing. Objective 3. To determine whether trunk injection of bactericides could decrease Las acquisition by Asian citrus psyllids (ACP).We are determining whether trunk injection of bactericides at three different doses could decrease Las acquisition by ACP in greenhouse and in the field. Objective 4. To monitor resistance development in Las against bactericides and evaluate potential side effects of trunk injection of bactericides. Las-specific primers were designed to target the putative binding sites of OTC in 16SrRNA gene of Las. Plant genomic DNA was extracted from citrus trees received OTC injection for three years. PCR were performed with the primers and DNA samples, and the products were purified and subjected to DNA sequencing. No mutation was identified yet. We have collected more samples from multiple citrus groves.
Our team meets every two weeks to discuss progress on this project. We have both greenhouse and field projects in progress.
In Gainesville, a greenhouse experiment is in progress to assess the effect of phosphate fertilization on citrate levels in phloem. In one treatment, we are spraying 2mM potassium phosphate at pH 6.5 on Valencia leaves. In another treament, we provide 2mM calcium phosphate at pH 6.5 to Valencia roots. The idea is that the plants fed calcium phosphate to roots will need to acidify the soil matrix with citrate in order to solubilize the phosphate and make it available. If we spray the leaves with potassium phosphate, no solubilization of phosphate will be required.
Samples from this experiment will be given to Nabil Killiny soon for citrate analysis in phloem.
At Lake Alfred, Nabil Killiny has extablished a terrific greenhouse experiment. He has grafted seedling to infect plants with CLas. He is supplying the plants with the two sources of phosphate as described above. In five months or so, we will assess CLas titer and examine HLB symptoms with the hopre that foliar potassium phosphate fertilization reduces titer and HLB symptoms while the root fertilization with calcium phosphate increases CLas titer and HLB sympotoms.
Meanwhile, the Polk Conty field trial was established and the first sampling is done. The field trials are led by Christopher Vincent. Nian Wang’s group is helping with sampling and the determination of CLas titer. These plants will be sprayed with foliar phosphate for the first time this week.
We have struggled to obtain a field site for this work in the flatlands region. After being unable to have a field trial with Southern Gardens and with a grower in the Indian River area, Dr. Vincent has found a site at the Southwest Florida REC in Immokolee. The experimental plots at that site will be et up next week. Initial sampling and the first spray will be done shortly there after.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require regulatory approval. Based on two year’s graft-inoculation assays in greenhouse with two HLB bacterial isolates and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees. The first group of the propagates on three different rootstock from the selections of Scott Grove’s seedling variants were grown at our research farm, Picos Farm, where the plants are under extreme high HLB disease pressure with very aggressive HLB pathogens. These new plantings (July, 2017; Nov, 2017; and May, 2018) showed different disease index, the longer the planting was, the higher the disease index, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infected plants. It is worth noting that the new HLB isolate from Picos Farm caused severe HLB disease on most of grapefruit selections of seedlings and bud sports in our latest greenhouse evaluation. Those selections were either resistant or tolerant to the previous HLB isolates we maintained in greenhouse. Prelimilary data showed some of the selections are better than the others with either lower disease index or better canopy growth. In general, the average infection rate was 20.85% for one group of grapefruit plants (470) 20 months after planting, and 5.46% for the other group of grapefruit plants (366) 15 months after planting. The second group of the propagates on three different root stocks mentioned above (Ca. 750 plants) have been budded and grown in our greenhouse, and over 400 plants in 12 combinations have been planted in Scott Grove, and the remainding plants will be planted in September. All of the propagates have been tested for the presence of Las. Propagates carring a low titer of Las will be planted separately and serve as the experiment for “cross-protection”.
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. We have completed construction of the rhizotrons and split the roots of and transplanted trees into the rhizotrons. Part of the delay was because we constructed new Aluminum framed rhizotrons with hollow framing to allow easy collection of irrigation leachate for nutrient analysis. This will also allow them to be sterilized and reused in the second rep without the risk of the frames rotting before the end of the experiment, which became a problem with the wood frames used in prior experiments. The trees for the first rep of the experiment will be inoculated within the week and irrigation water treatments will begin.
Project rationale and focus: The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Most recent quarter: Over 7000 infected ACP were used in the last quarter, in part to screen 450 trees, but also for other related uses. The Stover lab used 1700 ACP in no-choice inoculation of transgenic citrus. 2700 ACP were used for detached leaf assays in which leaves of putative CLas killing transgenics and related controls are exposed to CLas-infected ACP for 4 days, allowed 3 days for ACP-free metabolism and then assessed for CLas titer in leaves and the ACP. One thousand ACP were used in an assay in which CLas+ ACP are used to develop a uniform homogenate for rapid testing of putative CLas-disrupting peptides The Bowman lab has transitioned to use of grafted trees with a commercial scion in 2.5 liter pots. The first group of test plants will be removed from ACP inoculation the second week of July to begin post-inoculation evaluation. Subsequent groups of test plants for rootstock evaluation are being prepared. Previous quarter:The 35 day federal government shutdown, and the threat of a possible shutdown on Feb 15, directly disrupted our ability to initiate and conduct experiments using the CLas+ ACP colonies. In addition, considerable rehibilitation of colonies and supporting plants was necessary due to the minimal care that could be provided during the shutdown. Only 2400 CLas+ ACP were used for experiments in this quarter and were used for detached leaf assessments of plants expressing three different transgenic constructs. We anticipate a normal demand in the current quarter. Previously achieved in this project: As of December 21, 2018, a total of 14,111 plants had passed through the inoculation process. A total of 361,255 psyllids from colonies of CLas-infected ACP had been used in inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations were many used to refine inoculation procedures, which provided insight into the success of our inoculation methods and strategies for increasing success. After inoculations, plants were returned to the breeders and subsequently subjected to further inoculations when they are transplanted to the field. In addition to inoculating germplasm, infected psyllids were supplied to other researchers for other purposes. This side of the project grew over time, and detailed records were not maintained on how many were given out until 2018. More than 10,000 infected psyllids were supplied to the research community for an array of experiments during 2018. Recipients included researchers with USDA in Fort Pierce, Ithaca and Beltsville, UF in Gainesville, Cornell in Ithaca, University of California, and University of Nevada.
In this study, we will pursue the following specific objectives:1) GFP labeling of Candidatus Liberibacter asiaticus. 2) Elucidation of plant-Las interaction through real-time monitoring of Las movement and multiplication in planta using GFP labeled Las. 3) Investigate the effect of different control approaches on the dynamic population of Las in planta using GFP labeled Las. Previously, the reporter plasmid, pBAM1::R-PgyrA-GFP, composed of Tn5 and narrow host-range origin was constructed and therefore the GFP gene can be inserted into the genome of bacteria. However, it was only successfully transferred into a genome of Pseudomonas fluorescence with low transformation efficiency and failed with other bacteria including Escherichia coli DH5a, Sinorhizobium meliloti Rm1021, and Liberibacter crescens BT-1. Recently, pDH3::PgyrA-GFP was constructed which has a wide bacterial host range replicon, repW, but cannot be inserted into a genome. Transformation of E. coli by PEG mediated method with pDH3::PgyrA-GFP showed high transformation efficiency (~2 x 104 CFU/µg of DNA) than with previous reporter plasmid (failed). Following application with L. crescens BT-1 by electroporation was also successful (1.9 x 103 CFU/µg of DNA). Transformants and the GFP expression in L. crescens BT-1 were confirmed by PCR and fluorescent microscopic analysis, respectively. As L. crescens is a phylogenetically closest species to Ca. L. asiaticus, there is a possibility that pDH3::PgyrA-GFP would be useful for GFP labeling of Ca. L. asiaticus. We have further confirmed the Lcr-GFP using western blot. The GFP plasmid is being used to transform Las. To facilitate Las transformation, we have tested multiple novel methods of culturing. Las population was observed to decrease at the beginning, and increase slowly. Repeated experiments show similar pattern which suggest we might be able to acquire enough Las cells for transformation after further optimization. We are testing new methods for culturing Las. Especially, we are testing co-culturing Las with citrus tissue culture and psyllid tissue culture. Currently, we are in the process of establishing a pure psyllid cell culture. We have used two approaches to label L. crescens. Preliminary data showed one approach works for Las in vitro. We are testing whether we can label Las in vivo and observe its movement. 2) We have conducted Las movement and multiplication in planta based on qPCR method. We have tested approaches to prevent Las movement in planta. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. 3) We have been testing the effect of different control approaches including application with bactericides. One manuscript entitled: “Control of Citrus Huanglongbing via Trunk Injection of Plant Defense Activators and Antibiotics” has been published by Phytopathology. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. A manuscript entitled Targeted Early Detection of Citrus Huanglongbing Causal Agent ‘Candidatus Liberibacter asiaticus’ Before Symptom Expression has been published by Phytopathology. We determined the in planta minimum inhibitory concentration of oxytetracycline against Candidatus Liberibacter asiaticus effective for control of citrus Huanglongbing which has been accepted with revision by Phytopathology. In addition, we further characterized the movement of Las in planta and the treatment effect of antibiotics using trunk injection and foliar spray. Two manuscripts are being prepared.
July 2019 The objectives for this proposal are 1) Conduct field trials of new products and fungicide programs for PFD management as well as validation trials for the Citrus Advisory System (CAS); 2) Investigate the reasons for the movement of Postbloom fruit drop (PFD) to new areas and recent major outbreaks; 3) Evaluate methods for initial inoculum reduction on leaves so that early fungicide applications could be more effective and identify the constituents of the flower extracts using omics techniques. Objective 1 was mostly covered by project 16-010C and activities are also reported there. In 2019, a field trial was set up and treatments were applied in a Valencia grove in Ft. Meade. There were few predicted infection events this year and only one application was made based on the PFD-FAD or CAS model predictions. Button counts were collected. There were also two CAS model validation trials and button data were also collected for both of these. Fruit data should be collected by the end of July for all the trials. The experiments with the wind tunnel located at the ARS facilities in Ft. Pierce have commenced although access is still limited as progress is made for the USDA screening process. We hope to finish the screening process in the very near future for greater access. Preliminary trials with Colletotrichum acutatum in the wind tunnel indicates that the spores are able to move considerable distances in the wind alone. The modelling to indicate possible distances and gradient are very preliminary but indicate that some modifications to the experimental set up may be needed including increasing the number of repetitions since the variability was high. Work on modelling of leaf wetness to better predict PFD outbreaks in Florida is on-going. Five FAWN weather stations were selected for this work and are equiped with leaf wetness sensors. The recorded data was compared to the output of four leaf wetness estimation models, singly and in combination. The most accurate models were considered for modifying the citrus advisory system (CAS). Analyses to look at the number of recommended sprays and model accuracy are underway. Experiments to assess the effect of available fungicides on the secondary conidiation of C. acutatum on citrus leaves. In the intial experiment, pyraclostrobin was used and no difference was seen in the secondary conidiation regardless if sugar or water were used for stimulation of conidia. In subsequent experiments, ferbam was used instead. To stimulate sporulation, leaves were exposed to sterile water or 2.5% sucrose solutions with or without ferbam. Once the treatments were complete, the leaves were coated with nail polish and the conidia and appressoria stripped from the leaf surface and counted. Ferbam affected the viability of the spores but not the number produced. It also signficantly reduced the number of appressoria. These techniques are being used to assess the effect of the water, methanol, and ethyl acetate extracts of the flowers. Preliminary trials with the extracts are underway to make sure that the experimental procedures developed for the fungicides will be adequate or if modifications will be necessary. Flowers were collected and extracted using water, methanol and ethyl acetate. The yields of all the extracts have been calculated for future reference. All the extracts have been dried for testing their antifungal effects. Once we receive the antifungal effects of the extracts, we will start the composition analyses. Preliminary metabolomics analyses are planned once some data from the fungal tests are available.
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