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.
A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. In the last quarter, the most significant advances have been: 1) Planting of USDA Mthionin transgenics with 108 transgenic Hamlin grafted on wild type Carrizo (7 events represented), 81 wild type Hamlin grafted on transgenic Carrizo (16 events represented) and 16 non-transgenic controls. 2) Planting was made of transgenics from Zhonglin Mou of UF under Stover permit, with 19 trees of Duncan, each expressing one of four resistance genes from Arabidopsis, and 30 Hamlin expressing one of the genes, along with ten non-transgenic controls of each scion type. 3) Renewal and approval for BRS permit effective 9/1/19 through 8/31/20. 4) Continuation of an experiment on pollen flow from transgenic trees. FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. 5) Early-flowering transgenic Carrizo (flowered ex-vitro within five months of seed sowing, and used at 12 months) was used to pollinate some of the same FF-5-51-2 and some fruit appear to have set. 6) What should be the final samples from the C. Ramadugu-led Poncirus trial (#3 below) completed preparation and were shipped in ethanol to UC Riverside. Previously established at the site: 1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the Stover permit. 2) Under the Stover permit a replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF were planted. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 3) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. Likely 2019 will be the last year for data collection. 4) More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project. 5) A replicated Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant. 6) Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.). 7) In a project led by Fred Gmitter there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 9) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo. 10) Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site. New transgenics from Tim McNellis of PSU will be planted in the next quarter. 11) Availability of the test site for planting continues to be announced to researchers.
Two trials, (1) factorial combinations of perennial peanut in row middles with nematicide in irrigated zone and (2) efficacy against sting nematode of oxamyl, aldicarb, fluensulfone, fluopyram, fluazaindolizine, and an experimental compound, were treated with recommended rates of each nematicide. All nematicides except aldicarb and oxamyl were applied in dedicated irrigation lines by irrigating (microjet) for 30 minutes, injecting the nematicide for 60 minutes, followed by 30 additional minutes of irrigation. Prior to treatments, dye was injected sequentially into the manifold controlling each treatment to ensure the plumbing was treating all of the experimental trees. On May 30 and 31, two months after treatments were initiated, soil samples were taken from all trials and processed (sucrose centrifugation) for nematode determination. Two nematicides reduced sting nematode populations by more than 70%, with no significant effect yet by four compounds. Because there is evidence that some of the new compounds move in the soil very litte after application, the injection period will be extended to 2 hours in an effort to increase efficacy. Effects on fibrous root density are being processed. Treatments of most products will be resumed in the autumn. Perennial peanut is establishing reasonably well in all plots since the onset of the rainy season. The large effort of watering and weed management required of the sod laid during the spring dry season argues for starting such plots during the summer. Similarly, the sunn hemp crop did not establish well during the dry season and is will be replanted in early July. A second nematicide trial was initiated at a second young orchard by treating with aldicarb on 26 April, with all remaining treatments to be initiated in autumn after the plots are plumbed.
This project is an continuation of an objective of existing CRDF funded project (# 00124558 ; ended in March 2019) with some added treatments to be evaluated. The added treatments are:1. 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 These treatments have been initiated at all sites. The pretreatmentr data and first round of fertilizer application have been already made. The leaf and soil samples have been sent tout of nutrient analysis. Overally, the trees are looking good and they have a good crop loead. The trees seem uniform in symptom levels .The second application of the fertilizer will be made in late June-early July. A consumer taste panel was performed in April on 4 of the best performing treatments based on the yield.
The federal agency, USDA APHIS, will issue new guidance about the deregulation & environmental release of transgenics produced with plant pest sequences (35S promoter, Nos terminator, T-DNA borders) in the near future, which will significantly decrease the time & cost to release transgenics to the growers. This is extremely important for the citrus industry because Agrobacterium-mediated transgenics with plant pest sequences that are being field tested now could theoretically be commercialized, relatively soon. The FDA & EPA will still examine these plants. The final report of the external review of the transformation labs was received & it concluded that both transformation labs are necessary & do not have to be merged. This is encouraging since our objectives are completely different. However, now we are faced with what might be a formal split between the CRDF & UF over the role of direct support organizations. The Mature Citrus Facility produced ~85 transgenics this quarter, which is a significant increase in productivity & we are not sure what caused this increase. Use of the gfp reporter in some transformations, which makes transgenics easier to identify likely contributed somewhat to this increase, & the fact that it was spring when our productivity naturally increases probably contributed. We always seem to produce more transgenics during spring. However, not all will grow large enough for micrografting & only 57 transgenics have been micrografted thus far. Of these 57 shoots, 30 will definitely survive. I submitted a research proposal to the National Science Foundation (NSF). It will take ~6 months to review & ~7% of proposals are funded. In collaboration with another scientist, a letter of intent was submitted to USDA AFRI for another research proposal. I contacted many scientists to advertise our services but have heard back from few. In the future, after transgenics have been made for current customers, our prices will increase to better cover operating costs. Our biolistic transformation manuscript in In Vitro Cellular & Developmental Biology is finally in press. It took a long time for this journal to review & publish the manuscript. Currently we are working on other manuscripts with other UF CREC scientists. We have introduced additional new cultivars from the UF Plant Improvement Team & other breeders. EV2, WP Murcott, Valquarius, OLL4 scions have been introduced. OLL4 & Valquarius have very high Agrobacterium transformation efficiency. UF 15, UF 17, X639, US 942 rootstocks were introduced & are being tested in Agrobacterium transformations. We also recently introduced several grapefruit cultivars (Flame, Marsh, Ray Ruby & Duncan). In the past, grapefruit (Ray Ruby, Grosser’s Red Grapefruit, & Ruby Red Grapefruit) were recalcitrant using the standard Agrobacterium protocol.
The long term field trial continues with weekly psyllid counts and quarterly CLas infection testing. Treatments continue to have similar effects on ACP counts. Plants in both of the kaolin treatments continue to show higher growth rates than the other two treatments. The red treatment has the highest growth rate, trunk cross-sectional area, and canopy volume. Kaolin treated trees that are infected grow more than untreated-infected trees, but less than treated uninfected trees. The field trial will continue until the project ends, when we expect to have the first economic yield. We are now performing follow-up repetitions of the MS student’s thesis work. We anticipate publication submission of this work in the Fall.
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