Our survey of 8 ridge groves in Highlands County and 4 flatwoods groves in Hardee Co. with high bicarbonate stress continues. The survey is bimonthly to follow the recovery of these blocks and at harvest to compare 2015 season block yields 2.0 to 2.5 years after acid treatments began. Soil pH continues to range from 5.0-6.0 in the ridge and in the flatwoods groves has dropped after acidification treatments resumed in fall 2014. The additional survey of 5 grove locations initiated January 2015 in the flatwoods with relatively low bicarbonate stress continues. The root zone pH of these groves is less than 6.5 and fibrous root density higher than in high stress flatwoods groves surveyed in 2014. Phytophthora populations are damaging in 3 of the 5 groves in the new survey. To lower the pH or to increase the calcium status, the grower is considering Tiger Sulfur or gypsum amendments according to soil and leaf test.
For 2015 season, FireWall 50WP (50% streptomycin; Agrosource, Inc.) has been granted an EPA section 18 registration for control of citrus canker in Florida grapefruit. The label for FireWall restricts use to no more than two applications per season. As a condition for FireWall registration, EPA requires monitoring of Xanthomonas citri subsp. citri (Xcc) for streptomycin resistance in treated groves. The objective of this survey is to apply our published protocol (Behlau et al., 2012) for sampling canker-infected grapefruit leaves for isolation and detection of streptomycin resistant Xcc. The survey for 2015 season will be conducted in November 2015 and the report of results submitted to FDACS. Greenhouse trials to measure the residual systemic activity of streptomycin against Xcc in leaves after foliar spray confirm trans-cuticular and upward movement of streptomycin into new foliage via the xylem.
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing in Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, and an additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct are growing at Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. At Penn State, all the transgenic lines have been successfully propagated as vegetative cuttings. All of the lines growing at Penn State have been found to express the FLT-antiNodT fusion protein, with five of the seven lines expressing very high levels of the protein. We must continue to let these plants grow a bit more before starting the HLB resistance tests. We have initiated a collaboration with Dr. Janice Zale (University of Florida Mature Citrus Transformation Facility, Lake Alfred) to transform varieties important to the Florida citrus industry, including the ‘Valencia’ and ‘Hamlin’ sweet orange varieties and the ‘Citrumello’ rootstock with the FLT-antiNodT expression construct. We have immediately started transformations with the available transformation construct used to transform ‘Duncan’ grapefruit, in plasmid pTLab21. In addition, we are developing an FLB-antiNodT expression cassette in the transformation construct pBI121, which has a history of successful approval for transgenic plant development. In June, Dr. McNellis submitted a Stakeholder Relevance Statement to the USDA Specialty Crop Citrus Disease Research and Extension program to further develop this project. However, a full proposal was not invited. Dr. McNellis will present a poster describing the results of this project to date at the American Phytopathological Society conference in Pasadena, California, August 1-5, 2015.
Currently there are 9 total trails evaluating thermotherapy in the state. 8 of the trials are underway with the 9th yet to begin. 2 of the trials were treated by Dr. Eshani while the remaining 3 trials used private machines. Site 1 Cliff Whitaker – Lake County. This trial was done in conjunction with Dr. Ehsani. There are 20 trees being evaluated in 4 classification: steam, thyme oil, steam and thyme combined, and control. Each group has 5 trees. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, and fruit drop counts. Site 2 Larry Davis – Hardee County. I contacted Mr. Davis on 4/3/15 and set up the trial on 4/6/15. There are 24 trees being evaluated. All of the trees are steam treated, larry didn t want to leave any trees untreated. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, and fruit drop counts. Site 3 Premier Energy Raley grove in Dundee this trial was set up 5/21/15 and treatment was 5/28/15. There are 40 valencia on Carrizo trees being evaluated, 10 control and 30 treated. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts. Wheeler grove in Lake Wales This trial has 15 control trees and 15 treated trees. The trail was set up on 6/9/15 the treatment was done on 6/12/15. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts. Dunson grove in Haines City – this trial was set up on 6/8/15. Treatment has yet to be done. This grove is under a less intense management strategy. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts. Site 4 Daniel Scott St. Lucie County Trial A – 36 trees (Red grapefruit on Sour) this trial was a comparison of 3 different temps applied at different durations. The grower was trying to determine what combination of time and temperature worked best. Measurements being taken are DI, height, and PCR. Trial B 54 (Red grapefruit on Sour) this trial was a comparison of 3 different temps applied at different durations. The grower was trying to determine what combination of time and temperature worked best. Measurements being taken are DI, height, and PCR. Trail C 35 trees (ray ruby on sour) 20 treated trees and 15 control trees. We will take post treatment PMA samples on 7/23/15. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts. Site 5 Uncle Matt s Eddy Block in Winter Garden. This is the only trial that is being conducted in an organic grove. Most of the trees are in poor condition. If thermotherapy works here, I ll have hope for the whole industry. There are 10 control trees and 20 treated trees. The study trees are split into 4 groups: Large treated trees, Small treated trees, Large control trees, and Small control trees. Measurements being taken are DI, trunk circumference, height, canopy diameter, PCR, % leaf drop, and fruit drop counts.
The project has two objectives: (1) Increase citrus disease resistance by activating the natural SAR inducer-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we treated citrus plants with the natural SAR inducer using three different approaches: leaf infiltration, foliar spray, and soil drench. Three concentrations were tested: 1, 5, and 10 mM. For leaf infiltration, the infiltrated leaves were inoculated 1 day later with the canker bacterial pathogen; for foliar spray, treated leaves were inoculated 3 days later; and for soil drench, leaves on treated plants were inoculated 7 days later. For each treatment, 5 plants were used. Three leaves on each plant were inoculated and 6 inoculations on each leaf were conducted. A total of 90 inoculations were used for each treatment. Fourteen days after inoculation, numbers of lesions formed on the inoculated leaves were counted. Results showed that all concentrations of the SAR inducer induced strong resistance to citrus canker. The inoculated plants have been cut back. Systemic residual resistance will be tested on the new flushes. We are repeating this comprehensive testing experiment and will identify the most efficient treatment method. For objective 2, transgenic citrus plants expressing the Arabidopsis nonhost resistance genes have been propagated. The progenies are growing in greenhouse and will be tested for disease resistance.
Report for period ending 3/31/15 During the period of 1/1/15 to 3/31/15, Mr. Page assisted with the daily activities associated with CRDF funded field trials. During this reporting period, Mr. Page provided assistance on a daily basis through activities as described in the following: Stayed in constant contact with contracted crop consultants (CC’s) to make sure trials were on schedule, reports were to be submitted on time, and trouble shoot any potential obstacles that might arise in advance. As needed, supplies were ordered and delivered to the CC’s to ensure samples were collected and processed in a timely manner. During this period which coincided with fruit harvest season, Mr. Page worked with the CREC packinghouse manager to coordinate the delivery and analysis of fruit samples from field trials for fruit quality assessments. This involved making sure the proper analyses were planned and fruit was delivered on schedule. For trials where quantification of live vs. dead HLB bacterium assessment was required, coordination was made with the Wang lab (CREC) to ensure the proper protocols were being followed to ensure samples were collected and processed in the appropriate manner. During this time, the large-scale evaluation of new rootstock plantings was underway. Mr. Page worked with the grower-cooperators to make sure plots were planted and flagged to make sure data could be collected in a timely and efficient manner. This involved continued communication with the nurseries and growers to make sure trees were delivered and planted according to schedule. Multiple on-site visits were made to these locations with photo documentation of the entire process. For other ongoing CPDC projects, work continued collecting leaf/root samples, coordination of laboratory testing and fruit analysis was conducted as required. Because of the increasing number of trials and on-site visits required for monitoring and data collection, two new employees were hired to help conducted field trail evaluations.
Report for period ending 6/30/15 (Final Report) During the period of 4/1/15 to 6/30/15, Mr. Page assisted with the daily activities associated with CRDF funded field trials. During this reporting period, Mr. Page provided assistance on a daily basis through activities as described in the following: The major focus of field trials underway at this time were the new large-scale plantings being established. Numerous on-site visits were made as these plantings were being established to ensure plots were appropriately established and marked, tree measurements were made and PCR samples were taken. In one situation, a problem was noted with insufficient tree counts being received from the nursery. Mr Page worked to correct this short-fall in trees delivered to make sure planting stayed on schedule. With the assistance of his newly hired employees, Mr. Page has kept up to date with all the ongoing field trials, collecting tree growth data, PCR analyses, and root and fruit samples required by the established monitoring protocols. Additional trials are being established evaluating GRAS compounds and other treatments for which evaluations have been contracted through the CRDF CPDC. In the coming year, Mr. Page will continue to provide oversight to these ongoing projects to ensure that all required sample analyses, data collection and reports are submitted in a timely manner.
Report for period ending 12/31/14 During the period of 10/1/14 to 12/31/14, Mr. Page assisted with the daily activities associated with CRDF funded field trials. The general duties provided by Mr. Page on this project include: attending routine meetings with Drs. Browning and Syvertsen to provide updates on the status of trials, maintaining constant communication with crop consultants (CC’s) to ensure projects are moving forward, setup contracts with soil testing laboratory, sort out results from testing labs, submission of invoices, contact product reps for material samples, organization of spreadsheets for data analysis, scheduling submission dates for the CC s field trial data and on-site visits to field sites to make sure trials are on schedule and up-to-date. During this reporting period, Mr. Page provided oversight on the numerous ongoing CRDF-CPDC projects. Activities included organizing submission dates for CC’s to submit data, collecting root samples for analysis from CC’s and getting those samples to the appropriate laboratory for testing in a timely manner, working with researchers (in particular Nian Wang) to streamline sample submission for PCR testing, work with researchers to implement the proper canker evaluation protocols for ongoing trial work, travel to meet CC’s to pick up plant samples for testing as needed, on-site visits to field trials to photo-document ongoing trials, take tree growth data and collect leaf samples for PCR analysis.
Report for period ending 12/31/14 During the period of 10/1/14 to 12/31/14, Mr. Page assisted with the daily activities associated with CRDF funded field trials. The general duties provided by Mr. Page on this project include: attending routine meetings with Drs. Browning and Syvertsen to provide updates on the status of trials, maintaining constant communication with crop consultants (CC’s) to ensure projects are moving forward, setup contracts with soil testing laboratory, sort out results from testing labs, submission of invoices, contact product reps for material samples, organization of spreadsheets for data analysis, scheduling submission dates for the CC s field trial data and on-site visits to field sites to make sure trials are on schedule and up-to-date. During this reporting period, Mr. Page provided oversight on the numerous ongoing CRDF-CPDC projects. Activities included organizing submission dates for CC’s to submit data, collecting root samples for analysis from CC’s and getting those samples to the appropriate laboratory for testing in a timely manner, working with researchers (in particular Nian Wang) to streamline sample submission for PCR testing, work with researchers to implement the proper canker evaluation protocols for ongoing trial work, travel to meet CC’s to pick up plant samples for testing as needed, on-site visits to field trials to photo-document ongoing trials, take tree growth data and collect leaf samples for PCR analysis.
Our project aims to analyze gene regulators from Ca. Liberibacter asiaticus by expressing these in the related bacterium, Sinorhizobium meliloti. In previous reports we described initial success in cloning the CLas homolog of RpoH, and in expressing this to examine one phenotype. In our work from January-April 2015, we worked on optimizing expression. In the following section, the abbreviation Sm rpoH1 means the native S. meliloti rpoH gene, specifically the rpoH1 copy which is the most important rpoH gene for the S. meliloti stress response; La rpoH designates the CLas homolog with codon usage optimized for expression in S. meliloti; .rpoH1H2 is a host strain of S. meliloti in which both of the native copies of rpoH have been deleted. The pSRK-Gm vector expressing Sm rpoH1 and La rpoH has a lac promoter (inducible by IPTG) to control rpoH expression. 1. We have strains in which RpoH-controlled genes (targets) are fused with the uidA gene (GUS). We tested the Sm rpoH1 plasmid and optimized La rpoH plasmids in strains with rpoH target gene-uidA fusions in the .rpoH1H2 strain. a. Under inducing conditions (+IPTG), expression of the target gene fusions is much higher when the native S. meliloti rpoH genes are deleted. b. In addition, expression levels are higher with the La rpoH plasmid than with the Sm rpoH1 plasmid (we discussed possible reasons why in Dec 2014 report). c. We determined empirically that the optimum IPTG concentration for induction is 0.5 mM. 2. Another way to optimize expression is to test various ribosome binding site (RBS) sequences. We re-made Sm rpoH1 and codon-optimized La rpoH constructs in the same pSRK-Gm vector, but utilizing the vector RBS instead of the RBS inferred from S. meliloti genes predicted to be highly expressed (published by Schroeder et al., 2005, Appl.Env.Microbiol 71:5858). The goal of this test was to determine whether using a different RBS/cloning site would decrease the leakiness of the lac promoter (as discussed in Sept 2014 report). 3. The S. meliloti .rpoH1H2 mutant does not grow at 37 C. We tested the ability of constructs in (2) to restore growth of the .rpoH1H2 mutant at 37 C IPTG, and to carry out regulation of target genes with target gene-uidA fusion assays. a. In the heat stress assay (complementation of .rpoH1H2), non-induced controls (minus IPTG) show much less growth than induced (plus IPTG) strains. b. Basal (minus IPTG) GUS activity of rpoH target-uidA fusions is lower. c. Therefore, these new constructs are more tightly regulatable than the constructs using the S. meliloti RBS consensus. Several important lessons come from these results. First, we now know that native genes (such as Sm rpoH1 and rpoH2) can confound observation of introduced CLas genes. This may have implications for experimental design in the next group of experiments. Second, we know that the RBS can make a difference in whether we get tight off/on control with IPTG induction of the RpoH. These results will inform our next round of constructs and complementation tests.
In our work from April to July 2015, we moved forward to start molecular analysis of Liberibacter asiaticus rpoH effects on transcription. We also began the second major phase of work, in which we will study Liberibacter transcription regulators. We made use of new information about the effect of endogenous S. meliloti genes and the vector ribosome binding sequences (RBS) described in the March 2015 report. 1. Molecular analysis with the newly-characterized RpoH-expressing constructs: a. We have prepared RNA for S. meliloti strains containing the following plasmids: plasmid expressing Sm rpoH1; plasmid expressing optimized La rpoH; and the empty vector control, pSRK-Gm. These RNA preparations now will be processed for Affymetrix GeneChip analysis. 2. In our proposal, we aimed to study RpoH and also a set of putative transcription factors. Having completed construction of rpoH strains, we designed the optimized genes for the remaining six regulators: as before, we substituted codons of the native La rpoH gene for codons that are compatible with the GC-rich genome of S. meliloti. We ordered and received optimized DNA for these six genes. These were cloned into the pSRK-Gm vector, using the minimal background vector RBS. a. The codon-optimized La visNvisR genes were cloned as a single insert to maintain operon structure. We used the same intergenic spacer sequence between visN and visR that is present in S. meliloti. 3. We did not originally propose to create deletion strains lacking each orthologous S. meliloti regulator. However, because we found that the native rpoH genes interfered with our ability to observe the full effect of plasmid-based rpoH, we decided to construct strains with each corresponding regulator deleted. For example, we now plan to make a visNR deletion strain of S. meliloti in which to test regulation by the optimized La visNR gene; we will construct a S. meliloti ctrA deletion strain to host the La ctrA plasmid, etc. a. We are in the process of making these deletion strains using our customary sacB plasmid deletion method. In this method, a vector bearing a resistance marker and the sacB gene is used to clone DNA from regions flanking the gene to be deleted. After recombining into a wild-type S. meliloti host by a single cross-over event, the strain is plated on sucrose, which is lethal unless the sacB-containing vector has recombined a second time and been lost from the cell; among such double-crossover events, some restore the wild type gene and some have a clean deletion of the target sequence. Deletion strains are confirmed by performing PCR and sequencing. b. Results to date: We have single-crossover strains containing the sacB construct for all six chosen regulators. c. Of these six, two strains are complete and confirmed for deletion: the visNR deletion strain and ldtR deletion strain. d. We are presently screening for putative deletions of phrR1, phrR2, and lsr, and are planning a two-step construction of a ctrA deletion strain (needed since ctrA is required for basic viability).
A transgenic test site at the USDA/ARS USHRL Picos Farm in Ft. Pierce supports HLB/ACP/Citrus Canker resistance screening for the citrus research community. There are numerous experiments in place at this site where HLB, ACP, and citrus canker are widespread. The first trees have been in place for over five years. A number of successes have already been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, continues to replant with new advanced material, with ~200 new trees in April 2015 (Grosser, personal comm.). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material is being planted this spring that has shown great promise in the greenhouse (unpublished). A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 5 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Lee et al., in preparation), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). A new UF-Gmitter led association mapping study has just been initiated using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the sire in a project led by UC Riverside/USDA-ARS Riverside, in which half of the trees of each seed source were graft-inoculated prior to planting. A collaboration between UF, UCRiverside and ARS is well-underway with more than 1000 Poncirus-hybrid trees (including 100 citranges replicated) being evaluated to map genes for HLB/ACP resistance. Marked differences in initial HLB symptoms and Las titer were presented at the 2015 International HLB conference (Gmitter et al., unpublished). In July 2015 David Hall will be leading assessment of ACP colonization across the entire planting, and the Gmitter lab will map markers associated with reduced colonization. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. As of April 2014 at the Picos Test Site, several of these USDA hybrids had grown to a height of seven ft, with dense canopies and good fruit set, while sweet oranges are stunted (3 ft) with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population will be planted at the Picos Test Site in July in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting will also include a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been Las-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.).
Chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) are being tested. Carrizo transformed with a chimera AMP showed remarkable resistance in citrus canker compared to control. RT-qPCR showed 50% of 16 chimera transgenic Hamlin have relatively high gene expression, with 100x difference between high and low expressers. These promising transgenic lines were replicated by grafting for HLB challenge. Twenty transgenic Hamlin lines were confirmed to contain thionin gene by PCR, and six have high gene expression by RT-qPCR. Transgenic Hamlin lines expressing thionin were grafted onto Carrizo for HLB challenge. Replicated transgenic Transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon. Promising resistance to HLB was observed based on plant growth and phenotype. Las titer is being checked from root and new flush rough lemon leaves. Two new chimeral peptide from citrus genes only were developed and used to produce many Carrizo plants and Hamlin shoots. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Many putative transformants were generated on the selective media: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. There is only slight canker resistance by infiltration, but considerably resistance to spray inoculation. To confirm that high ROS production was not due to variability in Hamlin, we examined l 40 Hamlin seedlings and no or very low level ROS production was detected. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. Primers were designed for two citrus FLS2 orthologues. They showed low gene expression in transgenic and nontransgenic citrus. Results on FLS2 transgenics against canker disease were summarized and a manuscript was written and submitted to MPMI. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
Objective 1: Using the Hamlin/Swingle trees available at the Gapway Grove ACPS site in Auburndale, FL we were able to determine the following. 1) The vascular anatomy of Hamlin trees dramatically adapts to the irrigation program. Trees growing under the traditional grower irrigation program with 1-2 pulse irrigation events per week were, generally, much more resistant to the effects of drought and better able to maintain water transport. The daily minimum water potential was significantly less negative (i.e. less water stress) in the grower irrigation program compared to ACPS trees. 2) The ACPS trees, with abundant and regularly applied irrigation water, appear to have maximized the efficiency in delivering water to the canopy. ACPS xylem specific conductivity was nearly two fold higher than trees in the grower irrigation program, meaning that water could flow through these trees to the canopy at significantly higher volumes. This increase in conductivity came at the cost of resistance to drought induced embolism formation that decreases conductivity. 3) The increases in ACPS conductivity were largely attributable to slightly wider conduit diameters, changes in vessel grouping, and decreased pit membrane thickness, which is one of the major points of resistance to water flow in the xylem. 4) These data suggest a high degree of plasticity of Hamlin orange to irrigation programs, and the increased water transport capacity that developed in ACPS trees may be one of the main contributors to the faster growth rate and higher yield found in these blocks of trees. Objective 2: For this objective, a ~100% HLB-affected mature Hamlin grove with both Swingle and Carrizo rootstocks was used to determine whether plant growth regulators (PGRs) could be used to mitigate preharvest fruit drop. We applied 2,4-D (synthetic auxin) at a low (below labeled rate) 5 mg/L dose in 100 gal/acre foliar spray using an orchard airblast sprayer, starting from the time petal drop completed on 3/28/13, and repeating every six weeks. The 2,4-D sprays were timed to coincide with the four routine foliar nutrition sprays applied in the growing season to help correct nutrient deficiencies caused by HLB. Therefore the final treatment spray was applied in September. In this field experiment we also added EcoAgra , a commercially available plant-based surfactant based on nano-emulsion technology, with claims of improving plant health and vigor. The treatments were structured factorially, so that 2,4-D and EcoAgra were tested individually as well as in combination. We monitored fruit drop nine times with hand-raking of dropped fruit, every two weeks starting in September through harvest in December, and found no consistent significant differences between the different treatments. For example, statistical differences were found on only the third sampling date, where 2,4-D and EcoAgra both reduced fruit drop relative to the untreated control. At this point we do not recommend 2,4-D at the concentration and frequency applied in this study to be used to mitigate preharvest fruit drop, but these data should be considered within the scope of other PGR trials occurring simultaneously around the state, including those using 2,4-D at a single application, labeled rate. Part of the reason for lack of statistical significance in the total season-long fruit drop count is due to compensatory effects. A reduced fruit drop due to 2,4-D treatment early on like at the DROP[3] stage may have been compensated by a higher drop later on. This is a likely outcome since fruit retention represents cumulative stress to a tree. Only if everything works out perfectly with the remedy meaning total health improvement season-long will the tree likely retain its fruit in the long term as it would do if it was uninfected.
During the life of this project, 121 mature citrus transgenics were produced using genetic transformation with Agrobacterium. Sixty-six were transgenic for reporter genes and provided proof of concept that this protocol works in our hands. In the last 21 months, 55 transgenics with disease tolerance genes, most without reporters, were produced and micrografted onto rootstock. Agrobacterium transformation efficiencies were relatively low (3.47% positive shoots for constructs with reporters in scion, and 3.0% positive shoots for constructs with no reporters in scion and rootstock). Only 0.78% shoots/total explants plated were transgenic. Many more transgenics were probably produced, but were lost in the micrografting process. Attempts are being made to increase micrografting efficiencies. Although 9 vectors carrying disease tolerance genes were used in sizable transformation experiments, only 4 vectors yielded transgenics. The 55 mature citrus transgenics were produced with these 4 vectors to confer disease tolerance to HLB, canker, or both. These primary transgenics are being propagated into vegetative progeny to facilitate replicated field trials this fall. Numbers were low for Ray Ruby grapefruit (3 transgenics) and efforts are being made to optimize the tissue culture and transformation protocol for grapefruit. Experiments are underway to root mature scion because a larger scion could be easily micrografted onto rootstock with greater efficiencies. To increase our chance of success, we have been utilizing nurse cultures to supply additional nutrients to developing mature shoots. Additional vectors are being acquired from scientists around the country and worldwide. Budding is now done entirely in-house. A gene gun was purchased in July, 2014 to develop a high-throughput biolistics transformation system for mature citrus. Transient expression levels are relatively high, and a few stable transgenics have been produced. Optimizations for mature citrus have been hindered by the limited supply of mature scion in the growth room, which was primarily used for Agrobacterium transformations. In the future, we will purchase mature citrus from nurseries to continue optimizations. We have developed a high throughput screening system in which thousands of putative transgenics can be rapidly screened. A number of equipment expenditures were necessary to achieve this high level of efficiency. Equipment expenditures included a refrigerated centrifuge, a plate reader, a tissuelyser, a laminar flow bench and an incubator. A hybridization oven and dry baths were purchased for molecular analyses. Maintenance expenses for lighting, AC repair, sensors and expansion cards for RCWebview, and the water softener in the growth room are ongoing. This project depends upon a continual supply of healthy, viable rootstock seed and this was problematic last year. Rootstock of Swingle and Volkameriana had poor germination, and Macrophylla and Carrizo had disease/endophyte issues that negatively impacted budding of mature citrus and the tissue culture process.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
