Transformations of citrus plants with the FLT-antiNodT fusion protein expression construct are now underway. The transformations are being performed at the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The FLT-antiNodT expression cassette is being introduced into ‘Duncan’ grapefruit by Agrobacterium tumefaciens – mediated transformation. Transformations were begun on November 5, 2013. The transformation construct includes a green fluorescent protein (GFP) marker. Within a few weeks of initiating the transformation experiment, clusters of green-fluorescing citrus cells were observed, indicating that the FLT-antiNodT fusion protein transformation vector was working. This was important, because we had encountered significant difficulties in the development of the FLT-antiNodT fusion protein transformation vector. All indications are that the transformation vector is working as expected, which is good news. Transgenic shoots were successfully regenerated that are green-fluorescing and presumably carry the FLT-antiNodT fusion protein expression cassette in their genome. As of December 13, 2013, micro-grafting of transformed shoots onto recipient plants was begun. Successful regeneration of shoots containing the FLT-antiNodT fusion protein expression cassette is critically important. Sometimes certain proteins and transgenes might interfere with the normal plant biology in such a way that regeneration of normal plant tissues cannot occur. We are pleased to report that this has not been a problem so far for this project. All indications are that we should be able to obtain transgenic plants suitable for testing for HLB resistance. This project has gone a bit more slowly than initially anticipated. All steps of the project took more time than initially predicted, from antibody development and cloning to the development of the transformation construct. It is likely that we will take at least until August, 2014, to obtain transformed plants that are suitable for HLB resistance testing. We will also need time to do laboratory experiments characterizing the expression of the FLT-antiNodT fusion protein in the transgenic plants. Therefore, we fully anticipate needing to request a minimum of one year no-cost extension to this project. A no-cost extension of the project will be necessary to achieve the goals of the project fully.
This research is directed at determining the activity of Non-antibiotic tetracycline analogs against the causative agent of HLB. Through chemical changes within the family of tetracycline derivatives it has been determined that specific derivatives harbor increased potency against surrogate strains of Liberibacter used to assess antibioitic compounds in vitro. In a several series of derivatives analogs were found to possess 2 to 3 orders of activity against the test strain compared to oxytetracycline, and EPA registered antimicrobial agent, and a 1st generation tetracycline useful against Liberibacter and other alpha-proteobacteria. In these studies, conducted at the University of Florida, separate compound series have emerged, with limited or no activity against human Gram positive and negative pathogens, allowing their classification as Non-antibiotic compounds. Additionally, clinically used tetracyclines, doxycycline and minocycline, had no activity against the surrogate strain, delineating specific structure-activity properties in these non-antibiotic tetracyclines. Other relationships exist between potency and activity in vitro, and further synthetic studies will be done to further increase activity against the causative agent of HLB. Other studies using non-antibiotic tetracyclines have demonstrated that specific compounds and series are able to be formulated using specific systems for delivery and application to the tree bark systems, showing enhanced penetration and distribution throughout the citrus tree via phloem transport. In future studies 2 compounds have been readied for field trials, with current enhanced formulations, and other and more potent compounds will be synthesized and examined for enhanced anti-HLB activity.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. Since we are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, we changing our focus of building new constructs to controlling psyllids until we have more conclusion from the peptides under screen. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. A recent advance is that has greatly speeded up our screen is that we now can estimate when plants become infected with HLB and can tell whether a peptide is working more quickly.
Transformations of citrus plants with the FLT-antiNodT fusion protein expression construct are continuing at the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL. The FLT-antiNodT expression cassette has been introduced into ‘Duncan’ grapefruit by Agrobacterium tumefaciens – mediated transformation. Plants resistant to the kanamycin selection marker and expressing the green fluorescent protein have been regenerated successfully into plantlets. Plants are developing and appear to be healthy and normal, without any deleterious effects of the transgene noticed in the plant regeneration process so far. We anticipate that 1-foot tall plants will be available to begin testing as early as August of 2014. We are requesting a no-cost extension to the end of 2014 to have enough time to test the HLB resistance status of the trangenic plants.
This project is focused on the optimal deployment of a superinfecting Citrus tristeza virus (CTV)-based vector as a tool to be used in the field to prevent existing field trees from the development of the HLB disease and to treat trees that already established the disease. In order to provide protection against HLB, the superinfecting CTV vector will be carrying an anti-HLB gene (i.e. a gene of an effective antimicrobial peptide). The majority of trees in Florida are already infected with some CTV isolates. The main question for us is how these pre-existing isolates would affect the establishment of infection with the superinfecting vector and, thus, expression and the production level of an anti-HLB polyprotein. Several sets of experiments in which we are examining how preexisting infection with different CTV strains affects the ability of the superinfecting CTV vector to infect and get established in the same trees are ongoing. We are also examining the levels of multiplication of the superinfecting CTV vector in trees infected with different field isolates of CTV. We first graft-inoculated sweet orange trees with the T36,T30 and/or T68 isolate of CTV, singly or in mixtures (these isolates were propagated in our greenhouse) as well as with CTV-infected material obtained from the field trees (FS series isolates). Real time PCR analysis protocol is being optimized for quantification of multiplication of CTV genotypes in the inoculated trees. Trees with developed CTV infection along with uninfected control trees were challenged by graft-inoculation with the superinfecting vector carrying a GFP gene. The latter protein is used as a marker protein in this assay, which production represents a measure of vector multiplication. The trees are now being examined to evaluate level of replication of superinfecting virus. Tissue samples from the challenged trees are observed under the fluorescence microscope to evaluate the ability of the vector to superinfect trees that were earlier infected with the other isolates of the virus. Levels of GFP fluorescence are monitored and compared between samples from trees with and without preexisting CTV infection. Real time PCR quantification is also being employed to these tests. In these experiments we are using different citrus rootstock/scion combinations in order to find combinations that would support the highest levels of superinfecting vector multiplication and thus, highest levels of expression of the anti-HLB protein of interest from this vector. These combinations include trees of Valencia and Hamlin sweet oranges and Duncan and Ruby Red grapefruit on three different rootstocks: Swingle citrumelo, Carrizo citrange, and Citrus macrophylla.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we tried citrus leaves with NAD+ or water (negative control) and collected the treated leaves at 0 hr, 4 hr, and 24 hr. Three biological replicates were collected for each time point/treatment. RNA samples were extracted and subjected microarray analysis. The microarray data have been analyzed and the results showed that a group of salicylic acid pathway genes were upregulated in NAD+-treated samples. We also repeated one more time of the NAD+ treatment experiment in citrus and are currently analyzing the results. We are testing NAD+ analogs in Arabidopsis and will then use test them in citrus if effective. For objective 2, in the last quarter we started genetic transformation of citrus ‘Duncan’ grapefruit with the Agrobacteria carrying a T-DNA vector with non-host resistance genes. Several putative transformants were identified in the last quarter. In this quarter, about 20 independent lines have been generated for each construct. They transgenic are growing in the greenhouse. We expect to perform molecular characterization in the next quarter.
Clibasia_1180 (LdtR): A deeper analysis of the structure of the transcription factor was carried out by advanced in silico modeling. The ligand binding pocket was modeled with the different ligands docked, and successfully used to assess the relevance of each amino acids on ligand binding. Several protein mutants were in silico evaluated. The amount of energy required for each molecular interaction was estimated for each case. This approach was carried out in collaboration with IBM Computational Biology Center. Subsequently, in our laboratory, several new point mutations were introduced in LdtR . The five mutant transcription factors were successfully purified. We are currently assessing the effects of each mutation on the DNA binding capabilities as well as ligand binding recognition. The goal of this approach was to increase our fundamental knowledge on the ligand binding pocket to be able to rationally modify antimicrobial molecules improving their effectiveness (See previous report). The data obtained was used to design new chemical variants to be tested as antimicrobials. The binding constant of the new chemicals were in silico estimated (Lead Optimization). The best compounds made by design will be synthesized (4 new compounds). The rational modification of the ligand is expected to improve the interaction with the protein. These new compounds will soon be evaluated in our laboratory using the biological models previously described. Purification of a Transpeptidase. The evidence collected in our laboratory indicated that one of the main LdtR targets is a small protein systematically annotated as a transpeptidase. The gene encoding this protein was successfully cloned, the protein expressed and purified to homogeneity. This monomeric protein is involved in the biosynthesis and modification of the bacteria cell wall. The mechanical resistance of the cell wall is a consequence of the peptide crosslinking performed by the transpeptidase activity. The phenotype obtained in presence of the LdtR ligands and the catalytic nature of this enzyme suggested that this regulon is involved in Clas stress resistance. Collectively, the evidence obtained suggested that this specific system (LdtR ‘ transpeptidase axis) is a relevant biological target to design effective antimicrobial therapies against this bacterium. In parallel to this work a substantial progress was made regarding the characterization of an accessory transcription factor characteristic of Ca Liberibacter asiaticus. Biological assays suggested that this protein is involved in the expression of several genes actively transcribed when the cells are exposed with redox active chemicals (oxidative stress). Several mutant proteins were successfully isolated; these mutants will be used to evaluate the effectiveness of a few specific protein ligands identified with a combination of thermomelting techniques/two hybrid system study. Our molecular studies involving protein:protein interactions assays have identified the Clas RNA polymerase as a molecular partner of this transcriptional accessory protein. We hypothesize that inhibiting the activity of this important transcriptional accessory protein will have a direct and negative impact on the transcription rate of several Clas genes.
Our efforts over the last quarter have focused further on our transformation bottleneck using several approaches: 1. Stable transformation of citrus at UC Davis transformation facility. Previously we reported that we sent two of our constructs to the contract transformation laboratory at UC Davis. Carrizo transformants were obtained, albeit at lower efficiency than the transformation standard, but no transformants were recovered from Duncan grapefruit. These results are consistent with our own experiences, reflecting lower efficiencies of Duncan transformation over carrizo overall and in particular with our constructs. We will test the carrizo transformants and we have changed the vector backbones of our constructs to pCAMBIA 2201, which is routinely used in the Moore lab 2. Stable transformations in citrus using new vectors. New transformation experiments have now been carried out in Duncan grapefruit and carrizo now with 6 constructs in the pCAMBIA backbone. Plantlets are being transferred to soil, and initial PCR and GUS testing is in progress. Most plantlets are still too small for analysis, but this should be well underway next quarter. 3. Stable transformation of test systems – tobacco. UC Davis also made several lines of tobacco transformed with marker gene and resistance gene constructs. These have just been received in Gainesville and will be tested by PCR and pathogen assays. 4. Stable transformation of test systems – tomato We are testing a tomato system as a proof of concept. We used the same PIP14 promoter to drive expression of the avrBs4 gene. Expression of AvrBs4, a Xanthomonas effector, in tomato results in a hypersensitive reaction, this this construct will induce localized cell death when induced by pathogen effectors in Xanthomonas strains, as in our other systems. Explants of Bonny Best and large Red Cherry tomato cultivars were transformed to test the PIP14 construct efficiency via Agrobacterium mediated transformation. Transformants were recovered and PCR was conducted to assess transgene presence. With Bonny Best, several positive transformants were isolated. In addition, a few transformants showed a moderate resistance to Xanthomonas gardneri XV444, a strain that produces a TAL effector that can trigger our promoter construct. With Red Cherry, positive transformants were selected, and the resulting transgenics are being screened using PCR. We have some potential candidates that we are currently screening. 5. Transformation of mature citrus explants We will explore mature citrus transformation with the Lake Alfred group working on this effort.
The overall objective of this project is to develop soft nanoparticles (SNP) of antimicrobial agents for treatment of HLB infected citrus. Previously, we have reported the development of SNP using two essential oils, EO A and EO B. Microemulsion formulations have been prepared using surfactants with different hydrophilic-lipophilic balance and charges having droplet size ranging from 3 to 30 nm. Formulations containing EOA loading from 1 to 7% (w/w) and 1to 35% (w/w) for EO B were developed. The stability of the formulations developed was tested by dilution with water and phosphate buffer solution (PBS). The formulations were diluted at 10%, 5%, and 2% concentrations. While most formulations were stable, some made with ionic surfactant displayed instability when diluted with PBS. The developed EO formulations and their respective controls were tested for the anti-bacterial activity against the surrogate bacteria, Liberibacter crescens in Department of Microbiology and Cell Science (UF). The antibacterial efficacies of the SNPs were tested at three dilutions 1, 5 and 10% (v/v). All SNP formulations showed > 90 % inhibition at 1, 5 and 10 % (v/v) dilution. The controls solutions containing surfactants also showed high bacterial inhibition. While initially the SNPs were developed to achieve high EO loadings for maximum bacterial inhibition, it led to SNP formulations having relatively high surfactant concentration. The high concentration of the surfactants used is likely contributing to the anti bacterial efficacy of the EO SNPs. Further experiments are being performed to develop SNPs with lower concentrations of surfactants. Phytotoxicity of select formulations containing EO A and EO B were performed at Lake Alfred facilities (UF) following same protocols as used for testing SNP formulations developed previously. The formulations were tested at 1:1, 1:10 and 1:20 dilutions. All SNP formulations showed low phytotoxicity when applied at 1:20 dilutions. Antibacterial and phytotoxicity results indicate that with suitable dilutions the SNP formulations can be used to for performing foliar applications tests on citrus crops. Future plans include reducing the amount of surfactant used within formulations and study the effect on EO SNPs anti-bacterial efficacy and phytotoxicity. The study of foliar uptake of SNPs is planned to be investigated by preparing dye doped EO SNPs. In addition, field / greenhouse tests of select SNP formulations showing high bacterial inhibition and low phytotoxicity on HLB infected citrus plans will be planned in coming months.
Current status of the research: Objective 1: Generate functional EFR variants (EFR+) recognizing both elf18-Xac and elf18-CLas. In order to develop an efficient system of screening EFR mutants for their binding to elf18-CLas, and in vitro binding system was developed which could be used to screen a mutant phage display library. We demonstrated in vitro binding of both elf18-WT and elf18-CLas to fragments of EFR ectodomain, but not to the ones of related receptor FLS2. Binding of elf18-CLas was weaker than that of elf18-WT, but it was considered that improvements in binding achieved by phage display screening may not be evident considering the binding of WT EFR to elf18-CLas. In addition, recent structural information about FLS2 binding to flg22 indicates the involvement of BAK1 as a co-receptor directly binding the ligand. As the phage display system would not account for this interaction, it may yield mutants which would perturb the binding of BAK1. An alternative system based on split ubiquitin is currently in the process of being investigated, which will hopefully overcome these issues. Additional experiments have also been performed to determine which portion of elf18-CLas is non-functional, by making chimeric elf18 peptides with WT and CLas portions. Both WT-CLas and CLas-WT peptides fail to elicit ROS, indicating there are multiple issues with the function of the elf18-CLas peptide, and thus requiring further investigation. A collaboration is currently being set up with the laboratory of Prof. Chai at Tsinghua to obtain structural information surrounding elf18-WT and elf18-CLas binding to EFR, which would enable more straight-forward testable hypotheses. Objective 3: Generate transgenic citrus plants expressing both EFR+ and XA21-EFRchim. Vectors are currently being constructed in the pCAMBIA backbone, under the expression of the 35S/FMV promoter. These constructs will contain: EFR; XA21; both EFR and XA21; and EFR and XA21:EFR chimeric. Cloning of these constructs should be completed in the next few weeks and will then be passed on to the Moore laboratory for transformation in citrus.
In the previous three months Core Citrus Transformation Facility (CCTF) has continued to provide services for production of transgenic material to interested clients. In comparison with the third quarter of 2013, productivity was higher in the last quarter. Within this quarter, CCTF has received eleven orders. For five of those orders together with the other two from previous quarter, limited number of co-incubation experiments was performed as they required Valencia seedlings which are out of season. Other orders required the work with Duncan seedlings and experiments were performed at full capacity. Considerable amount of work was put into few orders placed in the first half of the 2013. In coordination with the client who ordered these plants, efforts are continuing to find out if the genes requested to be introduced into citrus have negative developmental effect that is hindering production. Additional six vectors and/or Agrobacterium strains received from the same client fall into the group were problems with the (common) PCR primers developed and are not solved yet. Since for all except two current orders, screen for putatively transgenic shoots is done based on PCR reaction, the work on the orders with questionable PCR primers is stalled. Plants that were produced within these three months are from the following orders: pN4-one plant, pN5-one plant, pN7-two plants, pX28- six plants, pX19- 23 plants, pTMN1- seven plants, and pMG105- two plants. Altogether 47 plants were produced and they were all Duncan grapefruit. Despite problematic nature of some recent orders, they are being placed at high volume which shows continued need for the CCTF services.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. A total of 4,472 transgenic plants have passed through inoculation process. A total of 89,810 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects. As an offshoot of the research, damage by western flower thrips was so severe that research was conducted to validate damage by this pest to developing flush and facultative predation on ACP, which led to the following publication: Hall, D. G. 2014. Interference by western flower thrips in rearing Asian citrus psyllid: damage to host plants and facultative predation. Crop Protection. (in press). A thrips predator, Orius insidiosus, proved to feed aggressively on immature ACP, thus would be incompatible for thrips control in an ACP rearing operation.
A experiment partially supported by an an enhancement CRDF 447 was planted to grapefruit at the South West Florida Research and Education Center in Immokalee, FL 14 May 2013 on 4 acres using a RCB design with 4 replicates and 2 main treatments: compost and no compost. Each of the 8 plots is divided into 2 subplots 3 rows wide 15 trees long and randomized to whiteface black (1.25 mil) or Imaflex reflective mulch as above. Insecticide drenches are applied according to IFAS recommendations in this trial. Thus far a base line HLB leaf sample, trunk diameter measurement, soil sample analysis, and leaf nutrient analysis has been collected. Sticky card traps and flush inspections are conducted every two weeks. Of 33 ACP captured on sticky cards 26 were in compost plots which are flushing strongly but only 6 in reflective mulch plots. Inspection of 7,013 flush shoots has produced 14 infested with ACP, 9 of which came from compost plots but none from reflective mulch plots. Another experiment supported by the enhancement was initiated at the Florida Research Center for Agricultural Sustainability, Inc (FLARES) in Vero BeachTo prepare for a synchronized tree planting and reflective mulch installation, the 15 acre test has been prepared in the following stages. The beds were initially mowed and new weed growth was treated twice; first with a systemic herbicide application and a second application with a contact herbicide. When the weeds within the 8 foot herbicide treatment area were completely dead, the resultant herbicide band was mowed a second time. Due to a large excess of dry mater on the surveyed tree rows, a prescribed burn was conducted to combust the dry vegetative matter. A subsequent regrowth of nutsedge weeds — which are known to penetrate the reflective mulch — was again treated with a systemic herbicide. Upon weed mortality due to the herbicide applications, the drill line of the trees rows was surveyed and a rotovator was used to till a 7 foot wide swath of loose soil to accommodate the installation of the reflective mulch. The irrigation water supply risers were located, pressure tested and marked for access and location. The layout for the four treatments has been randomized and entered into a spreadsheet with treatments assigned to their plots. Trees are on hold till the threat of predicted freeze events have passed (late February).
The objectives of this project are: 1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, soil moisture, soil nutrients, foliar nutrients, and eventually yield in newly planted citrus blocks, 2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters, 3. Evaluate the effectiveness of reflective mulch to repel ACP and reduce incidence of HLB, 4. Provide economic analysis of costs and projected benefits, and 5. Extend results to clientele. The first experiment was planted 3-4 July on 23 x 9 ft spacing in a 10-acre block at the A. Duda & Sons, Inc. farm in Hendry County south of LaBelle at 26.64315 degrees S. -81.45456 degrees W and 26 ft elevation. The experimental design of main plots is factorial RCB with 4 replicates and 4 treatments: insecticide alone, foliar nutrition alone, insecticide + nutrition, and untreated control. Each of 16 plots is split into two subplots 5 rows wide and 13 trees long, mulch or no mulch. Mulch provided by Imaflex Inc. is metalized (aluminized/reflective) polyethylene film of 3 mils thickness covered with a clear protective polyethylene coat. Metalized mulch was shown in preliminary evaluations on single plots to repel Asian citrus psyllid and together with a drip irrigation/fertigation system increase citrus growth rate over the unmulched control. ACP and other pests as well as beneficials are monitored every 2 weeks by flush inspection and sticky cards. To date, 1698 psyllids have been found on sticky cards of which greater than 70% are in no-mulch plots while only 11% have been found in plots that receive insecticides. Only 2% of the ACP found on sticky cards come from plots with both mulch and insecticides. Thus far over 47,000 flush have been observed of which 7,310 were infested with ACP. These are predominately in no-mulch plots with 65% or more of the total flush infested. Few (475) infested shoots have been found in plots receiving insecticides, and even fewer (93) found in plots with both mulch and insecticide applications. Leaf samples for HLB testing will be collected in the next two weeks as well as growth measurements. Monthly foliar nutrition sprayes were suspended in October and will resume in March. Leaf samples for nutrient analysis will be collected before the March foliar nutrition spray. Normal grove care operations continued. These include one herbicide application in October of glyphosate, Kocide was sprayed on October 17 for control of canker, and one application of Intrepid for leaf miner control. Envidor was applied for mite control on November 8. A second experiment partially supported by an amendment to CRDF 447 was planted to grapefruit at the South West Florida Research and Education Center in Immokalee, FL 14 May 2013 on 4 acres using a RCB design with 4 replicates and 2 main treatments: compost and no compost. Each of the 8 plots is divided into 2 subplots 3 rows wide 15 trees long and randomized to whiteface black (1.25 mil) or Imaflex reflective mulch as above. Insecticide drenches are applied according to IFAS recommendations in this trial. Thus far a base line HLB leaf sample, trunk diameter measurement, soil sample analysis, and leaf nutrient analysis has been collected. Sticky card traps and flush inspections are conducted every two weeks. Of 33 ACP captured on sticky cards 26 were in compost plots which are flushing strongly but only 6 in reflective mulch plots. Inspection of 7,013 flush shoots has produced 14 infested with ACP, 9 of which came from compost plots but none from reflective mulch plots. A third trial on 15 acres at the Florida Research Center for Agricultural Sustainability, Inc (FLARES) in Vero Beach is being prepared for planting next month after the danger of freezes. To date the beds have been treated with herbicide three times, mowed twice, burnt to remove excess dry matter and rotovated. The layout has been randomize and entered into a spreadsheet with treatments assigned to their plots.
Citrus scions continue to advance which have been transformed with diverse constructs including AMPs, hairpins to suppress PP-2 through RNAi (to test possible reduction in vascular blockage even when CLas is present), a citrus promoter driving citrus defensins (citGRP1 and citGRP2) designed by Bill Belknap of USDA/ARS, Albany, CA), and genes which may induce deciduousness in citrus. Putative transgenic plants of several PP-2 hairpins and of PP-2 directly are grafted in the greenhouse and growing for transgene verification, replication and testing. Over 40 putative transgenic plants transformed with citGRP1 were test by PCR and twenty two of them were confirmed with citGRP1 insertion. RNA was isolated from some of them and RT-PCR showed gene expression. Some transgenics with over-expression of citGRP1 increased resistance to canker by detached leaf assay and infiltration with Xanthomonas. About 10 transgenic Hamlin shoots with citGRP2 were rooted in the medium and nine of them were planted in soil. Over 60 transgenic Carrizo with GRP2 were transferred to soil. DNA was isolated from 20 of them and 19 of them are PCR positive. They will be used for canker resistance screening by detached leaf assay and infiltration. Belknap reports that potatoes transformed with citGRP2 are displaying considerable resistance to Zebra Chip in Washington state. Fifteen transgenic Carrizo and seven transgenic Hamlin with peach dormancy related gene MADS6 were planted in soil and they are ready for DNA isolation. In addition, numerous putative transformants are present on the selective media transformed with different constructs. A chimeral construct that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) is being tested. Many kanamycin resistant transformants were generated on the selective media. About twenty kanamycin resistant shoots are rooted in-vitro and three Hamlin transformants are in soil. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector (collaboration with Duan lab). 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 responding to HLB and other diseases. Many putative transformants were generated on the selective media. About ninety transgenic shoots were rooted with eighty Carrizo and ten Hamlin transformants planted in soil. DNA was isolated from 80 of them: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay and canker resistance test will be performed soon. Other targets identified in genomic analyses are also being pursued. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. Several D35S::D4E1 sweet oranges show initial growth in the field which exceeds that of controls. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
