Citrus trees transformed with a chimera AMP (thionin-D4E1) and the thionin alone showed remarkable resistance in citrus canker compared to control. These promising transgenic lines were replicated for HLB challenge. Replicated transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon buds. Las titer was checked from new flush rough lemon leaves at six month after grafting. Las titer from 18.6-36.5 was detected in 90% of transgenics expressing the chimera. Some transgenic lines expressing thonin had lower Las titer(most in 33.3-36.4 ranges). Transgenic root sample were further tested and most were detected with las titer from 30 to 35. Root samples from control plants and transgenic Carrizo expressing chimera and thionin were taken nine months after grating inoculation. Our results showed transgenic Carrizo expressing thionin significantly inhibited Las growth (0.5% of control level) compared to control and transgenic Carrizo expressing chimera. Antibody against thionin will be produced for Western detection. Two new chimeral peptides (second generation) were developed and used to produce many Carrizo plants and Hamlin shoots. Transgenic Carrizo plants carrying second generation AMPs were obtained. DNA was isolated from 46 plants and 40 of them are PCR positive. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. 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. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. Trees showed significant canker resistance to spray inoculation. Replicated Carrizo and Hamlin were challenged with ACP feeding. Leaves were taken six months after ACP feeding inoculation. DNA will be isolated and Las titer will be tested. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing AHLs to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Replicated transgenic Carrizo plants were challenged by ACP feeding. Las titer will be tested soon. Transgenic Hamlin were propagated by grafting for HLB challenge. 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 was used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only. A Las protein p235 with a nuclear-localization sequence has been identified and studied. Carrizo transformed with this gene displays leaf yellowing similar to that seen in HLB-affected trees. Gene expression levels, determined by RT-qPCR , correlated with HLB-like symptoms. P235 translational fusion with GFP shows the gene product targets to citrus chloroplasts. Transcription data were obtained by RNA-Seq. Data analysis and comparison are underway. Antibodies (ScFv) to the Las invA and TolC genes, and constructs to overproduce them, were created by John Hartung under an earlier CRDF project. We have transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv ready for testing. A series of AMP transgenics scions produced in the last several years continue to move forward in the testing pipeline. Many trees are in the field and some are growing well but are not immune to HLB. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and now in the field.
The Huanglongbing Diagnostic Lab at UF-IFAS-SWFREC has now been in operation for 8 years. As of March 2016, we have processed more than 38,000 grower samples. For the 2016 calendar year to date, we’ve received 917 samples from growers, which is on track towards a calendar year total very close to 2015 levels. The 3,995 growers samples processed during 2015 represented a 46% increase in the number of grower’s samples over the previous calendar year, which in itself had seen a 37% increase over 2013 numbers. These increases are likely due to the increased efforts to mitigate the HLB-associated tree stresses. Growers in this area, and most other regions, currently have one or more HLB mitigation program that they are evaluating. These growers are using the HLB lab to evaluate the effectiveness of their efforts. Another evidence of increased grower usage of the lab is seen in the fact that 60% of the individuals who submitted samples during 2015 were new clients who had not previously submitted samples. So far, new clients comprise 46% of submitters in 2016. Additionally, nearly 43,800 samples have been received for research for the entire period of diagnostic service, supported by grant funding of individual researchers. This brings the grand total to more than 81,800 plant samples processed. Grower samples are typically processed and reports returned within a two to four week time period. For this report, focusing on the quarter from March 2016, there were 855 growers samples processed and 1,071 research. Since the start of the current grant in July 2015, the lab has received 3,093 growers samples, which is even higher than the expected increases in sample volume. The HLB Diagnostic Lab continues to offer the service of detection of CLas in psyllids as funded in this grant. Current methods of sample processing have become streamlined and therefore seen no change in procedure.
Liberibacter crescens strain BT-1, has recently been cultured under laboratory conditions and is the model system for our studies. We had previously reported that we had initiated studies using the well-studied R2 tailocin to design fusions between N-terminal tail fiber region of the R2 tailocin and C-terminal portions of tail spike from BT-1 prophages. Tailocins are protein assemblages that function like the tails of phages, by adsorbing to the bacterial cell and then puncturing the cell envelope. Unlike phages, tailocins do not have a capsid and thus inject no DNA, instead relying on the membrane puncturing activity to kill the cell. Like phages, tailocins use tail fibers to recognize specific receptors on the target cell surface. Tailocins are thus potent and specific lethal nanoparticles. The assembly of active tailocin requires chaperones specific to the C-terminal and the availability of chaperone can limit tailocin production. In order to understand the protein folding necessary to assemble functional hybrid tailocins, we are fusing different N-terminal region-encoding portions of the tail fiber gene of the well-studied tailocin R2 to a series of C-terminal regions of the P2 tail fiber. This is being accomplished in- trans using a broad host vector with a tac promoter. This series of experiments will allow us to understand the fusion(s) points necessary to construct active tailocins. Using the developed overlay system that incorporates several modifications of medium BM7, we are testing broad host phages, tailocins and environmental samples to identify phages active against L. crescens.
The goal of this project is to find non-copper treatment options to control citrus canker, caused by Xanthomonas citri ssp. citri (Xcc). The hypothesis of the proposed research is that we can control citrus canker by manipulating the effector binding element (EBE) of citrus susceptibility gene CsLOB1, which is indispensable for citrus canker development upon Xcc infection. We have previously identified that CsLOB1 is the citrus susceptibility gene to Xcc. The dominant pathogenicity gene pthA4 of Xcc encodes a transcription activator-like (TAL) effector which recognizes the EBE in the promoter of CsLOB1 gene, induces gene expression of CsLOB1 and causes citrus canker symptoms. To test whether we can successfully modify the EBE in the promoter region of CsLOB1 gene, we first used Xcc-facilitated agroinfiltration to modify the PthA4-binding site in CsLOB1 promoter via Cas9/sgRNA system. Positive results have been obtained from the Cas9/sgRNA construct, which was introduced into Duncan grapefruit. We analyzed the Cas9/sgRNA-transformed Duncan grapefruit. The PthA4-binding site in CsLOB1 promoter was modified as expected. Currently we are using both Cas9/sgRNA and TALEN methods to modify EBE in sweet orange using transgenic approach. Transgenic Duncan and Valencia transformed by Cas9/sgRNA has been established. Totally four transgenic Duncan grapefruit lines have been acquired and confirmed. Mutation rate for the type I CsLOB1 promoter is up to 82%. GUS reporter assay indicated mutation of the EBE of type I CsLOB1 promoter reduces its induction by Xac. The transgenic lines are being grafted to be used for test against citrus canker. In the presence of wild type Xcc, transgenic Duncan grapefruit developed canker symptoms 5 days post inoculation similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not mutated Type I CsLOBP and Type II CsLOBP, was developed to evaluate whether canker symptoms, elicited by Xcc.pthA4:dCsLOB1.3, could be alleviated on Duncan transformants. Both #D18 and #D22 could resist against Xcc.pthA4:dCsLOB1.3, but not wild type Xcc. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by Xcc-derived PthA4 is enough to induce citrus canker disease and mutation of both alleles of CsLOB1, given that they could not be recognized by PthA4, is required to generate citrus canker resistant plants. The data has been published by Plant Biotechnology Journal Transgenic Valencia transformed by Cas9/sgRNA has been established in our lab. Three transformants have been verified by PCR. The PthA4-binding site in CsLOB1 promoter was modified as expected, only one transgenic line seems to be bi-allelic mutant. The EBE modifed transgenic line is being evaluated for resistance against Xac. One Cas9/sgRNA binary vector, which is designed to target CsLOB1 open reading frame, designated as GFP-Cas9/sgRNA:cslob1, was used to transform Duncan grapefruit epicotyls by Agrobacterium-mediated method. Several transgenic citrus lines were created, verified by PCR analysis and GFP detection. Cas9/sgRNA:cslob1-directed modification was verified on the targeted site, based on the direct sequencing of PCR products and the chromatograms of individual colony. Upon Xcc infection, some transgenic lines showed delayed canker symptom development. We are currently analyzing the genome modified plants using transgenic approaches including off-targets. To generate non-transgenic DNA free canker resistant citrus, Cas9 containing nucleus localization signal was overexpressed and purified. The purified Cas9 showed activity in cutting target sequence and will be used to generate canker resistant plants.
Previously, we have shown that the Genome of Candidatus Liberibacter asiaticus (CLas) possess luxR gene that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing”. However, the genome lacks the second component; luxI that produce Acyl-Homoserine Lactones (AHLs) suggesting that CLas has a solo LuxR system. In the current project, we have only one objective: study the effect of AHL-producing citrus plants on the pathogenicity of CLas. We have selected different Lux-I genes from different bacteria expressing structurally different AHLs. Due to the difficulties we faced in growing different bacteria to extract DNA for LuxI isolation, we changed our strategy to synthesizing these genes (G-Blocks). Synthesized genes include 1- Agrobacterium tumefaciens N-(B-oxo-octan-1-oyl)-L-homoserine lactone(Hwang et al., 1994) TraI Accession number#L22207.1 2- Pseudomonas fluorescens Six acyl-HSLs, including the 3-hydroxy forms, N-(3-hydroxy-hexanoyl)-L-homoserine lactone(3-OH-C6-HSL), N-(3-hydroxy-octanoyl)-L-homoserine lactone (3-OH-C8-HSL), and N-(3-hydroxy-decanoyl)-L-homoserine lactone (3-OH-C10-HSL); the alkanoyl forms hexanoyl-homoserine lactone (C6-HSL) and octanoyl-homoserine lactone (C8-HSL)(Khan et al., 2005) PhzI Accession number#AAC18898.1 3-Rhizobium leguminosarum bv. viciae 3-OH-C14:1-HSL (Wilkinson et al., 2002, Edwards et al., 2009) CinI Accession number#AF210630 4-Serratia liquifaciens N-(butyryl)-L- homoserine lactone (BHL) (Oulmassov et al., 2005) SwrI Accession number#U22823.1 5- Vibrio fischeri OHHL (Schaefer et al., 1996) LuxI Accession number#AAD48474.1 We already started the first step of expressing these genes by inserting them in CTV-based vector prior to the infiltration inside citrus trees. Our aim is to interfere with CLas signaling by expressing AHL which will enhance the bacterial aggregation and attachment and results in localization of the bacterium in certain branches.
Our project is based on the biology of quorum sensing of Candidatus Liberibacter asiaticus. The system here the two components system LuxR-AHL. The Genome of Candidatus Liberibacter asiaticus (CLas) revealed the presence of luxR that encodes LuxR protein, one of the two components typical of bacterial “quorum sensing” or cell-to-cell communication systems. Interestingly, the genome lacks the second components; luxI that produce Acyl-Homoserine Lactones (AHLs) suggesting that CLas has a solo LuxR system. In the current project, we will test the effect of AHL-producing citrus plants on the pathogencity of CLas. We have selected different Lux-I genes from different bacteria expressing different AHLs. In order to isolate the genes, we will obtain bacteria from the American type culture collection including Agrobacterium tumefaciens, Aeromonas hydrophilia, Agrobacterium vitis, Burkholderia cepacia, Chromobacterium violaceum, Panteoa stewartii, Pectobacterium carotovorum, Pseudomonas aeruginosa, Pseudomonas aeruginosa, Pseudomonas fluorescens, Rhizobium leguminosarum bv. Viciae, Rhizobium leguminosarum bv. Viciae, Rhizobium leguminosarum bv. Viciae, Rhodobacter sphaeroides, Serratia liquifaciens, Sinorhizobium meliloti, Vibrio anguillarum, Once we receive the bacteria we will culture them prior to insolate the genes by PCR. We will insert genes in CTV-based vector prior to the infiltration inside citrus trees. Our aim is to interfere with CLas signaling by expressing AHL which will enhance the bacterial aggregation and attachment and results in localization of the bacterium in certain branches.
This overall 3 year project was focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. In this quarter (Jan 2016 to March 2015), we continue to evaluate 1) the effect of Pen and SD on control of HLB disease by gravity bag infusion in the field; 2) the efficiency of effective chemical compounds (Pen, SDX, Pcy and Carv) against HLB disease by gravity bag infusion; 3) the effectiveness of a combination of chemotherapy, thermotherapy and nutrient therapy against HLB in the field trials; 4) the efficacy of the new adjuvants to improve the uptake of antimicrobials. The chemical compounds (Pen and EBI-602) and additional nutrients were applied to the heat-treated citrus for three times by foliage spray, using our optimized nano-delivery system. The preliminary results showed that Pen was the more effective to control Las bacterium than EBI-602. The disease severity index (SDI) decreased by 6% after applied with Pen. The integrated practices (antimicrobial treatment coupled with heat treatment and nutrition fertilization) could decrease the fruit drop by 10~20 %, increase the fruit and juice weight by 3~13 %, and decrease the ratio of brix to acid by 0.2~5.0 %. The preliminary results from the other five antimicrobials (SD, Pen, SDX, Pcy and Carv) applied by gravity bag infusion showed that there were not different in the Las bacterial titers among the treated antimicrobials. Compared to the untreated plants, all antimicrobials reduced the Las bacterial titers, especially PEN. Both SD and Pen reduced the DSI through two years application. In last quarter, we tried to evaluate two new adjuvants (Bio and MF200) for improving the effectiveness of Pen by foliar spray. The preliminary results indicated that Pens formulated in both Bio and MF200 decreased the Las bacterial titers a lots. Ten antimicrobials were prepared in two different concentrations of the nano formulations (0.1 % and 1.0 %) in the greenhouse test. The Ct values kept over 36.o in the PEN-treatment. In next quarter, we will keep our application. Pcy and Carv will be changed application from trunk-injection to foliar spray. One papers has been published in the Crop Protection.
OVERVIEW: The Budwood Certification Program continues to build and develop the Foundation and Increase tree collection. Demand for budwood continues to increase and is anticipated to exceed 250,000 this year. PROGRAM OPERATIONS Texas Citrus Budwood Certification Program: 1. Budwood sales during the period were 229,664. Rio Red Grapefruit, Olinda and Standard Valencia Oranges, and Improved Meyer Lemon were the major varieties requested. 2. Much needed maintenance has been conducted on the main screenhouses. The old double poly roof on Screenhouses 1 thru 4 was replaced in November. A new layer of insect screen followed by a new layer of poly followed by aluminet shade cloth was put in place. 3. New roll-up curtains were installed in January to help protect the trees in the screenhouses during inclimate weather. 4. The project to upgrade Phase I of the Screen Structures will begin in the early summer. All existing trees will be removed and the screen and wood frame will be replaced with new galvanized metal frame and new insect screen. New Foundation trees will be planted in the structure when completed. 5. The Budwood Program Database continues to expand and is a key component in the management of the Texas program. The database program contains all records and information for all budwood production and sales, all foundation and increase trees, test results, and chemical applications. This database has the ability for an unlimited range of reporting information on the Texas program, including budwood orders, supply management, sales, testing, pesticide and fertilizer applications, budget analysis, and records needed for all compliance with TDA and USDA regulations. Stephenville Foundation Screenhouse : 1. New, clean Foundation trees continue to be added to established Foundation trees at the Stephenville greenhouse location. There are currently 75 Foundation trees consisting of over 60 different citrus species. Additional trees will be added as they are propagated to bring the total capacity to 100. These trees are being maintained as a reserve source of clean material for the Texas citrus industry. Texas Germplasm Introduction Program 1. The quarantine facility for the Germplasm Introduction Program is nearing completion and will be ready for USDA and TDA certification as a quarantine facility this summer. 2. Mark VanNess (Program Manager) and Sonia Del Rio (Lab Technician) visited the California CCPP in the fall to observe and receive training in their shoot-tip grafting program. Mark and Sonia visited Florida’s Germplasm Introduction Program in January and March to receive training and observe the Florida program. The new Texas program will operate based on the practices learned from both the California and Florida programs. Diagnostic Lab Testing – Foundation/Increase Trees: 1. All Foundation and Increase trees located in Weslaco and the Stephenville greenhouse were tested for HLB in October, 2015, and will be tested again for HLB and CTV in April, 2016. All tests were negative. 2. All Foundation Trees underwent virus/viroid testing in the fall of 2015. All test on all viruses and viroids were negative.
The first quarter of 2016 was very strenuous for Core Citrus Transformation Facility (CCTF). Two out of five employees left the facility in January and were eventually replaced by the new members of staff. CREC Center Director informed CCTF of possible move of the lab to the new site in March. Although March 24th was anticipated date for the move, that did not happen and CCTF still operates from its present location. CCTF received unprecedented number of orders (26) within the last quarter. Such a high volume of incoming orders called for an additional increase in production capacity of CCTF. I have purchased necessary consumables and tools for this transition and have taken steps to gradually ramp-up the input of starting material for experiments. However, uncertainty associated with possible move to new location prevented search for additional employees. I expect new recruit to begin working in the month of April. Partial increase in work load in March was little overwhelming for the present labor force and resulted in loss of some transgenic shoots and plants. I hope that when all aspects of CCTF functioning stabilize, so will the level of our production. The newly announced date for the move to new location is June 17th and I will try to organize it in such a way so that it will affect productivity of CCTF to the least possible extent. Between January and April, CCTF produced 57 plants. These plants belong to newer orders placed within the last 12-15 months. Four of the produced plants were Valencia oranges, three were Pineapple sweet oranges, eight were Carrizo citrange, and the rest were Duncan grapefruit. Transgenic rootstock plants carrying NPR1 produced in our facility are still in our greenhouse. They are at the stage when they could easily be propagated by cuttings. I am awaiting further instructions on what to do with these plants.
This is the final report for Project 13-926.3C. Over the last two years, we have been able to confirm that Citrus Leafminer Mating Disruption is a viable control solution for Phyllocnistic citrella. Data from 734 Citrus Holdings, Indian River Exchange Packers and The Packers of Indian River, all distributed across the state, confirmed that the performance of DCEPT CLM is in fact tremendously beneficial to CLM control. Performance across all of the trials, including monitoring data and damage evaluations, was consistent. This evidences the consistent and predictable performance of pheromone based mating disruption. After evaluating all of the data from this project, we can confirm that DCEPT CLM will quickly reduce populations of CLM and maintain control for up to twelve weeks. For these twelve weeks, pheromone monitoring traps will capture little to no adult CLM males because the populations are being controlled by the pheromone being released by DCEPT CLM. Additionally, damage will follow the same trend as the traps and will be equal or lower than farms that utilize conventional spray programs that spray as often as every two weeks. We also believe that DCEPT CLM, as a sole control input, can control CLM using a single application without CLM targeted conventional insecticide sprays for up to twelve weeks. Going forward, for farmers that plan to proceed with a single application of DCEPT CLM, our recommendation to farmers will be to target DCEPT CLM applications at the most important ten to twelve weeks of the citrus growing season. This will allow the farmer to have flexibility and choice, one where they can mix and match control strategies. For example, a farmers can protect their citrus during the highest pressure portion of the season with a DCEPT CLM application and protect the rest of the season with conventional insecticide sprays. The farmer could also apply DCEPT CLM twice which will cover nearly the entire susceptible season of Florida citrus. Unlike insecticide treated blocks, DCEPT CLM applications will also maintain the major advantage of residual performance. Although DCEPT CLM will not be able to eliminate damage after twelve weeks in the field, it will still maintain residual performance that keeps CLM populations lower than blocks treated with conventional insecticides. For example, at The Packers of Indian River, blocks that were treated with DCEPT CLM were able to maintain monitoring trap captures three to four times lower than blocks treated with insecticides every two weeks for an additional four weeks. Lastly, these three trials confirmed that DCEPT CLM performance can be maintained at essentially the same levels with applications in farms with tree densities ranging from 125-175 trees per acre. This confirmation is important because it will allow Florida farmers with various tree densities to adopt CLM mating disruption. We will now recommend that all farmers proceed with a minimum application rate of 125 DCEPT CLM per acre.
This project (Hall-15-016) is an extension of a project that came to a close last summer (Hall-502). The driving force for this project is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. 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 citrus breeding and transformation efforts by Drs. Stover and Bowman. Briefly, individual plants to be inoculated 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, and finally the plants are transplanted to the field where evaluations of resistance continue. CRDF funds for the inoculation program 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 technician is assigned ~50% to the program. USDA provides for the program two small air-conditioned greenhouses, two walk-in chambers, and a large conventional greenhouse. Currently 18 individual colonies of infected psyllids are maintained. Some of the individual colonies are maintained on CLas-infected lemon plants while others are maintained on CLas-infected Citron plants. Update: Two technicians funded by the grant have been fully trained in establishing and maintaining colonies of infected psyllids, conducting qPCR assays on plant and psyllid samples, and running the inoculations. As of March 17, 2016, a total of 8,169 plants have passed through inoculation process. A total of 160,395 psyllids from colonies of CLas-infected ACP have been used in no-choice inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations are many plants inoculated over the past year to assess transmission rates, which has provided insight into the success of our inoculation methods and strategies for increasing success. Research concluded during September 2015 showed that seedling citrus with flush is significantly more prone to contracting the HLB pathogen than seedling citrus without flush: Hall, D. G., U. Albrecht, and K. D. Bowman. 2016. Transmission rates of ‘Ca. Liberibacter asiaticus’ by Asian citrus psyllid are enhanced by the presence and developmental stage of citrus flush. J. Econ. Entomol. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Current research indicates that the no-choice inoculation step used in our program is successful 75 to 95% of the time when approximately 75% of ACP placed on a plant test positive for CLas and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant).
We continue to produce transgenic, mature citrus trees and transfer them to scientists (Drs. Dutt, Louzada, McNellis, Mou, Wang) as soon the primary or secondary grafts heal. Mature scion transformation efficiencies have increased to 7.6%, and micrografting efficiencies have improved to 77%. Approximately 154 transgenics (primary transgenics and vegetative progeny) have been transferred to Dr. Dawson’s lab for additional testing, and another ~50 will be transferred next month. For out-of-state transport of transgenics, USDA APHIS permits were obtained by scientists prior to shipping. Shipping certification was also obtained through UF. Transgenic, mature citrus has been shipped to Dr. McNellis at Penn State. A manuscript was submitted to a scientific journal describing biolistic transformation of immature citrus rootstock, never previously reported in the literature. Biolistic transformation to produce transgenics will augment those produced with Agrobacterium. A rapid, high throughput, nondestructive MUG assay is being developed to screen whole putative transgenic citrus shoots for GUS expression. It is quantifiable and more sensitive than using X-Gluc as substrate. Fluorescence can be quantified on a plate reader, or visualized on a gel doc with known controls. It is anticipated that GUS expression will correlate to copy number similar to NPTII expression. It remains to be determined whether the shoots will survive immersion in the MUG substrate and subsequent micrografting, but minimal exposure to the substrate, followed by rinsing, might not be harmful. Data are being collected describing the method for potential publication. We are still optimizing the PMI selectable marker using biolistics and Agrobacterium transformations in immature and mature citrus transformation. The results so far look promising and shoot growth doesn’t appear to be negatively impacted like shoot growth on kanamycin medium. Sour orange and Volkameriana seed have been purchased for the growth room because seed of our preferred rootstock varieties have been sold-out. It remains to be determined if these varieties perform well in the growth room.
During the period of project 767 funding, we accomplished the following: 1) screened Liberibacter crescens against a wide variety of antimicrobials that we predicted to be phloem mobile (this was done independently of a contract from CRDF to assess compounds of CRDF’s choosing); 2) worked with Erik Mirkov of Texas A&M to determine the efficacy of oxytetracycline and streptomycin against tomato yellows disease caused by Ca. L. solanacearum; 3) encouraged and worked with NuFarm to test the efficacy of oxytetracycline in the field against HLB; 4) mutagenesis of L. crescens identified 314 genes that are essential for growth in culture, of those 238 have homologs in Ca. L. asiaticus and can be considered excellent candidates for antimicrobial development; 5) developed a list of seven antimicrobials that are excellent candidates as second generation compounds for treatment of HLB in the field. A paper describing our findings in activities one through three above is in preparation and we hope to submit it soon. A paper on activity four is in revision in Frontiers in Microbiology. The major findings from these activities are as follows: 1. Three classes of phloem-mobile antibiotics were found to be highly effective against L. crescens: the cephalosporins, the penicillins, and the tetracyclines. The cephalosporins are still used widely in medicine making regulatory approval for citrus use difficult. Penicillins often cause allergic reactions in humans making them difficult to use on a fruit tree crop. The tetracyclines appear to be a very viable option for HLB since oxytetracycline has already been approved for use on bacterial diseases of fruit tree crops. 2. Erik Mirkov of Texas A&M showed that oxytetracycline, but not streptomycin, is very effective against tomato yellows disease. Thus, we recommended that oxytetracycline be tested in the field for control of HLB. 3. NuFarm showed HLB symptom relief in field trials in 2014 and 2015 using oxytetracycline. Streptomycin was not effective in these trials. Since streptomycin is not predicted to be phloem-mobile, we were not surprised by the failure of streptomycin to relieve Liberibacter-induced disease symptoms on tomato and citrus. 4. Thanks to our work on the essential gene list of L. crescens, we now have 238 excellent targets in L. asiaticus for antimicrobial action. This list of genes will soon be in the public domain as a paper describing this work is expected to be published soon in the open access journal, Frontiers in Microbiology. 5. Based on all of the above and on the properties of many antimicrobials tested against L. crescens, we have proposed that a second generation of antimicrobials be tested in preparation for their use in HLB control. These include sulbactim and thiamphenicol and other compounds that require the involvement of other parties. We expect all of them to be effective but have varying degrees of regulatory hurdles. All need to be tested in the tomato yellows assay followed by testing in the field on infected citrus trees. Our view is that a second generation of antimicrobials needs to be available: a) to be used in rotation with oxytetracycline and streptomycin to slow the appearance of resistance to these compounds and b) to replace oxytetracycline and/or streptomycin when resistance dominates in the L. asiaticus population.
This proposal is aimed at following previous work in CRDF-710 and CRDF-818 with a series of precise experiments that will: 1. Elucidate the nature of the HLB signal(s) 2. Provide additional evidence on its transmission in terms of movement across tissues and between trees though underground organs. 3. Determine the progression of physical symptoms from its inception. 4. Examine the in-tree variation in CLas titer. All experiments were completed and final testing done in December. 1. To test for he unlikely, but increasing, possibility that HLB is transmitted by extracellular vectors, we isolated DNA from HLB leaves and inject these into 2 year old Valencia trees. The trees are being kept in a greenhouse and are under observation. Trees continue growing normally. Trees tested in September 11, one tree tested HLB+, though a high PCR value. Trees were retested again late December and all tested HLB-. 2. Experiments for objective 2 are well under way. Two trees (one healthy and one HLB+) were root grafted in three different locations and placed in special pots large enough to accommodate the 2 trees (5 pairs). The trees were placed in a greenhouse and kept under observation. PCR analyses were conducted once more in September 2016. At this time, 3 out of the 5 pairs of the initially healthy trees tested positive, although clear visible symptoms were not evident in all cases. By the end of December, 4 of originally healthy trees tested HLB+. One remains under observation. 3. Grafted trees with HLB material are being monitored weekly using Narrow-band imaging under polarized illumination. Although we continue to have issues with the background, we have established a standard curve and a correlation relationship between starch levels, PCR values, and polarized light readings. 4. Trees have been grafted for a substantial amount of time and some are showing HLB symptoms. However, given that analysis of this objective destroys the trees, only trees with clear symptoms are tested. PCR analyses was conducted in a total of three trees that showed symptoms and tested HLB+. In any of the trees, there was correlation between PCR values and leaf position. These possible results prompted the design of a new system for HLB determination with a much higher level of precision.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, 30 additional transgenic lines were analyzed for gene expression using qPCR. Of them, 10 were determined to be high expressers while the rest were medium to low in expression. Most lines tested negative for the transgene in the aerial leaf and stem while there was gene expression in the roots. In addition, a number of additional lines have been transferred from in vitro medium and acclimated to greenhouse conditions. We expect to begin propagation of the larger plants in the next quarter for challenge with Diaprepes neonates.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
