1) Assessed use of isolated leaf inoculation, and small plant destructive sampling: Isolated leaf inoculations do not readily distinguish between resistant and susceptible citrus selections, but may prove useful in identifying nearly immune material. Small plant destructive inoculation assays now permit us to distinguish between susceptible Valencia and resistant Carrizo after 12 weeks. This assay seems to be an efficient way to test transgenics that are expected to kill CLas and experiments are underway. 2) Data collection continues on transgenics. Transgenic plants expressing a modified thionin are promising for HLB resistance and they have been extensively propagated for testing in the greenhouse and the field. Transgenics expressing LuxI from Agrobacterium, and an array of ScFv transgenics (more in 5 below) have also been propagated for testing. 3) Two new chimeral peptides (second generation) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. 4) 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. 5) 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 putative transgenic Carrizo reflecting almost 400 independent transgenic events and 17 different ScFv, but only 69 events from 7 ScFv produced proven transgenics ready for testing. These have been replicated by rooting and will be exposed to no-choice CLas+ ACP followed by whole plant destructive assays. 6) To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was used to transform citrus. Trees expressing NbFLS2 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 was isolated and Las titer was tested. Our preliminary results show that transgenic trees expressing NbFLS2 can reduced Las titer. In-silico analyses are being conducted to develop citrus FLS2 optimized for sensing CLas flagellin. 7) Arabidopsis DMR6 (down mildew resistance 6)-like genes were downregulated in more tolerant Jackson compared to susceptible Marsh grapefruit. DMR6 acts as a suppressor of plant immunity and it is upregulated during pathogen infection. In a gene expression survey of DMR6 orthologs in Hamlin , Clementine , Carrizo , rough lemon, sour orange and citron, expression levels were significantly higher in all CLas-infected trees compared with healthy trees in each citrus genotype. We developed 2 RNA silencing (hairpinRNA) constructs aimed to silencing citrus DMR6 and DLO1 respectively. Citrus DMR6 is silenced in hairpin transgenic plants and with an average silencing efficiency of 41.4%. DMR6 silenced Carrizo plants exhibit moderate to strong activation of plant defense response genes. 8) Optimizing use of a SCAmpP (small circular amphipathatic peptide) platform, was conducted in collaboration with Dr. Belknap and Dr. Thomson of the Western Regional Research Center of USDA/ARS. SCAmpPs were recently identified and have tissue specific expression, including having the most abundant transcript in citrus phloem. Furthermore, members of the SCAmpP family have highly conserved gene architecture but vary markedly in the ultimate gene product. Variants of a tissue-specific SCAmpP were tested using GUS as a reporter gene: removal of the conserved intron reduced tissue specificity and deletion of non-transcribed 5 region reduced expression. Excellent phloem-specific expression is achieved in citrus when a target gene is substituted for the gene encoding the SCAmpP peptide. We are using this promoter aggressively in transgenic work 9) The third generation chimeral peptides were designed based on citrus thionins and citrus lipid binding proteins and plants have been transformed.
A test site for material proposed to have enhanced HLB resistance has been maintained at the USDA/ARS USHRL Picos Farm in Ft. Pierce, and has been open to requesting researchers for experimental plantings. As space is needed and experiments conclude, trees are pushed and replaced. Dr. Jude Grosser of UF has provided hundreds of transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser has planted an additional 100 tress including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. USHRL has a permit approved from APHIS to conduct field trials of their transgenic plants at this site, with several hundred transgenics in place. An MTA is in place to permit planting of Texas A&M transgenics produced by Erik Mirkov and he has trees expressing lectins under a USDA-ARS permit. Discussions have been ongoing with Eliezer Louzada of Texas A&M to plant his transgenics which have altered Ca metabolism to target canker, HLB and other diseases. Answers have been provided to numerous questions from regulators to facilitate field testing approval. APHIS BRS has visited the site on several occasions and confirmed that plantings are in compliance. We have provided permit details to assist plantings by other groups. A number of plants have been pushed as the analysis has been completed and new plantings made. 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 7 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 (Ramadugu et al., 2016), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Tolerance to HLB was studied, with citron-derived material showing special promise within the genus Citrus (Miles et al., 2016). A new UF-Gmitter led association mapping study is nearing completion using the same planting, to identify genes associated with HLB- and ACP-resistance. 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 (Yu et. Al., 2015). David Hall led an effort to characterize ACP colonization and while pure Poncirus had much lower colonization, all citranges were intermediate between Citrus and Poncirus. Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruit that approach commercial quality, were planted within the citrange site. Several of these USDA hybrids continue to grow well, with dense canopies and good fruit set, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population has been planted in a collaboration between UCR and ARS to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. An additional trial, led by ARS Riverside is assessing HLB response in a broad array of Poncirus-derived accessions.
The new Post-Doc to take over the responsibilities of continuing and finishing this project, which had been paralyzed since the departure of the previous pos-doc ,arrived in mid September. Preparations to get the project back on track had already begun in terms of plant material, chemicals, greenhouse space, irrigation, etc. Upon his arrival, we started the objectives listed below: Objective III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. Trees were treated with SL and soil amended with Phytophtora and or fungicide following the established sequence of events. Trees are being monitored and the second SL treatment will be applied soon. Objective IV: Effect of SL on the growth of beneficial mychorriza. Trees have been treated with SL and mycorriza applied to the soil as planned. The second SL treatment will take in early spring. Objective V: Effect of spray application of SL + other promising compounds on citrus in groves. Additional natural organic such as organic acids, sugars, amino acids, and few other compounds will be applied on potted trees. Trees are being sprayed bi-weekly with a 10 mM solution of individual components. In the meantime, data are being collected on the above treatments, two manuscripts are being prepared from previous work and we are gearing up to address the remaining objectives.
The new Post-Doc to take over the responsibilities of continuing and finishing this project, which had been paralyzed since the departure of the previous pos-doc ,arrived in mid September. Preparations to get the project back on track had already begun in terms of plant material, chemicals, greenhouse space, irrigation, etc. Upon his arrival, we started the objectives listed below: Objective III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. Trees were treated with SL and soil amended with Phytophtora and or fungicide following the established sequence of events. Trees are being monitored and the second SL treatment will be applied soon. Objective IV: Effect of SL on the growth of beneficial mychorriza. Trees have been treated with SL and mycorriza applied to the soil as planned. The second SL treatment will take in early spring. Objective V: Effect of spray application of SL + other promising compounds on citrus in groves. Additional natural organic such as organic acids, sugars, amino acids, and few other compounds will be applied on potted trees. Trees are being sprayed bi-weekly with a 10 mM solution of individual components. In the meantime, data are being collected on the above treatments, two manuscripts are being prepared from previous work and we are gearing up to address the remaining objectives.
The driving force for this project (Hall-15-016) is the need to evaluate citrus transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus breeders at USDA-ARS-USHRL, Fort Pierce Florida are 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 20 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 CLas 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: As of January 1, 2017, a total of 9,494 plants have passed through inoculation process. A total of 297,595 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 we recently published 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. 109: 558-563. doi: 10.1093/jee/tow009. Therefore, the program has been changed to ensure that plants to be inoculated have flush. Incidentally, Setamou et al. (2016, J. Econ. Entomol., 109: 1973-1978) published supporting information that transmission rates of CLas are increased when flush is present. The no-choice inoculation step used in our program has been projected to be successful an average of 79% of the time when approximately 70% of ACP placed on a plant test positive for CLas (Ct <36) and have CLas titers of around CT=26 to 29 (success contingent on flush being present on a plant). We are in the process of analyzing data from research comparing success rates using ACP colonies on lemon versus citron, and using ACP colonies from greenhouses versus walk-in chambers.
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. In this quarter, we specifically tested the SAR inducer-activated residual activity in potted citrus seedlings. The seedlings were treated by either root drench or foliar infiltration with the SAR inducer. After the first round of disease resistance test, the plants were cut back. Two months later, residual activity in the new flashes was tested. Results showed statistically significant reduction in the number of canker lesions formed in leaves on the plants pre-treated with the SAR inducer. Reduction in the number of lesions was, on average, from 15% at a low dose (0.25 mM) to 40-50% at higher doses of the SAR inducer (5-10 mM). These results demonstrate that the SAR inducer activates strong residual activity in new flushes at least two months after the pre-treatment. We also confirmed the priming effect of the SAR inducer. Briefly, leaves were treated with 1 mM of the SAR inducer. The treated leaves were infected with citrus canker bacterial pathogens 36 hours later. The infected leaf tissues were collected at 0, 4, 8, and 24 hours later, similarly as in the previous experiments. Expression of PAL1,NPR1, PR5, CM1, ICS1, CM1, CM2, and PLDg was analyzed by real-time qPCR. We confirmed that expression of PAL1, NPR1, PR5, CM1, and ICS1 was significantly enhanced by pre-treatment with the SAR inducer, corroborating that the SAR inducer indeed has strong priming effects in citrus.
During this reporting time period, we made the following progresses for the proposed objectives: We have finished construction of most of the genes proposed. We have started the transformation of both juvenile and adult citrus tissues with these genes to test their effects on transient and stable transformation efficiencies of citrus plants. We have just submitted a manuscript for consideration of publication. We have shipped some relevant genes to Dr. Janice Zale, The Mature Citrus Facility (MCF) of the University of Florida, to test their effects on the citrus cultivars her facility uses.
Data collection continues across numerous experiments. A number of publications from UF and USDA have included data from these plantings. USDA/APHIS BRS reviewed and approved the work at this site in December 2016. Discussions are ongoing about inclusion of transgenics from programs with no field planting experience. A 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 six years and new trees are being added every few months. 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. 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 has been planted that has shown great promise in the greenhouse and the permit has been updated to plant many new transgenics. 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 6 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 (Ramadugu et al, Plant Disease, 2016), and P. trifoliata also displayed reduced colonization by ACP (Westbrook et al., 2011). Marked tolerance to HLB is apparent in many accessions with citron in their pedigree (Miles et al., 2016). All replicates of one alleged “standard sour orange” looks remarkably healthy and may permit comparison of more susceptible and tolerant near-isogenic variants. A new UF-Gmitter led association mapping study has underway using the same planting, to identify genes associated with HLB- and ACP-resistance. A broader cross-section of Poncirus-derived genotypes are on the site 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 led 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. Several of these USDA hybrids have grown well, with dense canopies and good fruit set but copious mottle, while sweet oranges are stunted with very low vigor (Stover et al., unpublished). A Fairchild x Fortune mapping population was just planted at the Picos Test Site in an effort led by Mike Roose to identify genes associated with tolerance. This replicated planting includes a number of related hybrids (among them our easy peeling remarkably HLB-tolerant 5-51-2) and released related cultivars. Valencia on UF Grosser tetrazyg 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.).
In the quarter ending September 15, we focused on study of 6 Liberibacter regulators: RpoH, VisNR (a dimeric regulator encoded by two genes), LdtR, LsrB, PhrR, and CtrA. We chose the following target promoters, based on our previous global transcription studies: Regulator: target promoter(s): – RpoH: ibpA, groESL5 – LdtR: tacA, SMc04059, ldtP – VisNR: rem – CtrA: minCDE – LsrB: SMc01834, lrp3-lpsCDE – PhrR: SMc00404, ldtR To assess strength of expression, we constructed plasmids in which the target promoter fusion was on the same plasmid as the transcription factor gene. The fusions used enhanced GFP (F64L, S65T), with codon usage optimized for S. meliloti. On this modular plasmid, one can exchange regulatory gene, reporter gene, and terminators with straightforward cloning. For example, we made a plasmid in which CLas rpoH (controlled by lac promoter) was on the same plasmid as the ibpA-eGFP fusion. Therefore activity of CLas RpoH protein would be detected as fluorescence; inhibition of CLas RpoH would be evident by a decrease in fluorescence. Using plates with induction of the lac promoter, we examined the level of fluorescence for each regulator-promoter pair. The data from these tests shows us that of our six original transcription regulators, 3 gave robust fluorescence, and therefore would be suitable to look for inhibitory compounds. We chose the best-responding promoter target for each. The final constructs are: RpoH (target promoter ibpA); VisNR (target promoter rem); and LdtR (target promoter SMc04059). The test plates used were 96 well Costar black plates with clear bottom. In initial trials, cells were diluted to various concentrations, and we tried several culture media including two rich media (TY and LB supplemented with Mg and Ca ions) as well as minimal (defined) medium. To assay fluorescence, we used the following conditions: Excitation wavelength = 489 nm Emission wavelength = 509 nm. In addition to fluorescence we measured optical absorbance at 600 nm in order to detect inhibition of bacterial growth itself. This is important because we want to know whether the compounds inhibit the regulator (desired) or just kill the cell (undesired). As of September, we had begun the first test run for control strains.
Transgenic evaluation of antimicrobial peptides (AMPs) has so far has identified a modified thionin (Mthionin) that conferred resistance against HLB and canker when over-expressed in Carrizo plants (Hao et al, 2016 Front Plant Sci.). From the same transgenic Carrizo population, we screened another 37 positive plants by PCR validation of gene insertion and documented Mthionin transcript level of each plant by RT-qPCR. Multiple high, intermediate and low transgene expressing plants were selected and propagated using stem cuttings (a total of 250 cuttings). Once established these plants will be tested as rootstocks with sweet orange and grapefruit scions. compared with wild type Carrizo as a rootstock. An antibody has been developed for specific detection of Mthionin and a second to detect both Mthionin and citrus native thionin. Currently, we are evaluating binding capacity and sensitivity of these antibodies to antigen peptides and to full-length proteins (expressed by E.Coli). Later the antibodies will be used for access expression level and mobility of thionin protein between root stock and scion. Two additional Mthionin chimera genes (2nd generation of AMPs), Mthionin-D2A21 and Mthionin-lipid binding protein (LBP), have been used to produce transgenic Carrizo and Hamlin. So far we have obtained a number of transgenic Carrizo plants and made a propagation of about 100 for each transgene. These plants will be used for initial evaluation of HLB resistance through no choice ACP feeding inoculation and promising lines will be further propagated for field tests. The 3rd generation of AMPs includes two variants of modified citrus thionin genes combined with citrus LBP. Transformation of these two chimeras into Carrizo and Hamlin is underway. So far we have obtained a number of Carrizo regenerations. We are also in the process of the evaluation of HLB resistance in several Hamlin transgenic lines: we graft propagated transgenic Hamlin expressing Mthionin, D4E1 linker Mthionin, and LuxI, with wild type scion as the controls. These plants are established and inoculated by no choice feeding from 9 to 12 month ago. The bacterial titer tests showed that only very few plants were HLB positive, indicating the inoculation procedure was not successful. These plants were uniformly trimmed for new flush growth and will soon be re-inoculated with our improved protocols. The previous generations of transgenics remain in testing in the greenhouse and field.
From September through December 2016, we carried out all the control runs, and then the test runs for a high-throughput screen of compounds in the Stanford Chemical Screening facility. As of the end of December, we had screened over 115,000 compounds. As previously described, we looked at three Liberibacter transcription regulators: RpoH, VisNR, and LdtR, each with a corresponding target promoter driving enhanced GFP. To run the test, the reporter strains are grown for one day in selective media, in 384-well plates. We used minimal media to avoid background fluorescence found with rich media. At day 1, the test compounds are delivered to each well by pinning. Sealed lidded plates are grown for 17-19 hours. Each plate includes induced strains with or without test compound, uninduced bacteria (no compounds) and simple growth media. We have now screened 9 libraries, as follows. Each library name is followed by the number of unique compounds in the collection (number in parentheses). Diversity collections main screening libraries: Specs (30,106 unique compounds) Chembridge (23,865) ChemDiv (49,946) Kinase-targeted collections: ChemRX (9,706) Known Bioactive Collections: Sigma LOPAC qHTS (1,269) NIH Clinical Collection (377, tested in duplicate) Microsource Spectrum: MS Spectrum qHTS (1502) Biomol collections: ICCB (296) FDA (175) The preliminary number of inhibitory compounds is over 3600, but this number is an overestimate for several reasons. First, over 100 are simply toxic to all strains (inhibitory to growth as indicated decrease of over 50% compared to control). Second, the first pass of the screen had a very liberal interpretation of “inhibition”, asking for 30% inhibition of activity. Third, until the compounds are re-tested, it is unwise to draw any conclusions. We are presently retesting all compounds. With these cautions in mind, it is interesting to see the kinds of compounds we have found that inhibit activity of the Liberibacter protein transcriptional regulators. In our first screen Some are highly expensive chemotherapy drugs, which would not be practical for use in an agricultural situation but might provide interesting structural clues about inhibition. At the other end, we discovered inhibitory effects of some natural plant products such as certain flavonoids. We will have more precise data in the near future.
This project is a continuation of CRDF 447 to evaluate effects of Metalized Reflective Mulch (MRM) to protect newly planted trees from the Asian Citrus Psyllid (ACP) with new emphasis to determine if grapefruit trees planted into beds covered with Metalized Reflective Mulch (MRM) come into viable crop production at a younger age than in conventional plantings. Throughout this quarter ACP populations were monitored weekly and two Unmanned Aerial Vehicle (UAV) flights were made using cameras equipped for Normalized Difference Vegetation Index (NDVI) to determine the tree condition for each treatment. Conventional pesticide spray applications were applied based on scouting and according to IFAS guidelines. Similarly, irrigation events were made based tree and field conditions. Representative fruit samples, 20 from each treatment, were measured for fruit weight, fruit color, juice content and juice weight and juice analysis was determined for Brix, Acid, TSS/TA Ratio, and pounds solids. The MRM fruit were significantly higher in juice volume, juice weight, and fruit weight while the bare ground juice exhibited a higher TSS/TS ratio (at P value < 0.05 based on Duncan's multiple range test). Eighty clusters of four trees were randomly selected for each treatment and each tree was individually picked and run through a portable optical fruit sizing machine. Data collected from each tree included the total number of fruit and the weight and diameter of each individual fruit harvested. The diameter data were used to develop a fruit size distribution curve for each tree. The size distribution curve was used to calculate yield as boxes per tree based on state size standard for fresh grapefruit. The data afford by this method was statistically robust in that it encompassed 240 individually sampled trees yielding 195 boxes of grapefruit with the MRM treatment yielding 101% more boxes that the bare ground treatment. The trees receiving the compost treatment had a 38% increase in yield versus bare ground. Similarly the MRM treatment trees produced larger fruit than either compost or bare ground treatments. Leaf Analyses were performed for each of the three treatments all treatments were within optimum IFAS standards except that the bare ground treatment trees were slightly low in calcium. Based on these leaf nutrient concentrations and the above described crop yields, the fertility program, via fertigation, was deemed to be adequate for all three treatments. The MRM treatment continues to offer lower ACP counts for all life stages (eggs, nymphs and adults) based on weekly scouting. A detailed spreadsheet has been created to track all the caretaking expenses for the trial block including all spray foliar spray applications, herbicide treatments, fertilizer applications, soil drenches as well as the associated materials. These caretaking events are entered as they are accomplished and are combined to yield the total production cost for each treatment. Presentations of research results will be made to citrus growers and other scientists at the Florida Citrus Show on January 26, 2017
Our productivity significantly decreased after the move to the packing house while the AC in our lab was being fixed. There was bacterial contamination of our cultures, presumably due to autoclave issues, unsealed windows, or poor temperature control. Bacterial and fungal clean tests of mature citrus budwood from the growth facility in LB & LW broth, respectively, showed that all mother trees were clean, even the new cultivar introductions (B770, OLL8, Vernia, red grapefruit). We anticipate having to do two Agrobacterium transformations per week to make-up for lost time. Needless to say our efficiencies declined because of the move. Due to the aforementioned difficulties, Agrobacterium transformations with disease resistant genes was slowed. Only ~10 transgenics were produced and one did not survive micrografting. The results of the remainder are pending. Ten immature Swingle transgenics for Dr. Wang and Vladimir were micrografted because Vladimir had micrografting issues in the packing house. One shoot died & the results for the others are pending. We have found a cDNA that dramatically increases our mature scion transformation efficiencies and we are investigating whether it will increase efficiencies in all cultivars. An invention disclosure entitled, A method to increase organogenesis and transformation efficiencies in recalcitrant woody species such as mature citrus, was submitted to UF/IFAS Tech Transfer. There have been significant unanticipated growth room repair & maintenance expenditures during this last quarter. The water softener had to be rebuilt & a new one must be purchased next fiscal year. Without the water softener, hard water clogs the humidifiers & a white residue is deposited on plants making them unsuitable for transformation. The AC ducts in both growth rooms must be replaced because of filth deposited inside them over the years. The sprayer broke down & was repaired again. In the future, a new sprayer must be purchased to alleviate costly repairs. Lights & ballasts are an ongoing significant expenditure. We had to replace some of the shelving in the laboratory because our shelving disappeared after the move. The use of the PMI selectable marker after biolistics of immature and mature citrus continues. Different sucrose concentrations are required for shoot regeneration in mature vs immature citrus. Similarly, more sucrose is necessary for shoot development in scion than rootstock. Mannose concentrations must be manipulated accordingly. A manuscript (25% funded CRDF & 75% funded CRB) was submitted to PCTOC & is in review: Y. Acanda, M. Canton, H. Wu and J. Zale (XXXX) Kanamycin selection in bioreactors allows visual selection of transgenic citrus shoots, PCTOC.
The period between June and September 2016 was mostly spent on manuscript preparation, making presentations and preparing for the next series of objectives to be commenced in Nov, 2016. In the meantime a replacement scientist was sought given the departure of the previous Post Doc. In preparation for the next objectives, we proceeded to purchase healthy trees to be used as controls, pots and other needed greenhouse materials. We also re-designed the irrigation lines to conform with the new experimental design and modified the greenhouse benches. The objectives to be addresses are: III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. IV: Effect of SL on the growth of beneficial mychorriza. V: Effect of spray application of SL + other promising compounds on citrus in groves. Additional natural organic such as organic acids, sugars, amino acids, and few other compounds will be applied on potted trees.
We demonstrated 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. We used synthesized genes (G-Blocks). 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 successfully, created the vectors and infiltrated in Citrus macrophilla. The plants will be ready for evaluation by January 2017. 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.
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