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.
Recently we have made significant progress in the study of antibiotic resistance in Liberibacter using L. crescens as a model. A former postdoc in the lab, Kin Lai, was able to obtain five spontaneous mutants of L. crescens that were resistant to streptomycin at a frequency of about 1 in 100 million cells (the expected frequency). He was never able to generate spontaneous mutants of L. crescens to oxytetracycline. Kin left the lab and I gave the project to a very talented undergaraduate, Alexa Cohn. Alexa repeated the work of Kin and found the same results. Also like Kin, Alexa was unable to obtain any spontaneous resistant to oxytetracyline even after screening 10e13 cells. But Alexa took the work one step further and discovered something very interesting. She screened for mutants that were simultaneously resistant to streptomycin and oxytetracycline. She obtained two colonies that were resistant to both antimicrobials. Alexa then discovered that all eleven of our streptomycin-resistant mutants were also resistant to both antimicrobials. We are not in the process of getting sufficient DNA form each mutant to sequence each genome to identify the sites of mutation in each strain and then re-create those mutations in wild-type L. crescens using CRISPR technology. This result may have significant management implications. Two antimicrobial products are available to treat citrus for HLB. One contains only oxytetracycline. The other contains oxytetracycline and streptomycin. Our results suggest that treatment with only oxytetracycline is not likely to generate resistance very soon. In contrast, treatment with both antimicrobials may generate resistance to both antimicrobials quickly. We need to work with growers over the next six months to test this notion in the field. This can be done easily once we identify the source of the resistance mutation, which is likely within the rps12 gene. That will allow us to do rapid screening for resistance in the field by qPCR. Our culturing work has also suggested a means by which citrus greening disease might be controlled by a non-antimicrobial means. We have discovered that the preferred carbon source for L. crescens is either alpha-ketoglutarate or citric acid. Citric acid is a common constituent of citrus phloem while alpha-ketoglutarate is not. We are designing experiments to test the hypothesis that if we can nutritionally prevent phloem loading of citric acid, we can then starve Liberibacter in the phloem. We are first testing this nutritional approach in a greenhouse experiment that begins December 12, 2016. This experiment will require two months of nutritional treatment of the saplings followed by another month to extract the phloem, measure organic acids, and interpret the results. We still need a culture of Ca. L. asiaticus (CLas). We have a greatly simplified defined medium for L. crescens that allows the organism to grow much faster than on BM-7, the complex, undefined medium. The necessary changes to this medium are now being made that should encourage CLas growth. Testing of these media will be started within two weeks.
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. We repeated the concentration gradient experiments. A series of concentrations of the SAR inducer, including 0, 0.25, 0.5, 0.75, and 1 mM, were used to treat citrus plants by infiltration and soil drench. The infiltrated leaves and soil drenched plants were inoculated with canker bacterial pathogens 24 hours and 7 days later, respectively. Again, 5 plants were used for each treatment; three leaves on each plant were inoculated; 6 inoculations on each leaf were carried out, and a total of 90 inoculations were used for each treatment. Results confirmed that the strength of canker resistance is concentration dependent in the range between 0 to 1 mM. We also confirmed the systemic residual resistance activated by the SAR inducer. The SAR inducer-treated plants were cut back and leaves on the new flushes were tested for resistance to canker. As observed previously, canker disease symptom development was significantly delayed on the leaves on the new flushes. This result indicated that the SAR inducer not only activates resistance in the treated leaf tissues, but also in new flush leaves not treated with SAR inducer. In addition, experiments determining if the systemic residual resistance is conferred by the SAR inducer residue or products induced by the inducer are still ongoing.
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 order to understand how the SAR inducer activates disease resistance, we tested defense gene induction in the treated plants. Citrus leaves were infiltrated with 0. 0.25, 0.5, 1, 5, and 10 mM SAR inducer and the treated leaf tissues were collected at 0, 4, and 24 hours. Expression of a group of defense genes were analyzed by qPCR. These genes include PAL1,NPR1, PR5, CM1, ICS1, CM1, CM2, and PLDg. Results showed that the SAR inducer activated the expression of several defense genes such as NPR1, PR5, and CM1. We also tested if the SAR inducer treatment enhances pathogen induced defense gene expression. Citrus leaves were infiltrated with different concentrations of the SAR inducer. Thirty six hours later, the infiltrated leaves were inoculated with citrus canker bacterial pathogens. The inoculated leaf tissues were collected at 0, 4, 8, and 24 hours later. Expression of the above defense genes were analyzed by qPCR. We found that the bacterial pathogen induced expression of PAL1, NPR1, PR5, CM1, and ICS1 was significantly enhanced by the SAR inducer pretreatment. This results indicate that the SAR inducer can prime citrus plants for resistance to citrus canker. We have also started to test if the SAR inducer can elevate resistance or tolerance to HLB. We are currently testing the treatment conditions.
Between March and June 2016, we followed the proposed research plan by terminating few greenhouse and field experiments, collect and analyze data. I: Effect of drenching application of SL on HLB-infected trees. The soil in potted Valencia trees was drenched with SL at predetermined concentrations. As in the case for foliar applications, tree characteristics were noted and changes recorded on a bi-weekly basis. The second drenching treatment was applied in February as planned and data continued to be collected. The data indicated that drenching application was inefficient in spurring any physical changes as opposed to foliar applications. Despite the double treatment, application of SL via the root system is not efficient. II: Effect of spray application of SL on HLB-infected trees (Repeat experiment). This is a repeat of the experiments involving spraying SL on Valencia potted trees in the greenhouse. Tree characteristics were noted and changes recorded on a bi-weekly basis. Second spray treatment was applied in February and data on flowering, flushing and fruit set recorded biweekly. In parallel to the first year, foliar application of SL oh HLB-affected trees prompted rapid vegetative and reproductive flushes accompanied by the deposition of a wider band of phloem in the roots. This was observed during a routine study of root xylem which walls thicken under HLB. Xylem cell walls in HLB affected trees are considerably thicker then in healthy trees reducing the water conduits. III: Effect of SL + Fungicides on Phytophthora growth in HLB-infected trees. This treatment has been postponed until the arrival of a new scientist to take over the project. He is expected in October, 2016 and the experiments to commence on Nov 2016. IV: Effect of spray application of SL on other varieties of citrus in groves. ‘Midsweet’ was selected as a second variety to be tested for SL effect on tree health. Tree physical characteristics and fruit drop are being monitored throughout. Data on fruit drop was completed after application of spring treatment. The data was similar to those collected the first year. 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 were applied to trees. So far, diluted sucrose solutions have been effective in enhancing new flush in trees with advanced stages of HLB. Sucrose has been applied to trees in a monthly basis and some recovery has been observed. Treatments continue but improvement has slowed down.
The project has three objectives: (1) Confirm HLB resistance/tolerance in transgenic citrus lines. (2) Determine the chimerism of the HLB-resistant/tolerant transgenic lines. (3) Confirm HLB resistance in citrus putative mutants (nontransgenic lines). The following work has been carried out in this quarter: (1) Inoculated the promising candidate transgenic plants with CTV carrying the flower-promoting gene FT3. (2) Propagated the transgenic line HAM 13-3, DUN 57-25, DUN205-25c, and DUN 207-8. (3) Infected progenies of transgenic plants with Las-carrying psyllids. We have generated more transgenic plants expressing various defense genes. These transgenic plants are growing in greenhouse and will be tested once they are ready.
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