Objective 1. Identify key metabolites that are associated with rootstock traits. Summary of accomplishments: Rootstock seedlings were grown in the USDA greenhouses and included standard rootstock cultivars and rootstocks developed by the USDA breeding program and in great demand by the industry. To establish metabolite profiles on a set of cultivars with known horticultural traits, we used the four rootstocks Cleopatra, Swingle, Sour orange, and Ridge Pineapple, and assessed the metabolite profiles of leaves and roots at two different seedling stages under greenhouse conditions. This allows us to characterize rootstocks based on their metabolite composition, and to assess the stability of metabolite profiles at different developmental stages. For a subset of the data, analysis has been completed and several compounds have been identified that may be associated with rootstock traits. A manuscript has been prepared and submitted to the journal Plant Science. In addition to these four standard cultivars, other rootstock seedlings were sampled and leaf and root extracts were submitted for GC-TOF MS analysis at the West Coast Metabolomics Center (WCMC), UC-Davis. Data were received in March 2017 and are currently being processed and analyzed by the team. Preliminary results of a selected set of the most recent data were presented at the IRCHLB in Orlando in March 2017. Objective 2. Investigate the effect of grafting on metabolite profiles. Summary of accomplishments: The four standard rootstocks (Cleopatra, Swingle, Ridge, and Sour orange) and other rootstocks developed by the USDA breeding program were analyzed as grafted trees in combination with Valencia and other scions. Trees were grown in the greenhouse or under field conditions at different locations. Comparison of the metabolite profiles of roots and leaves in these grafted trees with profiles obtained for leaves and roots of rootstock seedlings allows us to analyze rootstock and scion interactions, and evaluate how they affect tolerance to HLB and other biotic or abiotic stresses. The different field conditions under which the plants are grown will also aid in identifying environmental effects on metabolite profiles. The latest data set from this part of the study was received in March 2017 and is currently being processed and analyzed. Analysis of a subset of the data has been completed and we are in the process of preparing a manuscript for publication. Objective 3. Establish metabolite profiles of trees on different rootstocks in response to HLB. Summary of accomplishments: A paper was published last year describing our initial research on metabolic profiles of rootstocks with different response to HLB (U. Albrecht, O. Fiehn, K.D. Bowman. 2016. Metabolic variations in different citrus rootstock cultivars associated with different responses to huanglongbing. Plant Physiology and Biochemistry 107:33-44). In addition, an experiment was initiated that included many hundred grafted trees grown in the USDA greenhouses. These trees are composed of Valencia grafted on a diverse array of standard and USDA rootstock cultivars. One set of trees was inoculated with Las using graft-inoculation; a second set of trees was mock-inoculated using the same procedure. Trees were analyzed by PCR for presence of Las in leaf and in root tissue at regular time intervals. A subset of trees was selected, and leaf and root tissues of these plants were collected for metabolite analysis. These samples have been extracted and were sent to WCMS in April 2017. Some of the selected plants are being used for continuing infection and metabolic studies. Experimental design, data collected, analysis, results, and interpretation are too complex to present here. Additional information is available on request.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. Grafted plants are currently growing, and will be tested for responsiveness to the elf18 ligand for EFR and for canker resistance. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and therefore the protocol is being optimized for PCR screening. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs were provided to the Lake Alfred transformation facility, but transformation attempts have so far been unsuccessful. Troubleshooting has indicated that it is likely that the constructs have negative effects in citrus, and therefore work on these constructs has been discontinued. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homologs were sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. For proof of concept, we are targeting mutating the native citrus Bs5 alleles while simultaneously replacing the gene with the effective resistance allele. Two editing constructs have been created, one targeting the two conserved leucines, and one targeting two sites in the second exon to create a deletion in Bs5. Both constructs have been verified to function by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties. These constructs have been prioritized for transformation into Carrizo citrange, and transformations are underway at UC Davis. Transformants with mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
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.
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.
Evaluation of existing cultivar/rootstock combinations for HLB resistance/tolerance has revealed potentially valuable tolerance but indicates that early HLB symptoms and earlier CLas titer are unrelated to growth and cropping. This suggests that tolerance verification requires 5+ years of field evaluation. Discovery and utilization of molecular markers may help to accelerate the selection of resistance/tolerance materials by screening young plants and avoiding pathogen inoculation. Plant defense elicited by pathogen-associated molecular patterns (PAMPs) is an important component of disease resistance. In previous studies, we have shown that canker resistance in citrus correlates with responsiveness to Xcc-flg22, the 22 amino acid active region from the flagellin of Xanthomonas citri ssp. citri, the causal agent of citrus canker (Shi, Febres et al. 2015). To study the association between HLB resistance/tolerance and citrus response to flg22 of HLB causal bacterium Candidatus Liberibacter asiaticus (CLas), we designed an RNA-seq experiment comparing transcriptome responses in HLB moderately tolerant Sun Chu Sha mandarin and susceptible Duncan grapefruit, to Xcc-flg22 and CLas-flg22 (project initiated with Gloria Moore at University of Florida). Recently data analysis revealed that a group of 86 genes were differentially regulated by CLas-flg22 in Sun Chu Sha mandarin but not by Duncan grapefruit and not associated with differential expression from Xcc-flg22, suggesting they may have roles in HLB tolerance. The 16 genes with highest differential expression were selected for RT-qPCR validation, and 10 genes were consistent with the RNA-seq results. To evaluate if these genes were associated with HLB tolerance, Cleopatra mandarin (similar to Sun Chu Sha ) and Duncan grapefruit plants were inoculated with CLas using psyllid infestation. CLas titer and gene expression were monitored biweekly for 10 weeks after inoculation. High bacterial titer (Ct<30) was observed at 2 weeks in Duncan but not until 6 weeks in Cleopatra . RT-qPCR results indicated that 5 of the studied genes were differentially expressed between the Cleopatra HLB-infected and the uninfected control plants, but not in Duncan . It is worth noting that the induction of these genes was detected before bacterial infection was detected. Although not fully annotated in the citrus genomic databases, the function of some of these genes include a peroxidase, gibberellin 2-beta-dioxygenase, glucan endo-1,3-beta-D-glucosidase and an F-box domain containing protein. We will continue to characterize the expression of these genes and their association to HLB tolerance in other citrus genotypes, and determine if they may serve as marker genes for selection of tolerant citrus material. Trees of seemingly HLB resistant/tolerant sweet orange-like hybrids and mandarin -types were propagated and replicated trials with standards and have been established in growers' fields, in cooperation with G. McCollum. A mapping population of Fortune x Fairchild has been planted (collaborating Roose and Gmitter) along with related material, in an effort to identify genes associated with tolerance in the mandarin phenotypic group. Seedlings with a range of pedigree contributions from Microcitrus have been received in a collaboration with M. Smith, Queensland Aus. citrus breeder, are being grown, and will be planted in the spring for field testing of HLB resistance. In October 2013, 34 unique genotypes (USDA hybrids) some of which appear to have tolerance to HLB, and 16 standard commercial varieties were exposed to an ACP no-choice feeding trial and transferred to the field at Ft. Pierce FL. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a quarterly basis. HLB is now widespread and trees of more vigorous scion types are generally the healthiest at this point in time. At three years after planting, there continues to be great variation between selections and it may take 2-3 more years to clearly distinguish tolerant material.
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.
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.
Our project is focused on the following objectives: 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. 2. Breeding of HLB tolerant/resistant processing sweet orange-like hybrids. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. DPI Parent Tree Entries: Three gauntlet rootstock selections, plus two others already showing evidence of HLB tolerance in field trials. Several scions selections, submitted primarily because of high levels of tolerance to HLB in the field, including Cybrid Dancy (reduced seed); 1 Red pummelo, 1 nearly seedless pink pummelo, and seedless Pummelette ; 3 seedless mandarins; the so-called McTeer Murcott (low seeded and showing HLB tolerance under heavy field pressure); a late maturing true sweet orange exhibiting HLB tolerance in multiple replicates; two HLB tolerant sweet orange-like hybrids; one seedy but extremely high quality mandarin. Gauntlet activities: Several new candidates have been entered into the program, grafted with hot budsticks and their tops propagated. Seventy five candidates were moved from the greenhouse to a psyllid hot house, and another 145 trees were planted in Picos Farm for final field screening, in collaboration with USDA researchers. Transgenic plantings: 198 sweet orange trees and 27 W. Murcott trees, containing a total of 7 different constructs for possible resistance to HLB, were also planted at Picos Farm in collaboration with USDA researchers. Field trials: Meetings were held with our collaborators from Lykes and Cutrale Citrus to review plans and activities in large scale field trials with these companies. Replacement trees were replanted, and seeds for a large trial with Hamlin orange were collected and transferred to the relevant nurseries. Other activities: Meetings were held with CRDF staff to facilitate communications and mutual understanding of our current research agenda. Several interactions took place of our team with recently hired Dr. Catherine Hatcher of CRDF. We have made several field visits to some of our locations in an effort to help her understand the nature and landscape of our citrus breeding program. We feel we have established a good working relationship and anticipate that this will facilitate the more rapid deployment of potential genetic solutions to HLB in the industry. Our field research manager, Dr. Paul Ling, resigned during this time frame and we have begun a search for his replacement. Several meetings and teleconferences were held with collaborators from the USDA, UCR, DPI, and Rucks Nursery to develop and implement plans for two MAC projects aiming to plant several large scale trials in Florida of tolerant rootstock and scion cultivars.
Objective 1. (Greenhouse experiment): seedlings of the required rootstocks: x639, Swingle, WGFT+50-7, UFR-3 and UFR-15 potted in round citripots were trained to a single stem for subsequent stick grafting (in our HLB approved air-conditioned greenhouse). Liners are now stick-grafting size, grafting will begin in February, when conditions support a good graft-take. Objective III: To evaluate the effect of complete, balanced and constant nutrition on HLB-affected mature trees (composition, delivery and economics). All the pretreatment and mid-year data on tree health, canopy volume, leaf nutrients analysis has been collected and is currently being analyzed. We have applied the fertilizer for year 1 at both the locations. The trees have received full dosage of fertilizer for macro and micronutrients. Data collection on fruit drop has started at both locations. Next fertilizer application will be made in February. April is anticipated harvest time. Ojective 5. (funded by Orie Lee, using donated fertilizer products): Alligator Vernia/Rough Lemon Enhanced Nutrition Experiment Treatments: 6 tree plots (randomized), 2 plots per treatment treatments 2 times per year. The third treatment was applied as follows (using all donated products from TigerSul, Harrell’s and Florikan). Permanent field plot signs were installed (w/ assistance from Frank Rogers). 1. Control no extra nutrition 2. Harrells St. Helena mix (2lbs per tree) 3. Harrells St. Helena mix (2lbs.)+ 2x TigerSul manganese (90 gm) 4. Harrells St. Helena mix (2lbs.) + 2x Florikan polycoated sodium borate (32 gm) 5. Harrells St. Helena mix (2lbs.) + 2x TigerSul manganese (90 gm) + 2x FL sodium borate (32 gm) 6. 4x TigerSul manganese (180 gm) 7. 4x Florikan polycoated sodium borate (64 gm) 8. 4xTigerSul manganese (180 gm) + 4x Florikan polycoated sodium borate (64 gm) Donated micronutrient treatments were also applied at the Hughes Post Office block (Haines City) – where there were yield increases ranging from a half-box to 3/4’s box per tree this past season on 100% HLB-infected trees, enhanced by specific treatments, especially those containing both boron and manganese. Trees look exceptional at present, and we expect to see a yield increase for the 2nd year in a row since we began the study.
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.
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.
During this reporting period July, August, and September, 2016), Dr. McNellis continued to work with USDA APHIS to obtain permitting to transfer a set of ‘Duncan’ grapefruit plants expressing the FLT-antiNodT fusion protein from Penn State University to Fort Detrick in Frederick, Maryland. These plants are to be tested for resistance to HLB using a psyllid-vectored inoculation system in a secure greenhouse. We anticipate approval during the next reporting period. In addition, Dr. McNellis’ team at Penn State continued to evaluate the solubility and stability of the FLT-antiNodT fusion protein in citrus extracts and presence of the FLT-antiNodT fusion protein in various plant tissues by protein gel immunoblotting. The FLT-antiNodT fusion protein appears to be produced and present in all tissues examined to date, although these tests are ongoing and will continue into the next reporting period. Dr. McNellis presented a poster describing the results of the project to date at the annual conference of the American Phytopathological Society in Tampa, FL, July 30 – August 4, 2016. A poster viewer at the conference had some suggestions as to how to determine whether the FLT-antiNodT fusion protein indeed binds to its target in vivo, and Dr. McNellis has developed an experimental plan for doing this, which will be initiated during the next reporting period.
Objective 1: Assess canker resistance conferred by the PAMP receptors EFR and XA21 Three constructs were used for genetic transformation of Duncan grapefruit and sweet orange as part of a previous grant: EFR, EFR coexpressed with XA21, and EFR coexpressed with an XA21:EFR chimera. Seven transgenics have survived and passed a PCR screen, and these have been grafted onto rootstocks. To ensure that there will be sufficient events to analyze to come to a conclusion about the effectiveness of these genes, we have initiated more transformations in Duncan grapefruit at the Core Citrus Transformation Facility at UF Lake Alfred. In addition, we have added the recently-identified Cold Shock Protein Receptor (CSPR) to the transformation queue. Selection is underway, but the GFP marker is not expressed in citrus, and the protocol may need to be optimized. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Two constructs were created to co-express Bs2 with other R genes that may serve as accessory factors for Bs2. These constructs have been provided to the Lake Alfred transformation facility, and selection of transformants in Duncan grapefruit is underway. Objective 3: Development of genome editing technologies (Cas9/CRISPR) for citrus improvement The initial target for gene editing is the citrus homolog of Bs5 of pepper. The recessive bs5 resistance allele contains a deletion of two conserved leucines. The citrus Bs5 homolog was sequenced from both Carrizo citrange and Duncan grapefruit, and conserved CRISPR targets were identified. A construct targeting a site overlapping the two conserved leucines has been tested by co-delivery into Nicotiana benthamiana leaves with another construct carrying the targeted DNA from Carrizo or Duncan varieties, and verified to function. A replacement recessive bs5 allele will be added, and this construct will be prioritized for transformation into Carrizo citrange for proof of concept. Resulting plants with biallelic mutations in Bs5 that contain the replacement bs5 allele will be selected and tested for canker resistance.
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