The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). All these propagates (ca. 250 trees) are growing well in greenhouse, and expected to go to field next month. These propagates will be tested by qPCR before going to fields. The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees.
Excellent progress was made in continuing the development of new hybrid rootstocks for the Florida citrus industry. Highest priority is being placed on work to develop hybrid rootstocks that can be made available for release to growers in the next three years, including selections from about 300 Supersour-type rootstock selections. It is expected that two of these Supersour rootstocks will be released in 2018, and at least two more in 2019-20, as further information is collected from ongoing field trials, and clean source material is available from from FDACS-DPI. During this quarter, one new replicated rootstock field trial was planted on East coast flatwoods site. The trial used Valencia scion and included statistically randomized replicates of 72 different rootstocks, with most rootstocks represented by 12 replications. Standard rootstocks included for comparison were Sour orange, Swingle, Cleopatra, and Ridge. Emphasis in the new trial was on SuperSour selections for which preliminary performance information is promising and that are in the DPI clean source program. Trees in the nursery were in the final stages of preparation for planting two additional field trials in the coming quarter. The trials will each include 8-12 replicates of about 60 new rootstocks, along with several standard rootstocks for comparison. Emphasis in these new trials is also on SuperSour selections for which other performance information is promising and that are in the DPI clean source program. Liners were prepared in the nursery by seedage and cuttings for additional field trials to be prepared for future plantings, based on new hybrid rootstock selections that look the best with HLB in preliminary field sites. About 600 additional seed source trees were planted into the field for these new promising selections, so that more seed will be available from the SuperSour rootstocks for commercial use after release. Research was conducted in collaboration with commercial nurseries and tissue culture propagation facilities to study and compare rootstock liners produced from seed, cuttings, and micropropagation. The rapid expansion in the demand for the newest rootstocks following release makes it clear that available seed for these new rootstocks will frequently be inadequate to satisfy commercial demands. Preliminary propagation information suggests that more US-942 rootstock will be propagated by micropagation than seed in the 2017-18 season. It is likely that cuttings and micropropagation of rootstocks will become increasing common. Noted differences in nursery performance of liners by propagation type, have prompted concerns about the influence of propagation type on field performance. We have initiated research to study these issues and address the concerns in a way that provides clear answers to growers and nurseries. One field trial containing rootstocks propagated by the three methods will be planted in the coming quarter. Performance data was collected from several field trials, including tree size, canopy health, and infection status by qPCR. Extensive cropping and fruit quality data will be collected from trials beginning the the next quarter. Analysis of results from previously established trials indicates that HLB introduces more complex variability to trials than that observed without HLB. This means that more than five or six statistical replicates will be needed to provide clear evidence of superior rootstock performance. Based on this observation, new USDA rootstock trials will usually include more than six statistical replications, regardless of the number of trees per replication. Using valid statistical comparisons is essential to develop reliable information about relative rootstock performance in field trials and should be the foundation of grower rootstock selection for new field plantings. I am working with the UF breeding team under two HLB-MAC projects to establish at least 13 additional rootstock field trials over the next 12 months, combining the best advanced new rootstocks from the USDA and UF breeding programs. The first of these trials will be field planted at the USDA Ft. Pierce Farm in the next quarter. Additional information is available on the USDA rootstock breeding project, on request.
Objective 1. Identify key metabolites that are associated with rootstock traits. Summary of accomplishments: Metabolite profiles of greenhouse-grown rootstock seedlings of eleven rootstock cultivars (Cleopatra, Swingle, Sour orange, Ridge Pineapple, Carrizo, US-802, US-812, US-896, US-897, US-942, US-1516) with known horticultural traits were assessed for primary metabolites possibly associated with traits. Analysis has been completed on a preliminary dataset. The data from an additional study to corroborate findings were recently received from West Coast Metabolomics Center (WCMC), UC-Davis, and are being used for detailed analysis by the USDA-IFAS team. Additional supporting studies are being conducted, including physiological assays and confirmation of identified compounds using chemical standards. The team is currently working on establishing these procedures to strengthen research findings. Objective 2. Investigate the effect of grafting on metabolite profiles. Summary of accomplishments: The four standard rootstocks (Cleopatra, Swingle, Ridge, and Sour orange) and seven additional rootstocks (Carrizo, US-802, US-812, US-896, US-897, US-942, US-1516) were analyzed as greenhouse-grown grafted trees in combination with Valencia. Metabolic profiles of leaves and roots of the grafted trees are being compared with those of leaves and roots from rootstock seedlings to assess rootstock effects on the scion and the possible implications for tree performance. Extensive additional metabolic data from our experiments were received back from the West Coast Metabolomics Center (WCMC), UC-Davis, and are being used for detailed analysis by the USDA-IFAS team. Due to the complicated nature of these data sets, this process is expected to take several months until ready for publication. In addition to the study of greenhouse-grown trees, analysis of metabolite profiles has been expanded to grafted trees grown under field conditions. A current data set including young trees with two different scion cultivars (Cara Cara and Hirado) in combination with the eleven rootstocks is in final process of analysis. A publication is expected to be submitted in the next few months. These studies will aid in identifying rootstock-scion interactions and the possible impacts on stress and disease tolerance under commercial conditions. Objective 3. Establish metabolite profiles of trees on different rootstocks in response to HLB. Summary of accomplishments: An experiment consisting of 300 grafted trees grown in the USDA greenhouses was completed. Trees were composed of Valencia grafted on a diverse array of standard and USDA rootstock cultivars and were either mock-inoculated or inoculated with Las. PCR analysis of Las bacterial titers of leaves and roots for the trees at different time intervals is in process. Leaf and root tissue of Las-infected and Las-uninfected plants were collected at 30 weeks after inoculation, and appropriate extractions completed. Samples from these experiments were sent to WCMS for GC-TOF-MS analysis of primary metabolites in April 2017, and the resulting metabolic data were recently received. A second set of samples from the experiment was recently sent to WCMS for analysis of secondary metabolites. Results from these analyses will be used by our USDA-IFAS team to conduct detailed analysis of metabolite profiles associated with HLB response. Publication of the results in a refereed journal will be completed, along with identification of practical utility of the information. Experimental design, data collected, analysis, results, and interpretation are too complex to present here. Additional information is available on request.
1) Trees have been in the ground for 3.75 years in a trial of 50 selections and cultivars on US-802 following no-choice ACP inoculation and several months in an ACP house. Standard growth measurements and disease ratings were initiated in July 2014 and will continue on a semi-annual basis. HLB is now widespread and the trees looking healthiest include a full sib of our best mandarin selection, and several of our best grapefruit-like hybrids. The one true grapefruit is the least healthy selection in the trial. There are eleven selections with a canopy volume 50% greater than Valencia and 28 with canopy volume >2X that of Flame. The best performers include hybrids containing Poncirus, and conventional hybrids which are predominately mandarin or pummelo. It may take 2-3 more years to clearly distinguish tolerant material. These trees are cropping this year, though Irma dropped a lot of fruit, and fruit will be used in a complementary project exploring synthesis of orange-like juice from HLB-tolerant types. 2) In June 2015 a field planting was established of: seedling trees of 133 Fortune x Fairchild hybrids from an earlier mapping study, seedlings of 27 Ponkan-like accessions, budded trees of 10 advanced ARS selections that are predominately mandarin, and budded trees of Fortune, Fairchild and Valencia. Data collection is underway. A NIFA grant proposal was submitted to mobilize tolerance for industry use. It is a collaboration between ARS, UF and UC Riverside. 3) Replicated trials in multiple locations are established of our best sweet-orange-like cultivars and mandarin-types. Volatiles from sweet-orange-like hybrids are so similar to sweet orange that likely can be legally named sweet orange. 4) RNA-seq compared transcriptome responses in HLB moderately tolerant Sun Chu Sha mandarin and susceptible Duncan grapefruit, to Xcc-flg22 and CLas-flg22 (most active epitope from the pathogen flagella; project initiated with Gloria Moore at University of Florida). Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with CLas, suggesting their involvement in HLB tolerance. In addition, several genes were similarly regulated by CLas-flg22 and CLas treatments. Genes identified awere recently published and are valuable for studying HLB tolerance mechanisms and potential for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy. Using these genes as markers, expression analysis from a group of mandarin hybrid in their responses to CLas-flg22 is underway. Highly and lowly responsive plants will be marked for long term observation of field tolerance. 5)A group of CLas effector (28) was predicted using bioinformatics tools. The study of transcriptional level of these effectors in infected citrus samples was initiated. Many of the effectors were successfully detected by RT-qPCR in citrus RNA, suggesting their roles in pathogen virulence and host response modulation. The expression level and profiling of these effectors were being investigated for its connection with host tolerance level, through comparative analysis between HLB tolerant and susceptible genotypes. 6) 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 soon for field testing of HLB resistance. 7) Our putative chimeras have not proven to be successful. We identified a chimera (Satsuma and Poncirus) from the Citrus genebank, arranged its importation, and we finally got permission to accept this material and maintain it in a quarantine death house. Cuttings of the chimera and each separate component (Owari and Poncirus) have been rooted and will be challenged by hot ACP feeding in the next quarters.
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. Recently we have had delays due to failures in ACP-inoculation and have reinitiated several challenges. 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. Rooted cutting of 167 Carrizo plants were obtained. A subset of 67 plants representing 13 independent events and wild types (4-5 replicates each) were inoculated by ACP infestation. All of the plants except 2 were confirmed CLas positive after a 2-week ACP exposure, and the titer between wild type and transgenic groups are similar at two weeks. The plants are maintained in the greenhouse for tests at 3, 9 and 12 months after inoculation. Transgenics expressing AMP D2A21 suppressed canker but not HLB with manuscript accepted for publication. 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 (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A group of 100 Carrizo plants were obtained as rooted cuttings and will be used for HLB testing. 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 citrus chloroplasts. Transcription data were obtained by RNA-Seq showing significant alteration in the transgenics. Publication submitted. 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 69 events from 7 ScFv with verified 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. Paper is published. In-silico analyses are being conducted to develop citrus FLS2 optimized for sensing CLas flagellin. 7) Arabidopsis DMR6 (downy 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 (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so gar) are ongoing. Comparison of reactive oxygen species in transgenic and nontransgenic plants treated with CLas-flg22 are underway, to determine if there is an enhancement of the broad-spectrum PAMP-triggered immunity . With targeted gene expression data, we will propagate selected plants based on the above-mentioned tests for HLB inoculations purpose. 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. Expression of a GUS marker gene was 500 x higher in midribs vs. laminar area. We are using this promoter aggressively in transgenic work 9) Third generation chimeral peptides were designed based on citrus thionins and citrus lipid binding proteins and plants have been transformed. Carrizo transformation of two constructs was completed and regenerated many seedlings. expression. A total of 43 Carrizo regenerations were confirmed being positive by PCR and highly expressed by RT-qPCR. Two constructs with above gene driven by double 35S promoter have 400 explants of Ray Ruby for each. 10) Two constructs with chimeral peptides containing citrus thionin and citrus proteinase were developed with both encoding genes are under by 35S promoter and SCAmpPs promoters. Transformation of those constructs are ongoing.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) New replicates of the transgenic lines that have been inoculated by CLas-infected psyllids were maintained in the greenhouse for symptom development. Background: We have generated transgenic Hamlin sweet orange and Duncan grapefruit and screened the transgenic lines for HLB resistance or tolerance. We did not find any resistant line but identified three independent lines (two Hamlin lines and one Duncan line) that exhibit robust tolerance to HLB. This result indicates that the Arabidopsis NPR1 gene is able to create HLB tolerance in citrus. Since we only have HLB-tolerant Hamlin and Duncan , we decided to transform the NPR1 gene into other cultivars including sweet orange Pineapple and Valencia as well as grapefruit Ray Ruby (These plants were generated by the mature tissue transformation lab). The following table shows the new lines that have been inoculated by CLas-infected psyllids and are maintained in the greenhouse for symptom development: Replicate Genotype Transgene Parental line A1 Ray Ruby NPR1 21 81 Pineapple NPR1 15 75 Pineapple NPR1 16 66 Pineapple NPR1 17 78 Pineapple NPR1 18 84 Valencia NPR1 20 76 Hamlin NPR1 8 73 Hamlin NPR1 10 67 Hamlin NPR1 11 70 Hamlin NPR1 12 87 Hamlin NPR1 13 79 Hamlin NPR1 14 (2) Propagated plants from those that have been treated with CTV-FT3 and have produced flowers for later heat treatment to remove CTV and CLas. Background: The three independent lines ( Duncan 57-28, Hamlin 13-3, and Hamlin 13-29) with robust tolerance to HLB have been treated with CTV that carrying the FT3 gene, which promotes conversion from juvenile to mature tissues. The three lines have all developed blooms. The flowering-promoting CTV and HLB bacterial pathogen (CLas) in the transgenic plants need to be removed before producing healthy progenies. The following table shows the new lines received from the transformation lab and will be replicated and screened for HLB responses: Transgenic line Genotype Transgene Maturation Replicates made 57-28 Duncan NPR1 Yes 4 13-3 Hamlin NPR1 Yes 6 13-29 Hamlin NPR1 Yes 3 (3) More transgenic lines received from the transformation lab were transplanted into bigger pots and will be analyzed for the transgenic protein accumulation. Background: We have not obtained multiple independent lines for Valencia and Ray Ruby and have requested more from the mature tissue transformation lab. The following table shows the new lines received from the transformation lab and will be replicated and screened for HLB responses: Transgenic line Genotype Transgene A99 Valencia NPR1 A100 Valencia SuperNPR1(a) A102 Valencia SuperNPR1 A101 Valencia SuperNPR1 A72 Valencia ELP3 (b) A73 Valencia ELP3 A97 Hamlin SupperNPR1 A98 Hamlin SupperNPR1 (a) SupperNPR1 is a more active version of the NPR1 gene. (b) ELP3 encodes a disease resistance regulator, which appears to also provide tolerance to HLB. A manuscript titled Overexpression of the Arabidopsis NPR1 protein in citrus confers tolerance to Huanglongbing has been written and submitted to the Journal of Citrus Pathology in this quarter.
During the period of April, May, and June of 2017, most of the progress was related to application for follow-up funding to characterize HLB resistance of plants generated in the present project. With assistance from Dr. Catherine Hatcher (CRDF), Dr. McNellis has coordinated with three faculty at the University of Florida to make arrangements for HLB resistance testing for the existing plants expressing anti-NodT scFv antibody (FT-scFv). These faculty are Drs. Ozgur Batuman, Liliana Cano, and Rhuanito Ferrarezi. They have the expertise to do CLas infections and PCR-based quantification of CLas in plant tissues. We plan to submit a pre-proposal to the CRDF for funding to support HLB resistance testing of the existing trees at Fort Pierce, propagated in Dr. Tim Gottwald’s lab at the USDA facility there. This will allow us to infect and perform quantitative bacterial population analysis in the citrus trees. We plan to submit this CRDF pre-proposal by the end of August, 2017. Dr. McNellis also attended and gave a presentation at the May 22-23, 2017, Forum on Citrus Breeding and Transformation for HLB resistance hosted by the National Academies of Sciences, Engineering, and Medicine in Irvine, CA. In addition, Dr. McNellis, along with Drs. Batuman, Cano, Ferrarezi, and Vladimir Orbovic (also University of Florida) submitted a pre-proposal to the USDA Citrus Disease Research and Extension Specialty Crop grant program on May 12, 2017, and were invited to submit a full proposal as of June 28, 2017. This proposal uses the present research results from this CRDF project as the preliminary data for the proposal, and builds upon results from the present work. We plan to submit the full proposal for the August 18, 2017, deadline.
Activities are reported by project objectives below. 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions. Prepared 120 gauntlet trees (all HLB-affected Valencia on individual rootstock candidates) for planting at USDA Picos. Completed stick-grafting of approximately 150 new rootstock candidates selected from 2015 crosses, and selected and stepped up approximately 120 hybrid rootstock candidates from 2016 crosses from the high pH, calcareous/Phytophthora soil screen, including hybrids of Sugar Belle with disease resistant parent trifoliate orange 50-7 and others. Planted a small trial of EV-1 and EV-2 early Valencias on putatively HLB-tolerant rootstocks in efforts to identify rootstocks that can prevent fruit drop with these selections. Planted a new field trial with >2700 trees near Ft. Meade including some of the UFRs, releases from California and Spain, and several CREC hybrids also planted in other locations. Worked together with Dr. Ferrarezi at the IRREC, and a host of IR growers and the IRCL to plan a new rootstock trial for the IR region, based on scion cultivars of importance to the fresh fruit producers of the region. 2. Breeding of HLB tolerant/resistant processing sweet oranges and orange-like hybrids. Planted approximately 300 trees of promising new OLL sweet orange seedling selections including OLL-DC-3-36 and OLL-DC-3-40 (original trees showed no HLB symptoms after 4 years in the field under heavy pressure). Continued micro-grafting recovered hybrids from 2016 scion crosses, including several combinations that should produce seedless sweet orange-like fruit with improved HLB tolerance. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. We continue to monitor our germplasm collection and breeding families for performance against HLB. We have initiated a genomic selection effort based on phenotypic assessments and using a high density SNP chip for genotyping. This may lead to identification of genetic resources to facilitate HLB-tolerant scion and rootstock breeding. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. Performed subjective HLB scoring on the 300+ gauntlet trees at the USDA Picos Farm; identified several new promising rootstock candidates. Rated trees in 5 different replicated rootstock trials throughout the state for HLB responses, and collected yield and juice quality information in 2 such locations. Other related activities: Worked with Dr. Ferrarezi to have IRREC land prepared for planting of propagated selected cybrid grapefruit clones (kumquat cytoplasm) of Flame, Ruby somaclone N11-11 and Marsh showing canker tolerance in greenhouse assays. The original N11-11 somaclone, derived from Ruby Red, has shown a strong recovery response from HLB, so these cybrids may be both canker and HLB tolerant. Planted a scion trial in the IR region with lemons from the CREC program, selected for high oil yields in the IR region; lemon s HLB-tolerance has generated industry interest as an alternative citrus product. The Rootstock Selection Guide, has had over 515,000 hits in its first 23 months. Finally, an exhaustive review was prepared of the UF-CREC filed program for CRDF BOD, RMC, and any other interested entities, and presented.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). All these propagates are growing well in greenhouse, and expected to go to field within five months. These propagates will be tested by qPCR before going to fields. The fruit quality (Brix, pH and % TA) of the four selected seedlings showed no significant difference from their maternal trees. The overall flavor and individual sugar components will be described in next progress report.
Good progress was made in continuing the development of new hybrid rootstocks for the Florida citrus industry. As requested by CRDF, highest priority is being placed on work to develop hybrid rootstocks that can be made available for release to growers in the next four years, including about 300 Supersour-type rootstock selections. It is expected that at least one of these rootstocks will be released within two years, with more to be released in the following years as further information is collected from ongoing field trials. During this quarter, one new replicated rootstock field trial was planted on a central ridge site. The trial used Hamlin scion and included 8 replicates of 50 different rootstocks. Standard rootstocks included for comparison were Sour orange, Swingle, Cleopatra, and Ridge. Emphasis in the new trial was on SuperSour selections for which preliminary performance information is promising and that are in the DPI clean source program. Trees in the nursery were in the final stages of preparation for planting three additional field trials in the coming quarter. The trials will each include 8-12 replicates of about 50 new rootstocks, along with several standard rootstocks for comparison. Emphasis in these new trials is also on SuperSour selections for which other performance information is promising and that are in the DPI clean source program. Liners were prepared in the nursery by seedage and cuttings for additional field trials to be prepared for future plantings, based on new hybrid rootstock selections that look the best with HLB in preliminary field sites. Additional seed source trees were prepared for field planting, so that more seed will be available from the SuperSour rootstocks for commercial use after release. Research was conducted in collaboration with commercial nurseries and tissue culture propagation facilities to study and compare rootstock liners produced from seed, cuttings, and micropropagation. The trend for rapid expansion in the use of new rootstocks following release makes it clear that available seed will frequently be inadequate to satisfy commercial demands for the newest rootstocks. It is likely that cuttings and micropropagation of rootstocks will become increasing common. Noted differences in nursery performance of liners by propagation type, have prompted concerns about the influence of propagation type on field performance. We have initiated research to study these issues and address the concerns in a constructive way. Analysis of results from previously established trials indicates that HLB introduces additional variability to trials that requires more than the previously effective five or six statistical replicates to provide clear evidence of rootstock performance. Based on this observation, new USDA rootstock trials will usually include more than six statistical replications, regardless of the number of trees per replication. Using valid statistical comparisons is essential to develop reliable information about relative rootstock performance in field trials and should be the foundation of grower rootstock selection for new field plantings. I am working with the UF breeding team under two HLB-MAC projects to establish at least 13 additional rootstock field trials over the next 12 months, combining the best advanced new rootstocks from the USDA and UF breeding programs. The first six of these trials are expected to be field planted with grower-cooperators in the next quarter. Additional information is available on the USDA rootstock breeding project, 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 putative transformants are being screened by RT-PCR. Objective 2: Introduction of the pepper Bs2 disease resistance gene into citrus Work on these constructs has been discontinued due to negative effects of the constructs in citrus. 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, with several rooted plants obtained so far. Molecular characterization of the putative transformants will be carried out at UC Berkeley. 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. Recently we have had delays due to failures in ACP-inoculation and have reinitiated several challenges. 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. . Rooted cutting of 167 Carrizo plants were obtained. A subset of 67 plants representing 13 independent events and wild types (4-5 replicates each) were inoculated by ACP infestation. All of the plants except 2 were confirmed CLas positive after a 2-week ACP exposure, and the titer between wild type and transgenic groups are similar at two weeks. The plants are maintained in the greenhouse for tests at 3, 9 and 12 months after inoculation. Transgenics expressing AMP D2A21 suppressed canker but not HLB with manuscript submitted for publication. 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 (citrus only genes) have been used to produce many Carrizo plants and shoots of Hamlin, Valencia and Ray Ruby. A group of 100 Carrizo plants were obtained as rooted cuttings and will be used for HLB testing. 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 citrus chloroplasts. Transcription data were obtained by RNA-Seq showing significant alteration in the transgenics. Publication submitted. 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 69 events from 7 ScFv with verified 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. Paper is published. In-silico analyses are being conducted to develop citrus FLS2 optimized for sensing CLas flagellin. 7) Arabidopsis DMR6 (downy 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 (28 independent so far) exhibit moderate to strong activation of plant defense response genes. Determination of silencing efficiency of DLO1 in transgenic plants (20 plants so gar) are ongoing. Comparison of reactive oxygen species in transgenic and nontransgenic plants treated with CLas-flg22 are underway, to determine if there is an enhancement of the broad-spectrum PAMP-triggered immunity . With targeted gene expression data, we will propagate selected plants based on the above-mentioned tests for HLB inoculations purpose. 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) Third generation chimeral peptides were designed based on citrus thionins and citrus lipid binding proteins and plants have been transformed. Carrizo transformation of two constructs was completed and regenerated many seedlings. About 40 of each group are being tested for transgene insertion and level of expression. Two constructs with above gene driven by double 35S promoter have 400 explants of Ray Ruby for each. 10) Two constructs with chimeral peptides containing citrus thionin and citrus proteinase were developed with both encoding genes are under by 35S promoter and SCAmpPs promoters. Transformation of those constructs are ongoing.
Objective 1. Identify key metabolites that are associated with rootstock traits. Summary of accomplishments: Metabolite profiles of greenhouse-grown rootstock seedlings of four standard rootstock cultivars (Cleopatra, Swingle, Sour orange, Ridge Pineapple) with known horticultural traits were assessed for primary metabolites possibly associated with traits. Analysis has been completed; however, additional studies are necessary to corroborate findings. These include physiological assays and confirmation of identified compounds using chemical standards. The team is currently working on establishing these procedures to strengthen research findings. In addition to these four standard rootstock cultivars, profiles of seven additional greenhouse-grown cultivars that are popular with the citrus industry (Carrizo, US-802, US-812, US-896, US-897, US-942, US-1516) are currently being analyzed. Extensive additional metabolic data from our experiments were received back from the West Coast Metabolomics Center (WCMC), UC-Davis, at the end of March 2017, and are being used for detailed analysis by the USDA-IFAS team. Objective 2. Investigate the effect of grafting on metabolite profiles. Summary of accomplishments: The four standard rootstocks (Cleopatra, Swingle, Ridge, and Sour orange) and seven additional rootstocks (Carrizo, US-802, US-812, US-896, US-897, US-942, US-1516) were analyzed as greenhouse-grown grafted trees in combination with Valencia. Metabolic profiles of leaves and roots of the grafted trees are being compared with those of leaves and roots from rootstock seedlings to assess rootstock effects on the scion and the possible implications for tree performance. Extensive additional metabolic data from our experiments were received back from the West Coast Metabolomics Center (WCMC), UC-Davis, at the end of March 2017, and are being used for detailed analysis by the USDA-IFAS team. Due to the complicated nature of these data sets, this process is expected to take several months until ready for publication. In addition to the study of greenhouse-grown trees, analysis of metabolite profiles has been expanded to grafted trees grown under field conditions. A current data set including young trees with two different scion cultivars (Cara Cara and Hirado) in combination with the four standard rootstocks (Cleopatra, Swingle, Ridge, and Sour orange) was received from WCMC at the end of March and is in the final process of analysis. A publication is expected to be submitted in the next few months. These studies will aid in identifying rootstock-scion interactions and the possible impacts on stress and disease tolerance under commercial conditions. Objective 3. Establish metabolite profiles of trees on different rootstocks in response to HLB. Summary of accomplishments: An experiment consisting of many hundred grafted trees grown in the USDA greenhouses was completed. Trees were composed of Valencia grafted on a diverse array of standard and USDA rootstock cultivars and were either mock-inoculated or inoculated with Las. PCR analysis of Las bacterial titers of leaves and roots for the trees at different time intervals is in process. Leaf and root tissue of Las-infected and Las-uninfected plants were collected at the end of the experiment, and appropriate extractions completed. Samples from these experiments were sent to WCMS for GC-TOF-MS analysis in April 2017, and the resulting metabolic data should be available in August, to be used by our USDA-IFAS team to conduct detailed analysis of metabolite profiles associated with HLB response. Experimental design, data collected, analysis, results, and interpretation are too complex to present here. Additional information is available on request.
Activities are reported by project objectives below. 1. Development of rootstocks that can impart HLB tolerance/resistance to grafted scions: A set of approximately 70 new rootstock candidates, stick-grafted with HLB-infected Valencia, were rotated into the hot psyllid house in cooperation with Dr. Nian Wang. The previous set of 50 rootstock candidates were removed from the hot psyllid house and prepared for field planting at the USDA Picos Farm in Fort Pierce. Twenty five new crosses were made using parents that have demonstrated HLB tolerance or have yielded offspring exhibiting high levels of tolerance. 2. Breeding of HLB tolerant/resistant processing sweet orange-like hybrids: At least 35 crosses were made this spring, to develop HLB-tolerant scion cultivars, using tolerant breeding lines with complementary characteristics. Fruit samples of some HLB-tolerant candidate selections were processed and pasteurized; several were found that produced good juice with no bitterness or off-flavors as typically found in mandarin hybrids. Three Valencia clones identified previously as rather tolerant in germplasm collection screening were observed still to be performing well; new anatomical and molecular studies have been initiated to determine underlying mechanisms of their tolerance; further, they have been entered into the DPI PTP for cleanup, and trees have been propagated directly for further field testing. Additionally, cybrid grapefruit clones (kumquat cytoplasm) of Flame, Ruby somaclone N11-11 and White Marsh showing significantly improved canker tolerance in greenhouse assays, were propagated for anticipated field planting at IRREC (~200 trees). Collected leaf samples from approximately 1000 transgenic trees and 300 gauntlet trees for qPCR to determine HLB infection status. Trees in both categories with good appearance were identified for further close observations. 3. Screening of the UF-CREC germplasm collection to identify and validate HLB tolerant or resistant selections. We continue to monitor our germplasm collection and breeding families for performance against HLB. We have initiated a genomic selection effort based on phenotypic assessments and using a high density SNP chip for genotyping. This may lead to identification of genetic resources to facilitate HLB-tolerant scion and rootstock breeding. 4. Advanced field trials, release and commercialization of promising HLB tolerant/resistant scion and rootstock cultivars. We reorganized our field data collection team, to focus on collecting data from our most critical trials. A new planting was made of seedless and easy to peel fresh mandarins, some exhibiting HLB-tolerance, in Pasco County. A large rootstock trial was planned and organized in the Peace River region. Completed yield and fruit quality data collection from the St. Helena Project. Preliminary analysis of the data shows an overall slight increase in lbs. solids over last year, with some rootstocks increasing in yield, and some decreasing. Trees on Swingle have shown a remarkable recovery. Collected yield and fruit quality data from 3 year old trees in the OLL sweet orange clone/rootstock trial near St. Cloud. Completed young tree assessments in the large new Camp Mack and Basinger rootstock trials with Lykes; Hamlin and Valencia trees on ~60 rootstock candidates were assessed for size, fruiting, overall health, and HLB disease symptoms. Other related activities: Minor revisions were made to the Rootstock Selection Guide, which has incidentally had over 389,000 hits in its first 17 months.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) Conducted two more rounds of cage experiments to further test if the transgenic plants inhibit psyllid reproduction. Results showed that none of the transgenes was able to significantly reduce psyllid progeny numbers in the cage experiment. (2) Newly generated replicates of the transgenic lines were inoculated in the psyllid room and nymph-infested plants were recorded and moved to the immediate greenhouse for symptom development.
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