A transgenic 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 over three years. Dr. Jude Grosser of UF has provided ~600 transgenic citrus plants expressing genes expected to provide HLB/canker resistance, which have been planted in the test site. Dr. Grosser planted an additional group of trees including preinoculated trees of sweet orange on a complex tetraploid rootstock that appeared to confer HLB resistance in an earlier test. Dr. Kim Bowman has planted several hundred rootstock genotypes, and Ed Stover 50 sweet oranges (400 trees due to replication) transformed with the antimicrobial peptide D4E1. Texas A&M Anti-ACP transgenics produced by Erik Mirkov and expressing the snow-drop Lectin (to suppress ACP) have been planted along with 150 sweet orange transgenics from USDA expressing the garlic lectin. Eliezer Louzada of Texas A&M has permission to plant his transgenics on this site, which have altered Ca metabolism to target canker, HLB and other diseases. More than 120 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) have been planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants are being monitored for CLas development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. Dr. Roose has completed initial genotyping on a sample of the test material using a “genotyping by sequencing” approach. So far, the 1/16th poncirus hybrid nicknamed Gnarlyglo is growing extraordinarily well. It is being used aggressively as a parent in conventional breeding. In a project led by Richard Lee, an array of seedlings from the Germplasm Repository are in place, with half preinoculated with Liberibacter. Additional plantings are welcome from the research community.
cDNA and genomic DNA sequences from the three FT citrus constructs were aligned with GeneBank’s published Citrus Unshiu sequences to properly asses the identity of the constructs being used to transform tobacco and citrus. The evolutionary history was inferred using the Neighbor-Joining method with a total of 10 nucleotide sequences. The results display an optimal tree revealing that FT3 is the most different FT. Also, the analysis shows that ciFT1 and ciFT2 cluster together and display high sequence similarity, indicating that these genes might in fact be alleles and not two separate genes. The one year study of the in vivo tracking of FT1, FT2, and FT3 in various citrus trees differing in age and phenotype is currently being repeated with higher concentrations of cDNA to solidify data and four candidate genes in the flowering pathway (FLD, FLC, ELF5, and AP1) have been added to the study to determine their involvement in flowering time and what effect they might have in the induction of the three FT genes. Results and statistical analysis from the quantitative real time PCR for FT1 and FT2 are being analyzed to further prove the relationship between FT1 and FT2 as being allelic. Tobacco ciFT3 transgenic lines from T1 and T2 generations where studied in order to determine the segregation pattern for ciFT3. GUS histochemical testing was done on germinated seedlings from four lines of T1 plants and four lines of T2 plants. Results show a that the transgene is passed on to offspring in a stable manner and in accordance with expected segregation ratios given a single gene insertion assuming heterozygous parents or more than one genetic insertion of the transgene. One T2 line in particular appears to be homozygous for the GUS gene, indicating that all offspring should have the ciFT3 gene which induces early flowering. This segregation data will be included in the transgenic tobacco manuscript currently being written. The endogenous ciFT3 promoter was successfully cloned to be used in the transcription activator-like (TAL) effector system inducible by methoxyfenozide that will activate the naturally present FT3 gene in citrus.
This project is built on the legacy of materials produced and field trials planted across the past several years. The objectives are to evaluate existing families and created germplasm in the field and in greenhouses for their responses to HLB and citrus canker, to carefully observe and document rootstock effects on severity and rates of progression of HLB symptoms, and to maintain the facilities and activities involved in the state-wide assessment activities. Assessments of HLB field tolerance have been carried out in the vast collection of raw germplasm that exists on UF and collaborating growers’ property. Twelve individual rootstock trials planted in SW Florida, the Indian River region, and along the Ridge have been carefully observed for performance against HLB. In several cases these observations were made in a quantifiable fashion, measuring tree growth and estimating severity of symptom expression. In a few rootstock trials with late season scions, we collected data to document yields, fruit and juice quality, and fruit drop, and to correlate the disease rankings with yield performance. We continued monitoring those already identified healthy, albeit infected trees, showing high yields of normal fruit; some of these have been included on the CRDF Rootstock Matrix. Rootstock seedlings of 100 accessions were previously grafted with HLB-positive Valencia budwood, and those growing out normal flush were selected and exposed to hot ACP populations in greenhouse conditions for one month. Under DPI permit, these trees were planted in a high-pressure, unsprayed grove on the east coast; we have continued to assess these trees. A total of 295 individual candidate rootstock hybrids (includes diploids and tetraploids from 10 different crosses) were selected from a screen for calcareous soil/Phytophthora tolerance; of these, 235 were successfully grafted with large budsticks of HLB-infected Valencia, most have flushed out, and many are showing no symptoms at present. Seed trees are being propagated simultaneously from these 235 individuals, by rooting the tops that have been removed upon grafting. Thirty HLB-Valencia grafted trees on candidate rootstock hybrids were rotated out of the ‘hot psyllid’ house (now ready for field planting), and another set of 50 were entered; these rootstocks include both 2x and 4x hybrids. As the citrus industry has become aware of the unusual performance of assorted rootstocks from our breeding program that are planted in field trials affected by HLB, there has come a huge interest in propagation and planting of these trees on a large scale in the various growing areas in the state. We had already provided substantial quantities of seed from some of these outstanding HLB-tolerant candidates to a major grower for propagation of trees to be planted out in large blocks (up to one acre in size per rootstock); additional seeds of other selected rootstocks have been provided to the same grower, as well as to another citrus nursery business, all for the purposes of large scale field trials. Rootstock liners are being grown now, and previously grown rootstocks are being budded with commercial scions. Information has been collected to protect the IP in preparation for the upcoming release of the new UF-CREC rootstocks on the CRDF Rootstock Matrix. Seed trees exist for some, but not all of these selections; for those which saw their source trees burned a few years ago during canker eradication efforts, we have lifted roots to stimulate shoot development so we can recover these potentially valuable rootstocks for increase and production. Materials have been sent to the DPI PTP for STG and indexing, so certified materials can be made available to nurseries and TC companies for rapid increase of those trees currently with inadequate seed supplies. Finally, trees have been produced for 2 new rootstock trials, one with grapefruit on the east coast, and a second with a mandarin scion in SW Florida.
HLB’s impacts have led to grower interest in advanced production and harvesting systems with the potential for early and sustainable yield, as well as ease of harvest and other management efficiencies. In the absence of a long-term HLB solution, grove life may be only 12-15 years. A different production approach is required, and higher density plantings with smaller trees managed with intensive cultural systems may be a solution. The goal of this project is to identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Existing field trials previously planted with size-controlling rootstock candidates were monitored for tree growth and disease incidence, including the portion of the St. Helena project planted with dwarfing selections, and a 40-acre Hamlin/Valencia cooperative rootstock trial with trees planted between 300-500/acre. The latter planting is 3 years old, and yield data were collected on the Hamlin portion of the planting last winter and recently from the Valencia portion. Seed trees for selected dwarfing rootstocks, already showing good performance, were propagated and some were planted, to support expanded trials in the future. Some new rootstocks from the CRDF Rootstock Matrix selected for their potential in high density plantings through good tree size control were entered into the DPI Parent Tree Program for cleanup by shoot tip grafting followed by indexing, to provide certified budwood of these rootstocks for commercial nurseries upon their release. A new rootstock trial with Ray Ruby grapefruit and containing several tree size control candidates coming from crosses made using Flying Dragon as a seed parent (to capture the dwarfing trait, crosses must be made in this direction) was planted this spring, and the remaining selections for the trial were grown off and planted in late summer 2013; more than 2000 trees total were planted. Seedlings of some other Flying Dragon hybrids have come through the ‘HLB gauntlet’ screening process (grafted with CLas-infected Valencia budsticks, and then cycled through a hot psyllid house, ending with no obvious HLB symptoms); these will be planted in the field, under a DPI permit for further observation.
A number of Poncirus and Citrus cultivars have been recently found to be tolerant to HLB. Microarray-based profiling of the transcriptomes of two cultivars with HLB tolerance (Poncirus hybrid US-897and rough lemon) and two cultivars without HLB tolerance have identified over 1,150 genes that are differentially expressed in HLB-tolerant cultivars. These genes constitute a valuable pool of potential candidate genes from which true HLB tolerance genes may be identified. Additional candidate genes have recently become available from an RNA-seq experiment using rough lemon and sweet orange in a comparison similar to what was done with the Affymetrix microarray work in our lab (Fan, et al., 2012). This project aims to screen these potential candidate genes using high throughput target capture, massively parallel sequencing of targeted gene regions, and genetic association and linkage analysis to find the most likely candidate gene(s) for HLB tolerance in Poncirus and rough lemon. After several rounds of candidate searches, we have recruited suitable post-doctoral research associates. One post-doc scheduled to arrive in January 2014 and another to begin shortly thereafter, so it is our intention to accelerate the project substantially at that time. In the meantime, sequencing of the Poncirus and rough lemon genomes are in process. Plant materials to support the project are being collected and grown, for downstream applications. We are in the process of compiling the list of nearly 1,300 candidate genes and downloading their sequences for designing a high throughput target capture system that will be used to target sequencing, genetic association and linkage analysis.
We have continued to monitor previously identified candidate survivor trees at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program. Most of the trees now have begun to display symptoms after more than 24 months of observation, though there remain a few still unaffected as of March 2014. Trees at an abandoned location in Palm Beach County, that retain reasonably good condition and freedom from obvious symptoms, were sampled for both budwood and root tissues. Budwood samples were propagated onto healthy rootstock seedlings at the CREC. In addition, root samples were provided. We were able to collect rootsprouts from some of the trees that were decapitated; we have propagated from these rootsprouts, by budding and by rooted cuttings. These small trees are growing of for further propagations to test their responses to HLB. . We extracted DNA from these sprouts and compared their DNA fingerprints with what we produced from feeder roots collected previously, as expected, the fingerprints were identical. New rootstock samples collected from trees were used to confirm nucellar embryony. Thus far, all except one of the rootstocks collected has been shown to be a nucellar seedling of the presumed rootstock. Routine fingerprinting of all scion varieties sampled has shown them all to be true to type; this does not discount the possibility of mutations for HLB tolerance/resistance. We have gathered information on several other possible survivors identified and it was decided to allow more time to determine whether the these trees warranted more careful analysis because they generally represented quite a few trees in each location. In other instances, the reported survivors actually exhibited more symptoms than would qualify as a ‘healthy’ survivor. Two hybrids of unknown origin that we have been observing for several years in CREC groves, and were found to be HLB-free have begun to display minor and scattered symptoms. Recent qPCR has also shown the presence of CLas, though at very low levels; hwever, these trees are exhibiting ever-increasing severity of disease. New reports of survivors have come in from Highlands, Lee, Collier, Indian River, Lake, and Marion Counties; several have been documented, and some have been visited by the PI or extension personnel; materials have not yet been collected, pending further assessment of disease in 2014.
We have continued to monitor previously identified candidate survivor trees at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program. Most of the trees now have begun to display symptoms after more than 18 months of observation, though there remain a few still unaffected as of December 2013. Trees at an abandoned location in Palm Beach County, that retain reasonably good condition and freedom from obvious symptoms, have been sampled for both budwood and root tissues. Budwood samples were propagated onto healthy rootstock seedlings at the CREC. In addition, root samples were provided. In the past three months we have collected rootsprouts from some of the trees that were decapitated; we have propagated from these rootsprouts, by budding and by rooted cuttings. We extracted DNA from these sprouts and compared their DNA fingerprints with what we produced from feeder roots collected previously, as expected, the fingerprints were identical. These rootstocks will be increased and grown off for future experiments, to test their responses to HLB. New rootstock samples collected from trees were used to confirm nucellar embryony. Thus far, all except one of the rootstocks collected has been shown to be a nucellar seedling of the presumed rootstock. Routine fingerprinting of all scion varieties sampled has shown them all to be true to type. We have had information on several other possible survivors collected, and in these cases it was decided to allow more time to determine whether the these trees warranted more careful analysis because they generally represented quite a few trees in each location. In other instances, the reported survivors actually exhibited more symptoms than would qualify as a ‘healthy’ survivor. Two hybrids of unknown origin that we have been observing for several years in CREC groves, and were found to be HLB-free have begun to display minor and scattered symptoms. Recent qPCR has also shown the presence of CLas, though at very low levels.
This three-year project is to continue the search and evaluation of citrus tree survivors found under high pressure of HLB and its pathogen, on the basis of additional visits to groves in severely HLB-affected production areas, primarily in Florida, but also in areas of southern China that we have visited previously. Past exploration in China has identified three such trees and at least one of these remains free of HLB after several years. We have previously propagated a few trees in Florida from Pineapple orange budwood collected in Martin County. We have continued to monitor previously identified candidate trees at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program. Some of the trees now have begun to display symptoms after more than 15 months of observation, but there remain some that are still unaffected as of September 2013. Trees at an abandoned location in Palm Beach County, that retain reasonably good condition and freedom from obvious symptoms, have been sampled for both budwood and root tissues. Budwood samples were propagated onto healthy rootstock seedlings at the CREC. In addition, root samples were provided. We extracted DNA from these, and from several of the other trees mentioned above, to characterize the rootstocks on which they are growing. In most cases we have been told what the original trees were supposed to have been grown on, and we wanted to be able to confirm whether the root system is the variety expected, or if it is a chance zygotic seedling. To this end, we have improved our DNA extraction procedures from root tissues, so we can now reliably fingerprint the rootstocks. We have done so for nearly all of the candidate trees on which we have been working, and up until now all except one of the rootstocks has been shown to be on nucellar seedlings of the presumed rootstock. Routine fingerprinting of all scion varieties sampled has shown them all to be true to type. We have had information on several other possible survivors collected, and in these cases it was decided to allow more time to determine whether the these trees warranted more careful analysis because they generally represented quite a few trees in each location. In other instances, the reported survivors actually exhibited more symptoms than would qualify as a ‘healthy’ survivor.
Certain citrus cultivars, such as Cleopatra mandarin, seem to be incapable of supporting the full developmental life cycles of psyllids. Preliminary experiments with hybrids in a Cleopatra-derived family, using caged psyllid nymphs on pesticide-free, field grown trees, indicated possible genetic transmission to some of the progeny. A total of 91 trees in three families produced by crossing Cleopatra mandarin with three male parents were selected from field plantings for the project and for future evaluations. We have propagated replicates from each individual for assessments of psyllid reproduction and feeding behavior in controlled greenhouse and laboratory conditions, and transferred these to Dr. Rogers for greenhouse and lab studies, that will be conducted once the trees have reached sufficient size. A first round of caged tree limb experiments using a subset of the total trees available was completed. The trees were selectively pruned to provide abundant young flush. Specific numbers of nymphs were placed onto each of two branches per tree with flush, and cages were carefully placed over the branches. Data on survival, numbers of adults, egg deposition, etc. were collected. These results are being compared to results from previous preliminary experiments conducted in the field. Several of the hybrids were unable to support adult psyllid development on both branches. A second round of field testing will be delayed until spring, to ensure good flush growth.
We have quantified HLB symptom rankings for all trees in the field planting, this past October; symptoms and RT-PCR have not been studied through the winter months, as several of the hybrids are semi-deciduous, which complicates phenotyping and accuracy of RT-PCR results. We have found at least one tree to be PCR+ for almost all selections assayed, though some of these have very high Ct values indicating very low concentrations of CLas DNA in such trees; these trees will be monitored going forward to see whether CT values decrease remain stable, or increase. The pure trifoliate orange types in the planting remain essentially PCR-, while control sweet orange trees are quite obviously affected by symptoms, and they also had low Ct values, indicating substantial CLas population levels. The hybrids appear to be segregating for tolerance, though not into clear and distinct categories. Leaf samples were collected from all individuals in the field trial. DNA was extracted from more than 840 individuals total for qPCR runs, which have been completed and summarized. The same trends we see in our monitoring subset data are seen in the total populations. We have propagated plants from the mapping population to provide materials for greenhouse phenotyping via graft inoculations, increasing both the number of individuals propagated as well as the number of replicates of each genotype. We cannot accommodate the entire population in replicated fashion because of greenhouse space limitations, so we have moved selected subsets of individuals in the greenhouse to compare with phenotypes observed in the field; these plants are growing to a size sufficient for the graft inoculations. We are increasing simultaneously the supply of available budwood for inoculations. We have produced a source of inoculum in selected citrus accessions, which was initiated from a field source. This strain of CLas was passed first through Carrizo citrange so that it is free of CTV, by virtue of the immunity of Carrizo to CTV; this was done to remove possible complications in the evaluations that may be associated with CTV-HLB interactions. The selected hosts grow quickly and attain high titer of CLas as evidenced by low CT values.
We aim in this project to genetically manipulate defense signaling networks to produce citrus cultivars with enhanced disease resistance. Defense signaling networks have been well elucidated in the model plant Arabidopsis but not yet in citrus. Salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) are key hubs on the defense networks and are known to regulate broad-spectrum disease resistance. With a previous CRDF support, the PI’s laboratory has identified ten citrus genes with potential roles as positive SA regulators. Characterization of these genes indicate that Arabidopsis can be used not only as an excellent reference to guide the discovery of citrus defense genes and but also as a powerful tool to test function of citrus genes. This new project will significantly expand the scope of defense genes to be studied by examining the roles of negative SA regulators and genes affecting JA and ET-mediated pathways in regulating citrus defense. We have three specific objectives in this proposal: 1) identify SA negative regulators and genes affecting JA- and ET-mediated defense in citrus; 2) test function of citrus genes for their disease resistance by overexpression in Arabidopsis; and 3) produce and evaluate transgenic citrus with altered expression of defense genes for resistance to HLB and other diseases. With this support, currently we have cloned six full-length genes with potential roles regulating SA, ET, and/or JA pathways to the binary vector pBIN19plusARS and transferred the constructs to Agrobacteria. All six Agro strains were sent to co-PI Dr. Bowman’s lab to initiate citrus transformation. In the mean time, we started the process of transforming Arabidopsis to overexpress these genes and to test their defense function. T0 transformed seeds have been harvested for some constructs and will be screened for transgenic plants followed by disease resistance tests. We aim to clone at least 10 citrus genes for testing their effectiveness in conferring disease resistance to HLB and citrus canker diseases. Additional gene cloning is underway. In addition, we are continuing to characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project (#129), although the support of the project has already been terminated.
We have continued to propagate plants from the mapping population to provide materials for greenhouse phenotyping via graft inoculations, increasing both the number of individuals propagated as well as the number of replicates of each genotype. We cannot accommodate the entire population in replicated fashion because of greenhouse space limitations, so we are preparing to test subsets of individuals in the greenhouse to compare with phenotypes observed in the field. Selected plants from several individual hybrids will be graft inoculated using infected budwood sticks in early spring 2014, once we have come through the winter-induced dormancy of some of the trifoliate hybrids. We have quantified HLB symptom rankings for all trees in the field planting, this past October. In addition, we have sampled our preselected subset of monitor trees, and the frequency of CLas detections is increasing compared to previous samplings. We have found at least one tree to be PCR+ for almost all selections assayed, though some of these have very high Ct values indicating very low concentrations of CLas DNA in such trees. The pure trifoliate orange types remain essentially PCR-, while control sweet orange trees are quite obviously affected by symptoms, and they also had low Ct values, indicating substantial CLas population levels. The hybrids appear to be segregating for tolerance, though not into clear and distinct categories. Finally, leaf samples were collected from all individuals in the field trial. DNA was extracted from more than 840 individuals total for qPCR runs, which have been completed. These results currently are being summarized. The same trends we see in our monitoring subset data are seen in the total populations. A post-doc has been hired to begin work on the project in January 2014.
We have continued to propagate plants from the mapping population to provide materials for greenhouse phenotyping via graft inoculations, increasing both the number of individuals propagated as well as the number of replicates of each genotype. These trees are being grown to a size suitable for inoculation with HLB infected budwood in an approved greenhouse structure at the CREC. We cannot accommodate the entire population in replicated fashion because of greenhouse space limitations, so we are preparing to test subsets of individuals in the greenhouse to compare with phenotypes observed in the field. We have continued to monitor all plants in the field for symptoms of HLB. The site where these are being grown is under very high pressure and the trees are not regularly sprayed for psyllid control. Unfortunately, the soils at this site are high pH and do not support vigorous tree growth, they produce nutrient deficiency symptoms, and the trees are Citrus x Poncirus hybrids; therefore recognizing obvious HLB symptoms is difficult. However, management at the site has improved and the trees are looking better than previously. In mid-September it was becoming more obvious that HLB was impacting several of the selections. We selected a subset of trees to develop baseline qPCR values as we proceed to assess infection by CLas over time. DNA was extracted from these trees as well as positive and negative controls, and qPCR was run. We were able to detect CLas in some but not all of the trees. We plan to revisit and record observations on symptom development in the next month when symptoms should be most easily seen, to run qPCR again on our subset of trees, and then to look at qPCR Ct values in the entire collection. Our GoldenGate SNP assay platform has been used to genotype the entire population; the data produced have been preliminarily analyzed, prior to developing a high density genetic linkage map, as proposed.
In this proposal our objective is to find citrus versions for the two proteins that make up the functional components of a chimeric antimicrobial protein (CAP) previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have successfully identified a suitable replacement for the first component, the human neutrophil elastase (HNE) that also serves as the surface binding component of CAP. Since HNE is a serine protease with elastase activity whose 3D structure has been determined we used the PDB database to find a suitable plant protein with the same 3D structure. Using the active site geometry of HNE is a consistent structural feature we focused on a set of 288 non-redundant plant derived proteins extracted from the PDB database to narrow our search criteria. The key feature of our search involved using CLASP to search for a match using the electrostatic properties and structural geometry of the three amino acids that make up the active site of HNE. We obtained a close match with the tomato PR14a protein. Using the tomato amino acid sequences we then searched for a similar citrus protein by searching through citrus genome information in Phytosome (http://www.phytozome.net). This was successful and we have identified a single protein that has the identical amino acid sequence in both Citrus sinensis (Cs) and Citrus clementina (Cc) genomes. We have focused the 165 amino acid P14a protein from Cs which we refer to as CsP14a. We have analyzed this sequence and have determined that it is a secreted protein and contains what appears to be a 25 amino acid signal sequence. We have utilized the 137 aa mature protein and successfully constructed two synthetic genes that encode this protein, one that just contains the coding region of CsP14a and the other is a chimeric version that contains the CsP14a coding region linked to the CecropinB (CecB) protein. We have included a signal peptide (22aa) that we have used before and know works really well at secreting proteins to the plant apoplast and xylem. This signal peptide has been added at the N-terminal of this protein and we have added a Flag Tag also at the N-terminal so that the protein can be easily detected and purified. The Flag tag will remain a part of the secreted protein after cleavage of the signal peptide. We are constructing two CaMV35S expression cassettes to express both these synthetic genes. We are using CLASP to identify a citrus replacement component for CecB. Since CB has no enzymatic activity, we could not use a well-constrained motif like an active site. We chose instead the structural motif Lys10, Lys11, Lys16, and Lys29 a unique feature of CecB. Our analysis has proved fruitful and we have identified a good plant candidate that has the same shape and that is highly conserved in plants.
Our accomplishments are: 1) Various young and mature citrus plants and also citrus seeds that are used for this project were purchased, planted and maintained in a greenhouse; 2) Sterile culture of citrus plant materials were established; 3) Construction of the proposed genes that should enhance shoot regeneration and embryogenesis has been started and is well underway.
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