Certain citrus cultivars, such as Cleopatra mandarin, have been reported 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. We will evaluate this effect, in replicated experiments conducted under controlled conditions. Identifying genetic control of the suppression of psyllid feeding and reproduction in host citrus plants potentially points to future strategies aimed at capitalizing on this phenomenon as another tool to mitigate HLB disease effects in integration with other genetic, cultural, and chemical control strategies. 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. Rootstock seedlings were produced previously, and these have been used to propagate replicates from each individual for future assessments of psyllid reproduction and feeding behavior in controlled greenhouse and laboratory conditions; these trees are being grown now using management approaches to have trees available this autumn for greenhouse and lab studies. A first round of caged tree limb experiments using a subset of the total trees available has been initiated. 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. will be collected. These experiments will be repeated at least two more times to confirm results.
Plants of many but not all individuals from within the mapping population have been propagated to provide materials for greenhouse phenotyping via graft inoculations. 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 taking the approach of testing subsets in the greenhouse and comparing this with phenotypes as seen in the replicated field planting. Recently, plants in the field were observed 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. We plan to revisit and record observations in the autumn when symptoms may be most easily seen. 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. The GoldenGate SNP assay platform, developed under another project for genotyping applications, was used and data produced have been preliminarily analyzed, that will lead to a relatively high density genetic linkage map as proposed. The visiting scholar we assigned to this project has continued in carrying out the scheduled activities of the project.
Plants of many but not all individuals from within the mapping population have been propagated to provide materials for greenhouse phenotyping via graft inoculations. We cannot accommodate the entire population in replicated fashion because of greenhouse space limitations, so we are taking the approach of testing subsets in the greenhouse and comparing this with phenotypes as seen in the replicated field planting. Plants in the field were observed for symptoms of HLB, but as many of these Citrus x Poncirus hybrids are deciduous or semi-deciduous it is virtually impossible to draw any conclusions about infection and disease expression during the winter months. DNA preparations have been made from the mapping population individuals, and we will use the GoldenGate platform developed under another project to genotype the population, as well as the parents. We had recruited a post-doctoral research associate to work on this project, but at the last minute we were informed that the candidate decided to accept another position, so we are continuing the search for a qualified individual. In the meantime, a visiting scholar with interests and experience in disease resistance and genetic mapping has been assisting in carrying out the scheduled activities of the project.
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. Last summer, we visited properties at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program, with the express purpose of identifying particularly healthy appearing trees that can be found in blocks as HLB symptoms are becoming more widespread and obvious. These trees have been noted and marked on maps, and we have revisited these specific trees several times. Some have begun to display more obvious symptoms, but there remain several that seem unaffected. We have collected DNA samples and run qPCR in efforts to detect CLas, and several of these remain PCR negative; these observations and qPCR tests have been repeated in spring 2013. We will continue to monitor these. A new location with promising trees was identified in Palm Beach County. These trees have been abandoned for more than 3 years, yet appeared to be rather healthy considering the lack of any management input in that time. The best looking trees were tagged, because the budwood that might have been collected was too thin for good propagation. Therefore, the grower cooperator agreed to remove weeds and apply fertilizer. Budwood samples were collected and 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 want 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 been working recently to improve the ability to extract DNA of sufficient quality for analysis, from feeder roots as well as scaffold root bark. We have been more successful in this using healthy feeder roots than with bark, but in both cases we have been able to amplify DNA by PCR. Meetings were held with CRDF staff regarding this project and the possibility of increasing the focus of effort. UF-IFAS extension personnel have been brought into the process, as the first line of contact and collection of information. A questionnaire has been developed for extension personnel to use when being made aware of healthy surviving trees in devastated grove situations. Collaboration and coordination with Dr. Nian Wang on the project, who will look at the soil microbiome beneath these surviving trees has been established.
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. Two of 5 original source trees were found to be qPCR negative, while > 90% of the other trees in the block were dead. These trees were maintained in a greenhouse at the CREC, but unfortunately have been lost. Last summer, we visited properties at the CREC, the GCREC, and some Polk County commercial groves where we have planted out materials from the CREC breeding program, with the express purpose of identifying particularly healthy appearing trees that can be found in blocks as HLB symptoms are becoming more widespread and obvious. These trees have been noted and marked on maps, and we have revisited these specific trees several times. Some have begun to display more obvious symptoms, but there remain several that seem unaffected. We have collected DNA samples and run qPCR in efforts to detect CLas, and several of these remain PCR negative. We will continue to monitor these. A new location with promising trees was identified in Palm Beach County. These trees have been abandoned for more than 3 years, yet appeared to be rather healthy considering the lack of any management input in that time. The best looking trees were tagged, because the budwood that might have been collected was too thin for good propagation. Therefore, the grower cooperator agreed to remove weeds and apply fertilizer, and budwood will be collected in the future for experimentation.
The haploid Clementine and sweet orange sequences have been assembled, annotated, and are available to the research community at Phytozome and at citrusgenomedb.org. The new Clementine v. 1.0, has finally been validated and made publicly available in USDOE’s JGI Phytozome v 9.0 in December 2012. This assembly is a vast improvement over the first version (v 0.9) that was made available to the research community in January 2011. The genome is organized into 9 super-scaffolds, representing the basic nine chromosomes of citrus. This was made possible by the ICGC collaborative genetic mapping effort, in which our lab was a primary contributor. (See Ollitrault et al. BMC Genomics, 2012, 13:593 DOI:10.1186/1471-2164-13-593). New citrus sequences were provided in February 2013 of some varieties underrepresented previously including Ponkan mandarin and sweet orange. These have been further analyzed to elaborate the phylogeny of sweet orange, Clementine, Ponkan and Willowleaf, and sour orange; all are admixtures of C. reticulata and C. maxima, in varying degrees. A new manuscript has been prepared and is under revisions by the numerous co-authors. Work proceeds on the other objectives of this project. New experiments with new vectors to attempt transient gene silencing of HLB and citrus canker-associated genetic targets identified from our microarray studies, have not yet been successful overall. We have continued the yeast-2-hybrid experiments, as an alternative approach to identifying the effects of target genes on plant phenotypes and disease response. Twenty of more than 100 predicted effectors have been tested and several appear to be having subtle effects on yeast growth; these genes are now being cloned into a more sensitive line of yeast to determine whether the subtle differences represent true effects. The so-called von Willenbrand factors predicted to be effectors by other researchers have not shown any effects in this system, but these will also be cloned into a more sensitive strain to confirm. We have used the GoldenGate assay platform developed using the genome sequences we have previously produced for hi-throughput genotyping of DNA from >150 individuals of a large mapping family potentially segregating for HLB resistance, and a second family segregating for fruit quality traits; mapping has been completed in both families. The genotyping by sequencing (GBS) project is proceeding; several new DNA samples have been prepared and will be sent to our collaborators for further testing followed by GBS. The RNA-seq project to uncover differences in gene expression over time between HLB-sensitive and tolerant citrus has made good progress. A first round of bioinformatics analysis has been completed and interpretation of results is in progress. Many significant pathways associated with disease response, metabolism, and cell death have been highlighted. Given the huge dataset available, and the informatics challenges, we are seeking additional input from other collaborators to maximize the output from this study. The end result will be a more complete understanding of the mechanisms of tolerance and sensitivity to CLas induced HLB disease.
The haploid Clementine and sweet orange sequences have been assembled, annotated, and are available to the research community at Phytozome and at citrusgenomedb.org. The new Clementine v. 1.0, has finally been validated and made publicly available in USDOE’s JGI Phytozome v 9.0 in December 2012. This assembly is a vast improvement over the first version (v 0.9) that was made available to the research community in January 2011. The genome is organized into 9 super-scaffolds, representing the basic nine chromosomes of citrus. This was made possible by the ICGC collaborative genetic mapping effort, in which our lab was a primary contributor. (See Ollitrault et al. BMC Genomics, 2012, 13:593 DOI:10.1186/1471-2164-13-593). New citrus sequences were provided in February 2013 of some varieties underrepresented previously including Ponkan mandarin and sweet orange. These have been further analyzed to elaborate the phylogeny of sweet orange, Clementine, Ponkan and Willowleaf, and sour orange; all are admixtures of C. reticulata and C. maxima, in varying degrees. A manuscript based on earlier results was submitted, but following reviewer recommendations, we have reanalyzed the new genome sequences and have refined the conclusions; a new manuscript is being prepared. Work proceeds on the other objectives of this project. New experiments with new vectors have been initiated to attempt transient gene silencing of HLB and citrus canker-associated genetic targets identified from our microarray studies. We have also initiated preliminary yeast-2-hybrid experiments, as an alternative approach to identifying the effects of target genes on plant phenotypes and disease response; one predicted HLB effector, among several predicted by analysis of the CLas genome, appears to interfere with the growth of the yeast strain being used, and these experiments will be repeated. We have used the GoldenGate assay platform developed using the genome sequences we have previously produced for hi-throughput genotyping of DNA from >150 individuals of a large mapping family potentially segregating for HLB resistance, and a second family segregating for fruit quality traits. We are pursuing collaboration with the Dvorak lab to anchor the sweet orange genome sequence to the linkage map, thus substantially improving the quality and utility of the previously produced assembly. The genotyping by sequencing (GBS) project is proceeding, once proof of concept was provided, and mapping is underway in a large segregating Citrus x Poncirus family, however some samples have failed and new DNA extractions are planned. The RNA-seq project to uncover differences in gene expression over time between HLB-sensitive and tolerant citrus has proceeded. RNA samples have been prepared from appropriate times in the disease process, from infected rough lemon and sweet orange, and healthy controls. Libraries were tested successfully, preliminary runs have enabled us to multiplex libraries and these libraries have now been sequenced, generating a substantial data set that will soon go through first round bioinformatics analysis.
McTeer trial – (3.5-year old SugarBelle trees on 15 rootstocks, nearly 100% HLB infected as of September (2011)- remediation program initiated in January by application of southern pine biochar and Harrell’s UF mix slow release fertilizer): In this ACPS/rootstock trial, HLB-impacted SugarBelle fruit did not drop from the tree (unlike sweet orange). Infected trees have set a nice crop again, and overall appear to be quite healthy. Assessment of the quality of the new fruit crop this year should be quite informative. UF-CRDF matrix rootstocks included in this trial are all performing well, showing better recovery than commercial control rootstocks. St. Helena trial (20 acre trial of more than 70 rootstocks, Vernia and Valquarius sweet orange scions, 12 acres of 5.5 year old trees, Harrell’s UF mix slow release fertilizer and daily irrigation). Harrell’s UF mix slow release mixed combined with the Schumann formula TigerSul (Zn, Fe and Mn) was applied in July, using a fertilizer spreader. Approximately 90 rootstock seed trees were planted in a new row going out towards the road; along with approximately 100 resets on new rootstock candidates in the East block. Greenhouse Experiments – Re-grafting of failed grafts of rootstock liners (test rootstocks and controls) was completed, most trees with successful first grafts have sprouted with very few showing any HLB symptoms. Nutrition of liners of rootstock Orange #15 (for the individual nutrient experiment) was initiated, and poly-coated individual nutrients were ordered from Florikam. Additional liners of Orange #15 were added to accommodate the addition of 3 individual TigerSul nutrients. Stick-grafting will begin in approximately one month. Protection of seed source trees: The release of new and improved rootstocks to the Florida Industry will require a large and stable source of viable nucellar seeds for our nurseries. Since seed source trees will be growing in the HLB environment, such trees should be protected from HLB. Transgenic Orange #16 tetrazyg plants transformed with GNA and P-GNA have been produced and have been micrografted onto Carrizo rootstock for rapid growth and development. Transgenic Orange #4 plants containing the GNA transgene produced in the first quarter are currently being clonally multiplied by cuttings using a mist bed.
Correlation of transgene expression with disease resistance response: Western blot analysis for plants containing LIMA and GNA was completed, and as expected, data shows a strong correlation between transgene expression and desired phenotype. This supports the dogma that fairly large populations of transgenic plants are necessary (for each transgene/cultivar) to obtain adequate transgene expression while maintaining cultivar integrity. Improved transformation methodology: 1. In efforts to reduce transgene mediated metabolic load on the plant, we have transformed Hamlin suspension cultures with constructs containing our reporter gene driven by either an embryo specific Carrot DC3 promoter or an embryo specific Arabidopsis At2S2 promoter. Transgenic plants expressed anthocyanin only in the embryogenic cells. Cotyledonary cells did not express anthocyanin indicating tissue specific activity of the promoter. Plants germinated from these embryos have been observed to lack anthocyanin production and have been micro grafted for growth. RT-PCR will be carried out on these plants to confirm inactivity of the visual selectable marker. 2. A binary vector containing Dual T-DNA borders for gene segregation and marker free transformation of citrus suspension cell transformation and selection have been constructed. Hamlin and W Murcott cells have been transformed with this construct. Preliminary data indicates that the transformation vector is functional and able to incorporate both T-DNAs into the plant genome. Somatic embryos containing this dual T-DNA cassette have been isolated and have been placed in embryo maturation medium for growth and development. Plants will be tested by once the plants are large enough. Subsequent experiments will confirm if negative selection pressure can differentiate between cells that contain the marker free T-DNA from the T-DNA containing the selectable positive/negative fusion marker cassette and if it can be removed from the citrus genome. 3. The binary vector for an inducible cre-lox based marker free selection has been constructed containing a heat inducible excision system containing the cre gene driven by a Soybean heat shock gene promoter. Tobacco tissue has been transformed with the construct and we are waiting for the plants to grow to a suitable size for heat shock treatment and excision. Renewal of transgenic field permit with APHIS: following completion and review of paperwork, our permit was renewed for 3 years; we now have permission to allow transgenic trees to flower and fruit; the Southern Gardens transgenic site was added to our permit. Transgenic testing: Nearly 200 new transgenic citrus trees were planted at the Picos USDA Farm site (resets and new tree positions). Paperwork is nearly complete to move HLB resistant transgenic trees from the SG hot psyllid house to field sites (petition approved by DPI). Two large sets of transgenic plants were propagated for testing in the SG hot psyllid house; they include new transgenes LIMA-B and SABP2 (the latter being a plant gene showing promise). We expect to move these plants to the SG psyllid challenge house within the next few weeks.
This is a continuing project to find economical approaches to citrus production in the presence of Huanglongbing (HLB). We are developing trees to be resistant or tolerant to the disease or to effectively repel the psyllid. First, we are attempting to identify genes that when expressed in citrus will control the greening bacterium or the psyllid. Secondly, we will express those genes in citrus. We are using two approaches. For the long term, these genes are being expressed in transgenic trees. However, because transgenic trees likely will not be available soon enough, we have developed the CTV vector as an interim approach to allow the industry to survive until resistant or tolerant trees are available. A major goal is to develop approaches that will allow young trees in the presence of HLB inoculum to grow to profitability. We also are using the CTV vector to express anti-HLB genes to treat trees in the field already infected with HLB. We have modified the CTV vector to produce higher levels of gene products to be screened. At this time we are continuing to screen possible peptide candidates in our psyllid containment room. We are now screening about 80 different genes or sequences for activity against HLB. We are starting to test the effect of two peptides or sequences in combination. We are attempting to develop methods to be able to screen genes faster. We are also working with other groups to screen possible compounds against psyllids on citrus. Several of these constructs use RNAi approaches to control psyllids. Preliminary results suggest that the RNAi approach against psyllids will work. We are screening a large number of transgenic plants for other labs. We are beginning to work with a team of researchers from the University of California Davis and Riverside campuses to express bacterial genes thought to possibly control Las. Since we are testing about 80 genes for induction of resistance or tolerance to HLB in citrus, we changing our focus to controlling psyllids until we have more conclusion from the peptides under screen.
This is a joint project between CREC and USDA, Fort Pierce. The objective of this project is to find poncirus hybrids that exist now that are sufficiently tolerant and of sufficient horticultural and juice quality to be used now for new planting in the presence of high levels of Huanglongbing (HLB) inoculum. We believe there is a good chance that there mature budwood exists with these properties that could be available immediately for new plantings. Although these trees are not likely to be equal in juice and horticultural qualities of the susceptible varieties of sweet oranges grown in Florida, with their tolerance to HLB they could be an acceptable crutch until better trees are developed. We surveyed the trees at the Whitney field station and found 5 lines that we thought could be acceptable for juice. Those have been propagated and are being screened for tolerance and horticultural properties. The hybrid plants are being incubated in the psyllid containment room to allow multiple psyllids to inoculate the plants with HLB. At this time, all 5 hybrids continue to appear to be tolerant to HLB.
Function of individual X. citri transcription activator like effectors (TALEs): We previously reported the development of an assay using stable transgenic Nicotiana benthiamiana plants containing a 4 EBE promoter:GUS construct to be tested for activation by a number of PthA homologs we have cloned from various X. citri strains. The PthA homolog-EBE specificity was were tested by creating transconjugants of X. campestris pv campestris 8004 transconjugants carrying the various PthAs and infiltrating into the stable transgenic Nicotiana benthiamiana plants. X. campestris pv campestris was used in lieu of X. citri for screening in Nicotiana because X. citri doesn’t infect Nicotiana. We observed strong gus activity in transconjugants containing pthA1(21.5 repeats), pthA2 (15.5 repeats) and pthA4 (17.5 repeats) from strain A44; pthA1- 17.5 repeats from Etrog; but not PthA4 homolog from Miami strain. Transformation and production of stable citrus lines: Because we have had difficulty recovering intact and functioning stable transgenic citrus lines, we have undertaken a number of efforts to overcome this bottleneck. Changes included testing a different transformation vector, different promoter and construct components, adjusting the transformation methodology, and adding Carrizo citrange, given it’s greater transformation efficiency, for comparison with ‘Duncan’ grapefruit and ‘Pineapple’ sweet orange. So far we have regenerated 312, 22 and 261 putative transgenic shoots from grapefruit, sweet orange and Carrizo, respectively and transferred to rooting media. Ongoing transformation experiments with previously used constructs have resulted in a number of putative transgenic shoots that have been rooted and transferred to soil in trays or 4′ pots. PCR screening of putative transgenic grapefruit and sweet orange plants regenerated from segments transformed with various constructs were analysed for 3 genes, 2 of which are present in the transgene of the constructs used (avrGF2 and nptII). A total of 54 plants were tested, but none of the plants tested contained the avrGF2 gene while 36 of the plants contained the nptII gene. We also tested for the presence of the virC gene which indicates bacterial contamination. One plant was positive for virC. Histochemical GUS screening was also carried out on putative transgenic grapefruit, sweet orange and Carrizo. Segments were scored based on the extent of the blue staining observed on the segments. GUS positive shoots were considered those segments that stained entirely blue while chimeric GUS shoots were those segments that stained less than 80%. No GUS positive shoots were observed for grapefruit or sweet orange cultivars for any of the constructs analyzed. Some grapefruit shoots were observed to be chimeric. Carrizo citrange showed the best results with several plants being GUS positive and a large number of plants chimeric for GUS. Another effort we have undertaken was to have an external contract transformation lab test our constructs along side their standard transformation control. In these experiments we provided a 14 EBE promoter construct in the original vector driving either AvrGf1 or GUS, for stable transformation of tobacco and Carrizo. The results so far indicate that our construct was considerable less successful in their hands for transformation compared to their control. These results suggest that our difficulties arise from the original vector used. Now that we have made new constructs we expect to have greater success in the production of stable transgenic lines
Construction of primary structure is completed. Electrical work and the entry foyer will be completed in the next quarter.
The project has two objectives: (1) Increase citrus disease resistance by activating the NAD+-mediated defense-signaling pathway. (2) Engineer non-host resistance in citrus to control citrus canker and HLB. For objective 1, we are planning a microarray experiment to identify genes that are induced by NAD+ in citrus. Microarray chips have been ordered. The first batch of RNA samples we prepared did not pass the quality control (QC). We are making the second batch of RNA samples. Microarray will be performed soon. For objective 2, two non-host resistance genes against citrus canker have been cloned into the T-DNA vector pBI1.4T, a vector with good transformation efficiency in citrus. The plasmids have been mobilized into Agrobacterium and will be used for citrus transformation.
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 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. Currently we have cloned 10 full-length genes in these categories in the entry vector pJET. Five of the genes were further cloned to the binary vector pBIN19plusARS and transferred to Agrobacteria. The Agro strains were sent to our collaborator 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. In addition, we are continuing to generate and/or characterize transgenic citrus plants expressing the SA positive regulators, as proposed in the previous project, although the support of this previous project has already been terminated. A paper describing the cloning and characterization of the citrus NDR1 ortholog was recently published in the journal Frontiers in Plant Science.
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