The person hired to work on the project has not arrived. The genes to be constructed were designed and the DNA sequences were verified. Also, the sources of citrus plant materials have been identified.
The first task in this proposal is to find citrus version of the two proteins that make up the functional design of a chimeric antimicrobial protein previously described by us (Dandekar et al., 2012 PNAS 109(10): 3721-3725). We have completed the discovery of the replacement of the first component the human neutrophil elastase (HNE) the surface binding component. Since HNE is a serine protease with elastasin as a substrate and whose crystal structure has been determined, we used 3D shape criteria and electrostatic properties to search the PDB data base for a plant protein with the identical active site structure. Focusing on a set of 288 non-redundant plant derived proteins from the entire PDB database, we used CLASP to search for a match using the electrostatic and structural features of amino acids that make up the active site. This was successful and the tomato PR14a was identified as an exact match. Using the tomato amino acid sequences we then searched for a similar citrus protein by searching citrus genome information in Phytosome (http://www.phytozome.net). This was successful and we have identified a single protein that is identical in the Citrus sinensis (Cs) and clementina (Cc) genomes. We are focusing on the 165 amino acid protein from Cs which we call CsP14a. We have analyzed this sequence and have determined that it is a secreted protein and contains a 25 amino acid signal sequence. We are focusing on the 137 aa mature protein and begun the construction of two genes that encode this protein. The first is a CaMV35S expression cassette that will be used to express just the mature form of the CsP14a protein, however, 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 is part of the secreted protein after cleavage of the signal peptide. The second version is like our CAP protein and has Cecropin B (CecB) at the C-terminal linked via a flexible linker as we have described earlier. This protein is designated CsP14a-CecB. These genes will be used for in planta expression of the proteins CsP14a and CsP14a-CecB that will be isolated and used to test their efficacy against the bacteria as we described in our last report.
USDA-ARS-USHRL, Fort Pierce Florida is producing thousands of scion or rootstock plants transformed to express peptides that might mitigate HLB. The more rapidly this germplasm can be evaluated, the sooner we will be able to identify transgenic strategies for controlling HLB. The purpose of this project is to support a high-throughput facility to evaluate transgenic citrus for HLB-resistance. This screening program supports two USHRL projects funded by CRDF for transforming citrus. Non-transgenic citrus can also be subjected to the screening program. CRDF funds are being used for the inoculation steps of the program. Briefly, individual plants are caged with infected psyllids for two weeks, and then housed for six months in a greenhouse with an open infestation of infected psyllids. Plants are then moved into a psyllid-free greenhouse and evaluated for growth, HLB-symptoms and Las titer. To date on this project, it funds a technician dedicated to the project, a career technician has been assigned part-time to oversee all aspects of the project, two small air-conditioned greenhouses for rearing psyllids are in use, and 18 individual CLas-infected ACP colonies located in these houses are being used for caged infestations. Additionally, we established new colonies in a walk-in chamber at USHRL to supplement production of hot ACP. A total of 3,805 transgenic plants have passed through inoculation process. A total of 76,160 psyllids have been used in no-choice inoculations. USDA-ARS is providing approximately $18,000 worth of PCR-testing annually to track CLas levels in psyllids and rearing plants. Additionally, steps to manage pest problems (spider mites, thrips and other unwanted insects) are costing an additional $1,400 annually for applications of M-Pede and Tetrasan and releases of beneficial insects.
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 have performed the designed microarray experiment to identify genes that are induced by NAD+ in citrus. The microarray data is currently under analysis. For objective 2, in last quarter we cloned two non-host resistance genes against citrus canker into the T-DNA vector pBI1.4T and mobilized the plasmids into Agrobacterium. In this quarter we have started genetic transformation of citrus ‘Duncan’ grapefruit with the Agrobacteria. Several putative transformants have been identified.
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. Plans have been made for new caged psyllid experiments in the field, to confirm previous observations, and to provide baseline information on responses of psyllids to specific individuals from within the families.
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. The project’s funding came available in November 2012, but as this is based on the foundation of the long-term breeding program, activities have proceeded on a continuing basis. Individual assessments of HLB field tolerance have been carried out in the vast collection of raw germplasm that exists on UF and collaborating growers’ property, from fall 2012 through June 2013. 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 rootstock trials with early, midseason, and late season scions, we have collected data to document yields, fruit and juice quality, and fruit drop, and to correlate the disease rankings with yield performance; there are striking examples of very healthy, albeit infected trees, showing high yields of normal fruit, depending upon the rootstock. 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. Newly produced rootstock hybrids have been entered into the same screen recently; removed seedling tops were rooted to provide seed source trees, after the infected scion budwood was grafted for testing. 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). CRDF has approached us to produce a list of our top candidates for such plantings with several other growers through a process that is being developed in conjunction with the CRDF and their Commercial Product Delivery Committee. This list has been created and includes several tetraploid rootstocks of complex parentage, diploid new citranges, and sour orange types, all of which have shown excellent production with few symptoms even though affected by HLB. 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 do we can recover these potentially valuable rootstocks for increase and production.
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. The project’s funding came available in November 2012, but as this is based on the foundation of the long-term breeding program, activities have proceeded on a continuing basis. Individual assessments of HLB field tolerance have been carried out in the vast collection of raw germplasm that exists on UF and collaborating growers’ property, from fall 2012 through early spring 2013, and this will continue through the coming months. 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 rootstock trials with earlier maturing scions (Hamlin orange, Sugar Belle mandarin hybrid, and grapefruit), we have collected data to document yields, fruit and juice quality, and fruit drop, and to correlate the disease rankings with yield performance; there are striking examples of very healthy, albeit infected trees, showing high yields of normal fruit, depending upon the rootstock. We have now completed yield and juice quality analysis in some rootstock trials that include Vernia and Valquarius sweet oranges, and plans are in place for performing the same data collection tasks in trials with Valencia scions. 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; nearly one year after planting, these trees were assessed and nearly 80% of them were found to be free of HLB symptoms. Newly produced rootstock hybrids have been prepared for the same screen in the coming season; removed seedling tops were rooted to provide seed source trees, after the infected scion budwood was grafted for testing. Seven rootstocks were entered into the DPI Parent Tree Program for cleanup via shoot-tip grafting, followed by indexing, to provide certified budwood to nurseries upon release. A new filed trial using sour orange type hybrids was planted near Lake Wales, and another rootstock trial with grapefruit scion was planted at the IRREC. More than 100 rootstock seed source trees were planted on UF properties. Four new sweet oranges were approved for release by the UF-IFAS Cultivar Release Committee, including one that appears to be less sensitive to HLB than other oranges.
HLB’s impacts have led to grower-directed interest in advanced production and harvesting systems with the potential for early and sustainable yield, 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. This project will identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Though funding became available 1 November 2012, the project was built on many years of previous efforts in rootstock development and field trial testing. Existing field trials were monitored for tree growth and disease incidence, including a portion of the St. Helena project planted with dwarfing selections, and a 40-acre Hamlin/Valencia cooperative [GFC] 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. Seed trees for selected dwarfing rootstocks, already showing good performance, are being propagated, to support expanded trials in the future. Four rootstocks 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 have been grown off in our greenhouses to be planted in late summer 2013; more than 2000 trees will have been planted when this is finished. Seed trees from 5 selected tree size controlling rootstocks have been propagated and planted to provide a seed source for use upon their release, which will likely occur in the next three years.
HLB’s impacts have led to grower-directed interest in advanced production and harvesting systems with the potential for early and sustainable yield, 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. This project will identify appropriate rootstocks among exiting field trials and those soon to be planted that are well suited to advanced citrus production and harvesting systems. Though funding became available 1 November 2012, the project was built on many years of previous efforts in rootstock development and field trial testing. Existing field trials were monitored for tree growth and disease incidence, including a portion of the St. Helena project planted with dwarfing selections, and a 40-acre Hamlin/Valencia cooperative [GFC] 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. The best yield was only ca. 0.5 boxes per tree across a whole bed planted to one rootstock, in this case C-35 citrange. Observations of severity and frequency of HLB symptoms were recorded on the dwarfing rootstocks at the St. Helena planting, prior to recording a third year of yield data in the coming months. Seed trees for selected dwarfing rootstocks, already showing good performance, are being propagated, to support expanded trials in the future. Four rootstocks 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. Two dwarfing rootstock hybrids have been budded with vigorous lemon scions to test their potential for tree size control using a very vigorous citrus scion type. 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) has very recently been planted.
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 highly valuable pool of potential candidate genes from which to identify these true HLB tolerance genes can be identified. 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. Toward this goal, we will sequence the genome of rough lemon, and together with the genome sequence of Poncirus currently being produced, we will design a target capture system based on the Agilent SureSelect system. The custom-designed gene capture system will be used with Illumina HiSeq 2000 to rapidly identify sequence variations in the candidate genes in Poncirus and Citrus genotypes and to identify genes most likely associated with HLB tolerance. We had recruited a post-doctoral research associate, but this person decided to decline our offer, and currently we are trying to identify another qualified candidate. In the meantime, sequencing of Poncirus has been initiated and plans have been made to sequence the rough lemon genome, as well. Some of the plant materials to support the project are being collected and grown, to be prepared for downstream applications.
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.
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