Chimeral constructs that should enhance AMP effectiveness (designed by Goutam Gupta of Los Alamos National Lab) are being tested. Carrizo transformed with a chimera AMP showed remarkable resistance in citrus canker compared to control. RT-qPCR showed 50% of 16 chimera transgenic Hamlin have relatively high gene expression, with 100x difference between high and low expressers. These promising transgenic lines were replicated by grafting for HLB challenge. Twenty transgenic Hamlin lines were confirmed to contain thionin gene by PCR, and six have high gene expression by RT-qPCR. Transgenic Hamlin lines expressing thionin were grafted onto Carrizo for HLB challenge. Replicated transgenic Transgenic Carrizo lines expressing thionin, chimera and control were grafted with HLB infected rough lemon. Promising resistance to HLB was observed based on plant growth and phenotype. Las titer is being checked from root and new flush rough lemon leaves. Two new chimeral peptide from citrus genes only were developed and used to produce many Carrizo plants and Hamlin shoots. To explore broad spectrum resistance, a flagellin receptor gene FLS2 from tobacco was cloned into pBinARSplus vector. Flagellins are frequently PAMPS (pathogenesis associated molecular patterns) in disease systems and CLas has a full flagellin gene despite having no flagella detected to date. The consensus FLS2 clone was obtained and used to transform Hamlin and Carrizo so that resistance transduction may be enhanced in citrus for HLB and other diseases. Many putative transformants were generated on the selective media: 38 Carrizo and 7 Hamlin are positive by PCR test. Reactive Oxygen Species (ROS) assay showed typical ROS reaction in three transgenic Hamlin indicating nbFLS is functional in citrus PAMP-triggered immunity. There is only slight canker resistance by infiltration, but considerably resistance to spray inoculation. To confirm that high ROS production was not due to variability in Hamlin, we examined l 40 Hamlin seedlings and no or very low level ROS production was detected. Two potential FLS2 orthologues were identified in Hamlin and their expression was shown much lower compare to nbFLS2. Primers were designed for two citrus FLS2 orthologues. They showed low gene expression in transgenic and nontransgenic citrus. Results on FLS2 transgenics against canker disease were summarized and a manuscript was written and submitted to MPMI. To disrupt HLB development by manipulating Las pathogenesis, a luxI homolog potentially producing a ligand to bind LuxR in Las was cloned into binary vector and transformed citrus. Both transformed Carrizo and Hamlin were obtained. Further investigation are underway. A series of transgenics scions produced in the last several years continue to move forward in the testing pipeline. A large number of ubiquitin::D4E1 and WDV::D4E1 plants and smaller numbers with other AMPs are replicated and in early stages of testing. In collaboration with Bill Belknap two new citrus-derived promoters have been tested using a GUS reporter gene and have been shown to have extraordinarily high levels of tissue-specific expression. The phloem-specific promoter is being used to create a construct for highly phloem specific expression of the chimeral peptide using citrus genes only.
During the life of this project, 121 mature citrus transgenics were produced using genetic transformation with Agrobacterium. Sixty-six were transgenic for reporter genes and provided proof of concept that this protocol works in our hands. In the last 21 months, 55 transgenics with disease tolerance genes, most without reporters, were produced and micrografted onto rootstock. Agrobacterium transformation efficiencies were relatively low (3.47% positive shoots for constructs with reporters in scion, and 3.0% positive shoots for constructs with no reporters in scion and rootstock). Only 0.78% shoots/total explants plated were transgenic. Many more transgenics were probably produced, but were lost in the micrografting process. Attempts are being made to increase micrografting efficiencies. Although 9 vectors carrying disease tolerance genes were used in sizable transformation experiments, only 4 vectors yielded transgenics. The 55 mature citrus transgenics were produced with these 4 vectors to confer disease tolerance to HLB, canker, or both. These primary transgenics are being propagated into vegetative progeny to facilitate replicated field trials this fall. Numbers were low for Ray Ruby grapefruit (3 transgenics) and efforts are being made to optimize the tissue culture and transformation protocol for grapefruit. Experiments are underway to root mature scion because a larger scion could be easily micrografted onto rootstock with greater efficiencies. To increase our chance of success, we have been utilizing nurse cultures to supply additional nutrients to developing mature shoots. Additional vectors are being acquired from scientists around the country and worldwide. Budding is now done entirely in-house. A gene gun was purchased in July, 2014 to develop a high-throughput biolistics transformation system for mature citrus. Transient expression levels are relatively high, and a few stable transgenics have been produced. Optimizations for mature citrus have been hindered by the limited supply of mature scion in the growth room, which was primarily used for Agrobacterium transformations. In the future, we will purchase mature citrus from nurseries to continue optimizations. We have developed a high throughput screening system in which thousands of putative transgenics can be rapidly screened. A number of equipment expenditures were necessary to achieve this high level of efficiency. Equipment expenditures included a refrigerated centrifuge, a plate reader, a tissuelyser, a laminar flow bench and an incubator. A hybridization oven and dry baths were purchased for molecular analyses. Maintenance expenses for lighting, AC repair, sensors and expansion cards for RCWebview, and the water softener in the growth room are ongoing. This project depends upon a continual supply of healthy, viable rootstock seed and this was problematic last year. Rootstock of Swingle and Volkameriana had poor germination, and Macrophylla and Carrizo had disease/endophyte issues that negatively impacted budding of mature citrus and the tissue culture process.
The driving force for this project was the need to evaluate citrus transformed to express proteins that might mitigate HLB, which required citrus be inoculated with CLas. Although citrus can be manually inoculated by grafting with infected budwood, a program using CLas-infected ACP was preferred primarily because this is what occurs in nature. Nine colonies of infected ACP for inoculations were initially established in a walk-in chamber. Additional colonies of infected ACP were later established in two small greenhouses and in a second walk-in chamber. For each individual colony, a potted lemon or citron tree graft-inoculated with CLas (testing PCR-positive and showing HLB symptoms) was placed into a cage and trimmed to stimulate flush; ACP were introduced and allowed to reproduce; and the colonies were maintained over time by regularly trimming plants and introducing additional ACP as needed. The source of CLas for these lemon and citron trees was the original HLB-infected tree found during 2005 in south Florida. Germplasm to be challenged for HLB resistance was supplied by USDA-ARS citrus breeders. To inoculate plants, individual plants were caged for 2 wk with 20 psyllids from one of the infected colonies, after which the plants were held together for six months in a greenhouse with an open infestation of infected psyllids. After this two-step inoculation procedure, the plants were returned to the breeders. Challenges associated with the program have included variability in percentages of ACP testing CLas-positive in a single colony over time and among different colonies at any given time. Consequently for the first inoculation step we always used ACP from colonies with the highest percentages of infected ACP, and we increased the number of colonies to increase numbers having high percentages of infected ACP. Lots of qPCR assays were required to monitor infected ACP colonies and infected source plants. Growth chamber colonies generally have had greater percentages of infected ACP than greenhouse colonies, probably due to more stable environmental conditions in chambers. Another challenge has been that, among ACP that test PCR-positive for CLas, relatively low transmission rates have been reported (e.g., 12% or less). Therefore, caging an individual plant with 20 psyllids from an infected colony may not always result in inoculation. Success of the first inoculation step would likely be increased by increasing the number of ACP per plant. Subsequently holding the plants for six months in the greenhouse with free-roaming infected psyllids increases the probability of inoculation. With respect to the second inoculation step, population levels of ACP in the greenhouse were sometimes severely reduced as a consequence of damage to oviposition sites by western flower thrips or spider mites. Also, sometimes population levels of ACP eggs and nymphs were severely reduced by western flower thrips, and further reductions in populations of nymphs were sometimes caused by Tamarixia parasitoids that invaded the open infestation of ACP. Effectiveness of the inoculation program has been slow to gauge. A series of experiments was initiated during 2014 specifically to evaluate inoculation success. Meanwhile, recent feedback from inoculations of rootstock material gave some insight. Eleven groups of rootstock material (3,105 plants total) were passed through the inoculation program during 2011-2014. The average percentage of infected ACP used in step one was 52% (range 24 to 80%). qPCR 12 to 19 months after the two-step inoculation process indicated an average of 62% success in inoculating citrus. Plants that escaped inoculation had another opportunity for inoculation after being transplanted to the field.
Core Citrus Transformation Facility (CCTF) continued to operate at the high level it did in the previous period and produced transgenic material in a timely and dependable manner. CCTF maintained its standing as reputable partner that delivers transgenic material in reasonable amount of time and continued to be sought for services. This quarter marks the end of three year period that saw significant but expected increase in number of orders. With six new orders received most recently, CCTF has just surpassed its 200th order and 96 of those were placed within the last three years. Clients requested transgenic Duncan grapefruit and Valencia orange in the newest orders. The number of plants produced in this quarter is 85 bringing a total to 730 plants for the duration of the funding period. Most of the produced plants were of Duncan grapefruit cultivar, followed by Carrizo rootstock, Valencia orange, some Citrus macrophylla, Swingle citrumelo, and Mexican lime. This strong bias towards Duncan grapefruit points towards need of researchers to get fast answers regarding candidate gene that could potentially render Citrus plants tolerant/resistant to diseases. Since Duncan grapefruit is highly susceptible to both Huanglongbing (HLB) and citrus canker, challenging transgenic plants expressing gene of interest with bacteria causing these two diseases will quickly reveal if that gene holds any promise or not. Throughout the duration of this project, CCTF processed about 400000 explants. Approximately 5% of explants were lost to contamination which brings the total number of explants producing data to 380000. Overall transformation efficiency expressed as the percentage of positive shoots out of all of those tested was about 3% which is low. One of the reasons for this is the nature of orders received. Amongst 96 received orders, there were 34 for which total of just a few transgenic plants were produced. This was a result of withdrawal of 10 orders, presence of sequence detrimental for shoot development in binary vector used for transformation in six orders, and for 18 orders transformation was either not possible or achieved at the very low rate. The second reason for low transformation rate is the quality of seeds/seedlings resulting from the effect HLB infection has on fruit growing on trees. In the immediate future, CCTF will dedicate increasing efforts to acquire fruit/seeds of higher quality than what it was used recently. The labor force and the income of CCTF remained relatively stable during the last three years and changes in facility s staff never compromised the level of operation. Number of orders presently serviced by the CCTF and those that were either announced or anticipated in the near future is clear indication of sustaining interest of researchers in transgenic citrus plants. They also assure CCTF will stay busy for the years to come.
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. 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 have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
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. 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 have developed methods to be able to screen genes faster. Finally, we have found a few peptides that protect plants under the high disease pressure in our containment room with large numbers of infected psyllids. We now are examine combinations of peptides for more activity. We recently examined all of the peptides constructs for stability. The earliest constructs have been in plants for about nine years. Almost all of the constructs still retain the peptide sequences. One of the peptides in the field test remained stable for four years. All of these constructs had the peptide gene inserted between the coat protein genes, which is positioned sixth from the 3′ terminus. However, we have found that much more foreign protein can be made from genes positioned nearer the 3′ terminus. Based on that we built constructs with the peptide gene next to the 3′ terminus. These constructs produced much greater amounts of peptide and provided more tolerance to Las. Unfortunately, they are less stable. So now we are rebuilding constructs with the peptide gene inserted at an intermediate site hoping for a better compromise of amounts of production and stability. We have produced a large amount of inoculum for a large field test via Southern Gardens Citrus. We are screening a large number of transgenic plants in collaboration with Dr. Zhonglin Mou, Department of Microbiology and Cell Science in Gainesville, to test transgenic plants over-expressing plant defense genes. We are propagating a progeny set of plants of the promising candidates for a final greenhouse test.
Our project aims to provide durable long term resistance to Diaprepes using a plant based insecticidal transgene approach. In this quarter, as proof of concept to determine the root specific nature of the promoters (RB7, C1867 or SLREO), we have incorporated the promoter-gus sequences into N. benthamiana and several plantlets have been regenerated. These constructs were also incorporated into Carrizo citrange in the previous quarter, but we have had a severe Mould mite infestation in our tissue culture room which destroyed most the promoter-gus citrus plantlets. A repeat experiment to produce fresh carrizo citrange plants expressing the root specific promoters were carried out in this quarter and plantlets are being regenerated. The Carrizo citrange plants expressing a root specific promoter – insecticidal gene construct (GNA, APA or ASAL genes individually or stacked with the CpTI gene) were not affected by the mite infestation and several plants have been hardened and transferred to the greenhouse for growth.
The goal of this project was to create canker-resistant citrus through a strategy of an engineered cell death response to Xanthomonas citri pv. citri TAL effectors. During the project, we defined sequences of 14 distinct effector binding elements (EBE) recognized by Xcc TAL effectors. Constructs were created that incorporated these EBEs into the Bs3 promoter driving a pathogen effector gene (either avrGf1 or avrGf2) that triggers a hypersensitive cell death reaction in several citrus cultivars. Two key constructs were one with all 14 binding sites (14EBE) and one with four binding sites (4EBE) that was expected to be bound by at least two TAL effectors from each X. citri strain characterized. These constructs were shown to function as expected in a transient assay in citrus leaves: they produced a hypersensitive response and reduced bacterial growth when co-inoculated with a Xanthomonas strain carrying a TAL effector recognizing one of the EBEs. In the absence of pathogen inoculation, the promoters are tightly “off” in the transient assay. However, despite the tight regulation of these constructs in a transient assay and multiple attempts to produce stable transformants in Duncan grapefruit or sweet orange, we have been unable to recover transgenic lines. We also tested Carrizo citrange, a citrus variety that does not exhibit a hypersensitive response to AvrGf1 or AvrGf2, and we were able to recover transformants. These results suggest cryptic induction of the construct at some point during the transformation process, causing transformants to be selected against. A second promoter derived from the citrus Lateral Organ Boundaries (LOB) gene also failed to produce transformants when driving AvrGf2 expression. Therefore, despite the theoretical promise of the approach, more work would be needed to define a promoter and executor gene combination that could be efficacious in citrus.
The transgenic plants to be developed for this project are now growing in two different locations in secure greenhouses and growth chambers. Seven independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped from the Citrus Transformation Facility at the University of Florida Citrus Research and Education Center at Lake Alfred, FL, to Dr. McNellis’ lab at the Pennsylvania State University at University Park, PA, in early October, 2014. An additional eight independently-transformed citrus plants carrying the FLT-antiNodT fusion protein expression construct were shipped to Dr. Tim Gottwald’s lab at the United States Horticultural Laboratory in Fort Pierce, Florida. The plants at both locations are growing well. At Penn State, all the transgenic lines are being propagated as vegetative cuttings. We have achieved over 50% success rate in rooting cuttings from the plants, and rooted cuttings are now growing for all of the independent lines. The rooting process for cuttings takes 3-4 months in our facility at Penn State. We have performed two protein immunoblots with an antibody that will detect the FLT-antiNodT fusion protein (anti-cmyc antibody). We detected protein of the correct size (~50 kD) as a single band without degradation products in two different lines, and not in control plant samples. These results are very promising and are consistent with successful expression of full-length FLT-antiNodT fusion protein in ‘Duncan’ grapefruit. During the next quarter, we will be repeating these experiments with all of the transgenic lines and using improved protein extraction techniques to develop better western blot images and documentation.
Three Valencia-type clones (N7-11, B7R9T35 and B7R9T36) with excellent fruit quality were discovered in our collection (older trees) that have no HLB symptoms and are PCR-negative for Liberibacter. Trees of these clones are being propagated for further study. We continued to monitor performance, assess HLB severity, tree growth and yields at 15 different field sites throughout Florida. Yield and fruit quality data were collected from the St. Helena trial (last week of January); overall yields increased 42% over last year (where yields were down 18%), as HLB incidence in the trial increased from 56% to 92%. This remarkable increase in yield was primarily attributed to the addition of TigerSul micronutrients to the CRF mix and elevating the boron and manganese in the CRF. Also, we are importing seeds from Sicily of some of the winners at St. Helena and some new candidates of identical parentage for future trials. Gauntlet rootstock screening: Approximaly 75 HLB+ Valencia budstick-grafted hybrid rootstocks already passed through the ‘hot psyllid’ house were prepared (metal tags and tree wraps) for field planting at Picos Farm in April. Three parental combinations from 2013 crosses produced numerous hybrids that are growing off beautiful trees from grafted HLB+ Valencia budsticks: C2-5-12 pummelo x C. latipes (papeda); Amblycarpa+HBPummelo x Sour orange+rangpur; and Amblycarpa+HBPummelo x White #1. Stick-grafting and production of rooted cuttings from 2013 gauntlet crosses was nearly completed. The two early-maturing (December) Valencia somaclones identified, B7-70 and SF14W-65, have been cleaned up and a few pathogen-free trees were provided by the Parent Tree Program (DPI). A plan was developed for rapid pathogen-free budwood scaleup; liners of vigorous rootstock US-802 were obtained, and a robust micro-grafting and traditional grafting program is underway. We plan to have a population of budwood increase trees available for nurseries at the time of the official release announcement. We completed collection of botanical data on these clones as necessary for release and patenting, and both clones will be put up for official release this summer (with an expected late fall release). 2015 Crosses: Featured scion crosses included: crosses of the most HLB-tolerant mandarins and pummelos identified in our collection; interploid crosses of the most HLB-tolerant mandarin/sweet orange diploid and tetraploid hybrids (this includes use of the HLB-tolerant monoembryonic diploid and tetraploid cybrid 304 (large fruit with sweet orange-like juice amenable to processing). Featured rootstock crosses included a very tolerant and complex pummelo-sour-mandarin hybrid crosses with itself and some onto FD and FD curved phenotype hybrids; crosses of salinity and HLB-tolerant diploid sour orange-type hybrids; and crosses of the most HLB-tolerant tetraploid rootstock hybrids. MAC Projects: Seed of 12 promising potentially HLB-tolerant rootstock selections were provided to Ruck’s Nursery for large-scale tree propagation and testing as part of the MAC project in cooperation with Kim Bowman (USDA). Seed of 10 different potentially HLB-tolerant rootstocks were provided to Briteleaf Nursery for large-scale tree propagation and testing for the CREC MAC project (M. Rogers) to test our best new sweet oranges under an optimized production system on the new Lake Alfred city property just north of the CREC, obtained in the Hughes inheritance property swap.
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. 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 have continued to be observed, including the portion of the St. Helena project planted with dwarfing selections. Yield and fruit quality data were collected from the St. Helena trial (last week of January); overall yields increased 42% over last year (where yields were down 18%), as HLB incidence in the trial increased from 56% to 92%. This remarkable increase in yield was primarily attributed to the addition of TigerSul micronutrients to the CRF mix and elevating the boron and manganese in the CRF. Many of the tree-size controlling rootstocks in the trial produced fruit with higher lbs. solids and juice color than the more vigorous rootstocks; thus this information will be utilized in the economic analysis for the 2015 Field Day handout, and will help to identify the best HLB-tolerant rootstocks for ACPS along with recommended planting densities. Collection of botanical data on two high-performing tree-size controlling somatic hybrid rootstocks (sour orange+trifoliate orange 50-7; and White grapefruit+trifoliate orange 50-7) in the trial was completed as necessary for patenting and Fast Track release later in 2015. After 7 years, all 86 trees on the White grapefruit+50-7 rootstock in the St. Helena trial remain healthy and productive. New Trials: Planting of a major rootstock trial with Lykes is underway (>20,000 trees to be planted at high densities at two sites, Basinger and Camp Mack). This trial includes 19 tree-size controlling rootstocks from the CREC, as well as additional tree-size controlling rootstocks from the Forner Spanish breeding program and from the California breeding program (C-Series). Progress was also made with propagation of trees for the major rootstock trial with Cutrale, which will also include several new tree-size controlling rootstocks. ACPS Data collection: Yield and fruit quality data were collected in February from the ‘Hamlin’ section of a large ACPS rootstock trial in Indiantown being conducted in collaboration with Gardinier Florida Citrus (w/ Lee Jones). Collection of yield and fruit quality data from the ‘Valencia’ part of the trial began on March 30th, and was completed April 4th. Data is currently being analyzed. Several new potentially HLB-tolerant rootstocks that have capacity to dwarf trees for use in ACPS (diploid and tetraploid Flying Dragon hybrids) were identified that are amenable to seed propagation. Seed was planted for use in subsequent trials.
Little resistance to HLB, associated with infection by Candidatus Liberibacter asiaticus (CLas), is found within commercial citrus varieties, though Poncirus trifoliata has been described as resistant or tolerant. An intergeneric F1 population was developed from the crossing of Citrus sinensis and P. trifoliata. The objective of this study was to construct a high-density linkage map for further mapping HLB resistance QTLs. A composite of 182 intergeneric progenies derived from four crosses between C. sinensis cultivars. Sanford, Ridge Pineapple, Fiwicke, or Ruby as the maternal parents, and P. trifoliata cultivars Argentina or Flying Dragon as the paternal parents, were used for genotyping by the Illumina GoldenGate Assay. Totally 1536 SNP markers were developed from the sweet orange BAC library. Genotyping data were analyzed by the GenomeStudio (Illumina), and a linkage map was created by JoinMap 4.1 using the two-way pseudo-testcross mapping strategy and regression mapping algorithm. Individuals from this same population were replicated and planted in the field with no ACP control, to assess symptom development, as well as to use qPCR to track CLas levels, over the course of the study. A preselected subset was used as a monitoring population to quantify HLB symptom severity, relative vigor (stem diameter), and citrus canker severity. Combining all results of qPCR monitoring across the two years of the project, 5 F1 hybrids have been for the most part CLas-free, except for occasional low-level and inconsistent detections of CLas in a small number of the replicates. One F1 hybrid has always tested negative for CLas, among all the individual replicates across all sample dates. The variation of the population mean Ct value across all F1 progeny showed good normal distribution, on which the Ct values of susceptible and resistant parents were located at each end respectively. Analysis of genotyping data showed that 99% of the markers were heterozygous in maternal parents (C. sinensis), but only 6% in paternal parents (P. trifoliata), and 5% in both. Finally 698 high-quality SNP markers were mapped to nine linkage groups for sweet orange, but only 42 could be mapped to seven linkage groups for the paternal parents. A comparison of the genetic maps to the published sweet orange (C. sinensis) genome revealed both conservation and variations. The completed linkage map is still being utilized to associate with the phenotyping data for QTL mapping and cloning of HLB resistance genes in the future. At the current time we can only map QTLs for sensitivity from sweet orange; but with the availability of the Poncirus genome and/or access to GBS data from our collaborators, we will soon be able to map tolerance/resistance QTLs from the male parents. We have also conducted artificial inoculation experiments in the greenhouse with a subset of individuals from the mapping population, to compare natural inoculation in the field with the more severe test of graft inoculation. Although funding for the project has ceased, we intend to continue the work to its logical conclusions, as possible.
We routinely 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, including those more recently identified. Some of the previously identified selections have succumb to HLB and we have removed these materials from the program. We continue to grow out and propagate recovered rootsprouts and scion materials collected, to produce groups of trees that can be tested for their responses to HLB. A recent decision by FDACS/DPI, that researchers may propagate and plant in the field trees grown from non-certified sources, will help us move some of the selections we have propagated into the field for longer-term evaluations. Materials collected from a grove in Lake County that appeared to be completely free of HLB symptoms were propagated and increased. PCR results have been negative; these plant materials were placed in a growth room with CLas+ ACP populations to attempt natural inoculation to assess their responses; however, as Dr. Brlansky retired and the growth room was transferred to another researcher, we were forced to terminate the experiment prematurely. Materials previously collected from a legacy Parson Brown tree found in Marion County, and apparently free of HLB, have been propagated, and are being grown off for field planting. We visited several newly identified candidate trees, but in most cases materials were not collected because the trees actually were more symptomatic than we had been led to believe. However, we have learned of two new locations with reportedly very healthy trees surrounded by rather severe HLB diseased trees, one in Osceola County and the other in St. Lucie County; we will visit these in January or February 2015. We plan to revisit trees from which samples were collected previously to determine their current status and performance; scion and rootstock materials will be collected, tested, and propagated, if they remain in very healthy condition.
In preliminary studies on ACP biology, it was observed that there was a notable suppression of successful development from eggs to adults, on Cleopatra mandarin (Citrus reshni; CM) trees both growing in the field and also in greenhouse experiments. Based on these results, we looked at ACP viability in a field-grown population of CM-derived hybrids from the UF-CREC breeding program, in Lake Alfred. The trees were 18 years old and growing on their own roots. 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. Routine insecticide applications were withheld for a period of 2 months prior to the experiment. Large branches on the south sides of the trees were severely pruned, to stimulate a synchronized and vigorous flush of new shoot growth to introduce ACP. Pairs of mated ACP were caged on each shoot and after three weeks, the number of adults that developed on each was counted. Of 30 individual hybrids that were used, only 9 of these supported the development of new adults; the remaining 21 had absolutely no surviving adults. This experiment was repeated with the same results. We propagated replicates from each individual for observations of psyllid reproduction in controlled greenhouse and laboratory conditions. Additional trees were propagated from additional numbers of the hybrids from the families to provide increased numbers of replicates for the ACP reproduction and feeding behavior studies. We conducted several rounds of experiments in the greenhouse. Previously, ACP were able to reproduce on only 3 of the 26 plants screened (ACP did not reproduce on 88% of the plants screened). Since then we have screened seventy-three additional individual plants for the ability of ACP to reproduce on them. Of these, ACP were unable to complete their life cycle on 52 of these plants (71% did not support ACP reproduction). Our results indicated that several of the replicated hybrids were unable to support adult psyllid development. These results were compared to results from previous experiments conducted in the field, and essentially demonstrated the consistency of the response of ACP to specific individual hybrids. In addition, new candidate plants have been identified from other studies of HLB impact on diverse citrus germplasm, which have shown either no impact or dramatically delayed infection, and new hybrid families have been produced from some of these for possible future studies. These include certain mandarins as well as other complex citrus hybrids. It appears that suppression of ACP by Cleopatra mandarin and hybrids derived from it is a heritable characteristic, thereby opening new possibilities to understand the genetic control and underlying mechanisms of the characteristic. It remains to be determined whether these new candidates likewise possess genetics that may be transmitted to offspring and whether the mechanisms underlying the phenomenon are the same or different. Such information might lead to new strategies to minimize the HLB-vectoring capacity of ACP.
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 of new scion and rootstock performance with a focus on HLB responses. Assessments of HLB field tolerance are continuously carried out in the vast collection of raw germplasm that we maintain, and new selections have been identified, and several previously found continue to hold up to HLB; additional evidence is accumulating supporting what may be differential sensitivity to HLB among sweet orange clones from the CREC program. We monitored performance, assessed HLB severity, tree growth and yields at 15 different field sites throughout Florida. A new set of 25 HLB+ Valencia budstick-grafted hybrid rootstocks was rotated into the ‘hot psyllid’ house, following selection based on freedom from symptoms in the greenhouse test. Another set of approximately 50 hybrid rootstocks were removed from the ‘hot psyllid’ house and prepared for field planting (spring of 2015); another set of 50 was rotated into the ‘hot psyllid’ house. Several new clones were entered into the DPI Parent Tree Program, including a cybrid sweet orange with exceptional juice quality and preliminary evidence of HLB tolerance, two early maturing Valencia somaclones, and twelve new promising rootstock hybrids with preliminary evidence of HLB tolerance. The two Valencia somaclones identified, B7-70 and SF14W-65 had 16 ratio and 11.5 brix in mid-November (6 year old trees on rough lemon at St. Cloud), when Hamlins were still at 12 ratio. B7-70 appears to be slightly earlier than SF14W-65. Both clones have been significantly earlier than Valquarius the past two years. These Valencia clones have potential to replace Hamlin which would significantly increase the quality of our NFC and concentrate juices. Observations were made at a rootstock trial in Vero Beach from where several of the UF rootstocks already approved for release have been selected; a field day was held in October to highlight these and other promising rootstocks at this location. Another large field day was held at the St. Helena planting in December. Seedlings grown from seed collected from more than 3 dozen new candidate rootstocks were evaluated for trueness to type, and new seeds have just been collected from additional new candidates not evaluated previously. We recovered seed from 2014 diploid and tetraploid rootstock crosses (20 successful crosses), and these were planted in calcareous soil inoculated with two species of Phytophthora. Seed was extracted from 8 UFR FAST TRACK rootstocks and distributed to participating nurseries. Seed was also extracted from 16 additional new rootstocks showing potential for HLB tolerance and provided to nurseries for propagations of planned large-scale field trials.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
