Project rationale and focus:The driving force for this three-year project is the need to evaluate citrus germplasm for tolerance to HLB, including germplasm transformed to express proteins that might mitigate HLB, which requires citrus be inoculated with CLas. Citrus can be bud-inoculated, but since the disease is naturally spread by the Asian citrus psyllid, the use of psyllids for inoculations more closely resembles “natural infection”, while bud-inoculations might overwhelm some defense responses. CRDF funds supported high-throughput inoculations to evaluate HLB resistance in citrus germplasm developed by Drs. Ed Stover and Kim Bowman. The funds cover the costs associated with establishing and maintaining colonies of infected psyllids; equipment such as insect cages; PCR supplies for assays on psyllid and plant samples from infected colonies; and two GS-7 USDA technicians. A career base-funded USDA technician is also assigned ~50% to the program. USDA provides greenhouses, walk-in chambers and laboratory space to accommodate rearing and inoculations. Most recent quarter:The 35 day federal government shutdown, and the threat of a possible shutdown on Feb 15, directly disrupted our ability to initiate and conduct experiments using the CLas+ ACP colonies. In addition, considerable rehibilitation of colonies and supporting plants was necessary due to the minimal care that could be provided during the shutdown. Only 2400 CLas+ ACP were used for experiments in this quarter and were used for detached leaf assessments of plants expressing three different transgenic constructs. We anticipate a normal demand in the current quarter. Previously achieved in this project: As of December 21, 2018, a total of 14,111 plants had passed through the inoculation process. A total of 361,255 psyllids from colonies of CLas-infected ACP had been used in inoculations. Not included in these counts of inoculated plants and psyllids used in inoculations were many used to refine inoculation procedures, which provided insight into the success of our inoculation methods and strategies for increasing success. After inoculations, plants were returned to the breeders and subsequently subjected to further inoculations when they are transplanted to the field. In addition to inoculating germplasm, infected psyllids were supplied to other researchers for other purposes. This side of the project grew over time, and detailed records were not maintained on how many were given out until 2018. More than 10,000 infected psyllids were supplied to the research community for an array of experiments during 2018. Recipients included researchers with USDA in Fort Pierce, Ithaca and Beltsville, UF in Gainesville, Cornell in Ithaca, University of California, and University of Nevada.
The objectives of this study are to identify optimal pH range for root function and minimize root turnover on HLB-affected rootstocks and how uneven pH levels in the root zone (e.g. irrigated vs. row middle portions of root system) affect the overall health of the tree. This is being done in a split root system in the greenhouse where pH of different parts of the root system can be controlled an maintained. We are in the final stages of rhizotron construction to build enough for the experiments. Rhizotron construction was slightly delayed because of the late Valencia harvest this year for other projects combined with an unexpected loss of a staff member that will soon be replaced. The Masters student has assisted a member of Tripti Vashisth’s lab with the 2nd repetition of the experiment that created the foundation of this project to become familiar with techniques that will be important for maintaining pH and collecting data. We expect to initiate treatments before the end of May.
All transgenic rootstocks (Swingle and Carrizo) expressing the AtNPR1 gene, developed using mature tissue transformation and juvenile transformation have been evaluated using qPCR. Several of the promising lines have also been evaluated for the trans-protein production using western blotting techniques. Cuttings of several lines have been propagated and budded with non-transgenic ‘Valencia’ sweet orange. Many others are currently being propagated in the mist bed. To produce seed source trees, trees are being produced by budding onto US802 rootstock. These will be planted in a USDA-APHIS approved field site when ready. Transgenic rootstocks expressing other potent plant derived transgene(s) are also being produced using the mature tissue transformation method.
Citrus Transformation Facility (CTF) continued its operation without interruptions. In the first three months of 2019, CTF received nine orders. Five of these orders were for production of transgenic Duncan grapefruit plants and four for production of transgenic Valencia plants. Within this period, CTF produced 77 transgenic citrus plants. Out of this number, 42 were Duncan grapefruit plants, 19 Mex lime plants, 11 Pomelo plants, four Kumquat plants, and one Pineapple sweet orange plant. These plants are result of work on 15 different orders.In February, CTF purchased one bin of Duncan grapefruits and stored them in the cold room for the supply of seeds. For experiments requiring Valencia seedlings, we are picking Valencia fruit from the trees on the CREC property to get seeds. The seeds of other cultivars are obtained through purchase from Lyn Citrus nursery in California or by picking fruit from DPI Arboretum in Winter Haven.One of the employees with the lowest FTE has additionally decreased her attendance and will leave the lab at the end of June.
We continue to produce Agrobacterium-mediated mature transgenics for customers, test different approaches for increasing efficiency, & develop biolistics as a tool in citrus. During this quarter, 11 mature transgenics were produced with Agrobacterium & have been micrografted, & a remaining 42 positive shoots were produced using Agrobacterium & must still be micrografted once/if they get larger. These two genetic constructs had GFP & were transformed into Hamlin (one of our better cultivars for transformation). An inordinate number of transgenics were produced, which is very unusal in mature citrus & must have been the result of cultivar x genetic construct interaction. The GFP reporter makes identification of transgenics easy. A new initiative from the CREC Director is to produce mature citrus transgenics with stacked disease resistance genes using biolistics for faster deregulation. We have been able to produce transgenics for Dr. Mou using a promising, new disease resistance gene & the genetic construct did not rearrange in mature citrus. (Unfortunately it rearranged for Dr. Orbovic in immature citrus). We used a RecA- Agro strain because we suspected there might be a problem. In a couple of transgenics, the gene of interest (GOI) was lost, but the majority of transgenics have the transgene. This is very similar to the deletion of the GOI from Dr. McNellis’ genetic construct using the mature citrus transformation protocol. During that instance, Dr. Orbovic was able to produce immature transgenics that had the GOI, while it rearranged in mature transformation, even using a RecA- Agro strain. One advantage of biolistics over Agro transformation is that the GOI does not rearrange as easily as in Agro. A manuscript was accepted & revised for In Vitro Cellular & Developmental Biology-Plant. This manuscript showed that improved selection with phosphomannose isomerase after biolistics in immature citrus drastically increased transformation efficiency after bombardment (a mean of ~2 transgenics per paired shot). We are now testing phosphomannose isomerase (PMI) as a new selectable marker in mature citrus. Once the optimal mannose/sucrose concentrations have been determined in mature citrus, we will test PMI selection after Agrobacterium-mediated transformation. We are testing a new protocol for precipitating DNA onto gold particles for biolistics that increased efficiency in other species. This protocol will also be useful for gene editing using biolistic transformation. We must buy a new soil sterilizer & a new ultra low -80C freezer. Both are essential to this program. An external review of the two transformation labs was conducted March 28 & 29 & presumably there will be a report issued at some time in the future.
This project is a continuation of previously funded CRDF grants to TWO BLADES focused on utilizing multiple strategies to produce canker-resistant citrus plants. The project has focused on transforming Duncan grapefruit with genes that express EFR or a gene construct designated ProBs314EBE:avrGf2 that is activated by citrus canker bacteria virulence factors. Objective 1. To determine if Bs3-generated transgenic grapefruit plants are resistant to diverse strains of the citrus canker bacterium or to alternate target susceptibility genes in greenhouse experiments and to the citrus canker bacterium in field experiments in Fort Pierce. We have tested the transgenic Duncan grapefruit containing the Bs3-executor transgene for resistance to an array of strains representing a worldwide collection. All of the strains were shown to elicit a hypersensitive reaction when infiltrated with a bacterial suspension of Xanthomona citri into young leaves of the transgenic citrus plant but a susceptible reaction when infiltrated into non-transgenic susceptible Duncan grapefruit leaves. We are continuing further experiments to confirm the resistance in the greenhouse. Furthermore, we will quantify bacterial populations in transgenic and susceptible trees to demonstrate if the transgene is activated without the TAL effector that is predicted to activate this gene. We are also confirming activation of the transgene using a realtime protocol to determine if the transgene is activated properly. During the past three months we have placed our constuct in a different vector that is acceptable for future transgenic purposes. The previous constructs contained an additional selectable marker that allowed for identifying putative transgenics with a higher success rate. Given that there was concern about the additional marker, the new construct contains only NPT as a selectable marker. The construct was recently sent to Dr. Vladimir Orbovic, who is in the process of transforming grapefruit and sweet orange. We hope to have additional transgenics later this year. We have also grafted our lone transgenic plant onto two rootstocks (812 and Sour Orange) and are in the process of planting in the field at Fort Pierce in collaboration with Dr. Ed Stover. They will be going into the field within the next couple of weeks. We have also identified two other possible transgenics from plants received from Dr. Vladimir Orbovic that contain the Bs3-executor transgene. One of the putatve transgenics has a growth defect and in all likelihood will be of no use in future experiments. The other putative transgenic tree which appears to contain the gene may be useful for future testing. Objective 2. To determine if EFR-generated transgenic grapefruit plants are resistant to the citrus canker bacterium in field experiments in Fort Pierce. We have grafted our two most promising EFR transgenic plants (based on ROS activity) onto two rootstocks (812 and Sour Orange) and are in the process of planting in the field at Fort Pierce in collaboration Dr. Ed Stover. They will be going into the field within the next couple of weeks. We have additional transgenics from plants received from Dr. Vladimir Orbovic that need to be tested for the presence of EFR. Objective 3. To determine if bs5-generated transgenic Carrizo plants are resistant to X. citri and to generate transgenic grapefruit carrying bs5. Currently the trees are being grown and then will be grafted onto rootstocks for further testing.
Objective 1, Mthionin Constructs: Assessment of the Mthionin transgenic lines is continuing apace. Detached leaf assays, with CLas+ ACP feeding, have been conducted and lines with the most promising results have begun greenhouse studies. These studies (With 9 Carrizo lines and 4 Hamlin lines, 98 total plants with controls) include graft inoculation of Carrizo rooted cuttings with CLas+ rough lemon, no-choice caged ACP inoculation of Carrizo rooted cuttings, and no-choice caged ACP inoculation of grafted Hamlin on Carrizo with all combinations of WT and transgenic.The first field plantings with Mthionin transgenic Carrizo (45 plants) have been made with leaves of non-transgenic rough lemon on transgenics showing higher average CLas CT, significantly decreased leaf mottle and significantly increased health values after 6 months. Plants for follow up field plantings of transgenic Hamlin on WT Carrizo (112 plants), WT Hamlin on transgenic Carrizo (84 plants), WT Ray Ruby on transgenic Carrizo (118 plants) and WT Valencia on transgenic Carrizo (118 plants) with WT controls are being propagated.Seeds for additional scion variety transformations have been collected and germinated. Shoots will be developed enough to yield epicotyl tissue for Mthionin construct transformations in 2 (Hamlin), 4 (Ray Ruby) and 6 (Valencia) weeks. Objective 2, Citrus Chimera Constructs: Detached leaf assays, with CLas+ ACP feeding, were conducted on lines representing chimera constructs TPK, PKT, CT-CII, TBL, LBP/’74’, `73′, and `188′. Multiple lines from several constructs were moved forward into greenhouse studies based on these results as noted below. Definitive results for TPK, PKT, CII, and TBL were hindered by low inoculation rates. Assays for these constructs are being repeated to identify which lines of each are best suited for greenhouse studies. Detached leaf feeding assay protocols have also been adjusted to improve sensitivity (See section 4)No-choice caged ACP inoculation has been conducted on 8 lines of citrus Thionin-lipid binding protein chimeras (`73′, and ’74’). Three month data has been collected, while many plants are yet to show CLas DNA amplification, there is a statistically significant reduction (13x) in CLas titer for transgenics vs WT in the CLas+ plants. An additional 475 rooted cuttings have been propagated from chimera constructs (6 lines of `188′, 7 lines of `74′ and 12 lines of `73′) for the next round of ACP inoculation trials. Objective 3, ScFv Constructs: Greenhouse studies on the 5 scFv lines in the 1st round of ACP-inoculation has been completed with the best performing lines showing significantly reduced CLas titer over the 12 month period (up to 250x reduction) and a much higher incidence of no CLas rDNA amplification in all tissue types at the conclusion of the study. The best lines have been used as rootstock for WT Ray Ruby scions and will be moved to the field once the graft union is strong enough. An additional 129 rooted cuttings are propagated for additional grafts and field plantings. ACP inoculations were conducted on 150 more plants from 12 scFv lines. Data from 3 months post inoculation has been collected, but too few plants are testing positive at this time for a conclusive analysis. An additional 370 rooted cuttings have been propagated from the remaining scFv constructs/lines to be tested and will soon be mature enough for ACP inoculation. Objective 4, Screening Development and Validation: Details of the high throughput ACP homogenate assay, and its use for selecting lytic peptides for activity against CLas, has been submitted for publication and remains in use for early screening of therapeutics in the lab. The detached leaf ACP-feeding assay has undergone several small revisions to improve sensitivity and maintain consistent inoculation; increasing from 10 to 20 ACP per leaf, decreasing the feeding period (7 days to 3) and adding a 4 day incubation period between feeding and tissue collection.An array of phloem specific citrus genes has been selected for investigation as potential reference genes to improve detached tissue and plant sampling techniques. The use of a phloem specific endogene would allow for samples to be normalized to phloem cells instead of total citrus cells, more accurately evaluating bacterial titer and potential therapeutic effects with the phloem limited CLas. Objective 5, Transgene Characterization: Transgenic Carrizo lines expressing His6 tagged variants of chimeric proteins TBL (15 lines), BLT (15 lines), TPK (17 lines), and PKT (20 lines) have been generated and confirmed for transgene expression by RT-qPCR. These plants will be used for generating data on the movement and distribution of transgene products in parallel to antibody based approaches.
Three field trials were initiated in two orchards near Frostproof FL. Both had 15-month-old Valencia trees on Kuharski rootstock planted at 10×20 foot spacing. Trees exhibit variable growth and health and are infested by sting nematode. The first trial to determine the effect of perennial peanut in middles with or without oxamyl in rows was designed as a randomized complete block within four rows. Treatments comprise 4 adjacent trees replicated 4 times (complete factorial arrangement of untreated, peanut, oxamyl, peanut + oxamyl). We purchased the peanut as sod that was installed on 5 March and is being watered by hand 3 times weekly until established. The second trial compares the efficacy against sting nematode of oxamyl, aldicarb, fluensulfone, fluopyram, fluazaindolizine, and an experimental compound (Syngenta) in a randomized complete block design with eight, four-tree replicates. All plots were plumbed across 16 rows to be treated by injecting the materials into a single, central manifold feeding the appropriate microjets. Two products per day are being applied with the irrigation cylcle and the first treatments of all products will be completed this week. The third trial is in a nearby orchard in which sunn hemp has been sown in tree middles in such a way that eight, four-tree replicates will be arranged in a randomized complete block design testing effects on sting nematode and tree health of the complete factorial treatments of untreated, oxamyl treated, sunn hemp treated, and oxamyl + sunn hemp. Sunn hemp was planted on 7 March. Trunk girth of all experimental trees in all trials was measured on 6-7 March.
The research agreement contract was finalized in January, 2019, and funds became available in February, 2019. Dr. McNellis initiated a search for a graduate student to perform the work through the Department of Plant Pathology and Environmental Microbiology graduate program and the Plant Biology graduate program at Penn State. A suitable candidate was interviewed in February, 2019, and an offer of admission was made in March, 2019. A rotation student in the Intercollege Program in Plant Biology at Penn State also expressed interest in the project and began a rotation in Dr. McNellis’ lab on March 5, 2019, which will continue for at least the remainder of the spring semester. It is likely that one or both these students may become available to work on the project. The grapefruit trees expressing the FT-scFv anti-HLB antibody protein were continuously maintained at Penn State and at the USHRL in Ft. Pierce, FL, and continue to grow normally and are ready for analysis.
Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars. This is the 1st year, 1st quarter progress report; our grant started December 1, 2018. In the last three months, we have processed all the paperwork needed to establish the grant and begin spending funds at ARS. We have identified a qualified and interested post-doctoral researcher, Dr. Tamara D. Collum, who we will be hiring with grant funding. However, all ARS hiring actions, even those using soft funds, are currently on hold at the Department of Agriculture level. Now that the department has a full-year budget, we hope this hold will be lifted shortly so we can bring Tami on board in the next couple weeks. Objective 1 (development of transgenic constructs) is close to completion and work has begun in the Stover lab on objective 2 (production of transgenic citrus lines). For objective 6 (Additional Approach: Phloem limited citrus tristeza virus vectors will be used to express the His-FLAG-tagged ribosomal protein in healthy and CLas infected citrus) inserts have been assembled and sent to Dr. Dawson’s lab for inclusion in CTV vectors and subsequent introduction into citrus.
The purpose of this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Achieving this outcome will require progress in the following three tasks: (1) to compare the delivery efficacy of bactericides with three application methods (foliar spraying, truck injection, and root administration) based on the uptake and dynamic movement/distribution of the bactericide within the tree; (2) to clarify the systemic movement and transportation mechanisms of bactericides within the phloem of tree; and (3) to investigate the effects of citrus variety and age on the delivery efficacy of bactericides. This project requires a combination of greenhouse studies and field trials. Prior to conducting these experiments, a sensitive and accurate quantifying method of bactericides (oxytetracycline and streptomycin) in citrus tissues is needed. This project officially started on December 1, 2019. This is the first quarterly progress report covering 12/01 to 02/29, 2019. During this period we have started and/or completed the following work/research tasks: 1. A project team meeting was held to plan this year’s research activities and define the responsibilities of PI, Co-PI, participants, and collaboration partners. 2. A work plan with detailed information on experimental design, measurements and analytical methods was developed and will be followed by all the participants. 3. Preparation for project research, including purchase of chemicals, labware and small equipment was completed. 4. Samples of citrus leaf, stem, and fruit were collected from fields. These samples were treated with antibiotics to be studied and used for testing and validating existing methods for detecting trace amounts of antibiotics in plant tissues and fruit. The work planned for the next quarter: The major goal of research for the first six months are to develop an improved robust analytical method for detecting and quantifying antibiotic concentration in citrus samples. The following research will be conducted in the 2nd quarter to accomplish this goal. 1. Laboratory experiments and analyses to test existing methods for detecting antibiotics in citrus plant tissues and fruit samples. 2. Continue to collect plant tissue and fruit samples from citrus fields where oxytetracycline and/or streptomycin were frequently applied. 3. Laboratory experiments and analyses to improve antibiotic extraction and cleanup procedure based on the results from preliminary experiments. 4. Laboratory experiments and analyses to establish a reliable methods for extraction and analyzing antibiotics in citrus samples using LC-MS/MS. 5. To culture citrus plants according to the work plan for the greenhouse experiments.
The objectives of this proposal are: 1) conduct a field trial using the selected grapefruit seedlings to ensure the productivity of the trees in Florida where HLB is endemic; and 2) evaluate the quality of the fruit produced. Achievement of these goals will produce a more resistant/tolerant variety that could be available in the near future since its use would not require the regulatory approval. Based on two year’s graft-inoculation assays in greenhouse with two HLB isolates and the performance of individual seedlings in the field, four lines of the seedlings (with greater HLB resistance/tolerance) were selected for further propagation on three different rootstock (commercial sour orange, newly selected USDA-sour orange and 942). The fruit quality (Brix, sucrose, glucose and fructose, soluble solids, pH, % TA and total ascorbic acid) of the four selected seedlings showed no significant difference from their maternal trees. First group of the propagates on three different rootstock from the selections of Scott Grove’s seedling variants were grown in our research farm, Picos farm, where the plants are under extreme high HLB disease pressure with very aggressive HLB pathogens. These new plantings (July, 2017; Nov, 2017; and May, 2018) showed different disease index, the longer the planting was, the higher the disease index. It is worth noting that the new HLB isolate from Picos farm caused severe HLB disease on most of grapefruit selections of seedlings and bud sports in our latest greenhouse evaluation. Those selections were either resistant or tolerant to the previous HLB isolates we maintained in greenhouse. By the end of this year, we will expect to draw a better conclusion if the selected seedling variants display better resistance/tolerance to HLB pathogens in the Picos Research Farm. Second group of the propagates on three different root stocks (Ca. 750 plants) have been budded and grown in our greenhouse, and are expected to be planted in Scott grove within 3 months.
During this reporting time period, we have conducted the following studies. 1) To use genome editing technologies to produce gene-edited and transgene-free plants derived from mature citrus tissues, one challenge is to reduce chimeric plants that are composed of both edited and non-edited cells. We have been working to develop an endogenous chemical resistance to reduce chimeras. We have identified a citrus candidate gene and constructed a vector for the resistance to reduce chimeric plants. We have used Agrobacterium cells hosting these genes to infect mature and juvenile citrus tissues. Our first set experiment for creating the chemical resistance in citrus cells has not been completed because the chemical resistant cells from the infected citrus tissues have not been obvious. We may need to wait for additional weeks to see any resistance. We have also been continuing to repeat some previously observed results using the proposed genes because we have observed some inconsistent results regarding their effects when used in mature citrus tissues. We have also continued to test the effects of some chemicals on transient and stable expression activities and also regeneration efficiencies in mature citrus tissues. We have observed some improvement of shoot regenerated from calli derived from mature tissues of citrus. We are also using fresh young shoot tissues propagated from mature citrus shoots for regeneration and transformation studies. However, we need more tissues for testing because the effects are not always consistent from one experiments to another when mature citrus tissues are used. In addition to improving the efficiencies of traditional methods for mature citrus tissues, we have been working to develop an in planta transformation method for mature citrus tissues. In-planta transformation means that genetic transformation of can be achieved without going through tissue culture-based regeneration of shoots or embryos. In-planta transformation methods have been successfully developed for a small number of higher plants that are difficult to transform using traditional tissue culture-based methods. We have successfully used an in planta transformation method to produce transgenic and gene-edited plants using juvenile tissues of citrus. For mature citrus tissue transformation, we have done two testing so far but encountered some problems. One problem is that we need lots of shoots from mature citrus trees to work out important parameters of the method. We have worked with Mr. Phillip Rucks and Ms. Beth Lamb (Phillip Rucks Citrus Nursery in Frostproof, FL) who have kindly provided and will provide us significant amounts of mature citrus shoots for our experiments.
A number of successes have been documented at the Picos Test Site funded through the CRDF. The UF Grosser transgenic effort has identified promising material, eliminated failures, and continues to replant with new advanced material (Grosser, personal comm.). Using trees planted at the test site, transgenic overexpression of an Arabidopsis defense gene was reported to enhance citrus HLB resistance (Dutt et al., 2015). The ARS Stover transgenic program has trees from many constructs at the test site and is seeing some modest differences so far, but new material planted this spring that has shown great promise in the greenhouse (Hao, Stover and Gupta, 2016). A trial of more than 85 seedling populations from accessions of Citrus and citrus relatives (provided as seeds from the US National Clonal Germplasm Repository in Riverside, CA) has been underway for 6 years in the Picos Test Site. P. trifoliata, Microcitrus, and Eremocitrus are among the few genotypes in the citrus gene pool that continue to show substantial resistance to HLB (Ramadugu et al., 2016), P. trifoliata displayed reduced colonization by ACP (Westbrook et al., 2011), and measures of HLB-tolerance were associated with percentage citron in accession pedigrees (Miles et al., 2017). A UF-Gmitter led association mapping study is underway using the same planting, to identify loci/genes associated with HLB- and ACP-resistance. A broad cross-section of other Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside. More than 100 citranges, from a well-characterized mapping population, and other trifoliate hybrids (+ sweet orange standards) were planted in a replicated trial in collaboration with Fred Gmitter of UF and Mikeal Roose of UCRiverside. Plants were monitored for CLas titer development and HLB symptoms. Data from this trial should provide information on markers and perhaps genes associated with HLB resistance, for use in transgenic and conventional breeding. A manuscript reporting identified HLB resistance associated QTLs has been published (Huang et al., 2018). David Hall assessed ACP colonization on a subset of plants and further documented host morphological traits associated with ACP-colonization in Poncirus (Hall et al., 2017a&b). Several USDA citrus hybrids/genotypes with Poncirus in the pedigree have fruits that approach commercial quality, were planted within the citrange site. As of April 2014 at the Picos Test Site, several of these USDA hybrids had grown to a height of seven ft (one now released as US SunDragon), with dense canopies and good fruit set, while sweet oranges were stunted (3 ft) with very low vigor. These differences largely continue and the observations have encouraged aggressive use of this and other trifoliate hybrids as parents (Stover et al., unpublished). A Fairchild x Fortune mapping population was planted at the Picos Test Site in an effort led by Mike Roose to identify loci/genes associated with tolerance. This replicated planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. HLB phenotyping and growth data have been collected and genotyping will be conducted under a new NIFA grant. Valencia on UF Grosser tertazyg rootstocks have been at the Picos Test Site for several years, having been CLas-inoculated before planting, and several continue to show excellent growth compared to standard controls (Grosser, personal comm.). Numerous promising transgenics identified by the Stover lab in the last two years have been propagated and will be planted in the test site. New transgenics from Jeffrey Jones and Zhonglin Mou of UF, Tim McNellis of PSU will be planted in the next month. Availability of this resource will continue to b
Two Las repressors from Objective 1, and a Wolbachia repressor from Objective 2 were all confirmed as functional transcriptional regulators of Las phage genes and with DNA binding sites within key Las phage promoter regions. These three repressors are therefore potential chemical targets for inhibitors that may control HLB. In addition, a likely Las virulence effector, a secreted peroxiredoxin enzyme, was identified in Objective 3. This enzyme appears to prevent citrus host phloem cells from killing Las and also blocks systemic host responses to Las. This secreted enzyme is also a high value potential chemical target Two publications have appeared and a third is in press covering these Objectives. . Despite repeated attempts, the fluorimetric assay proved unusable for chemical library screening of the three small transcriptional regulatory proteins. All three repressors are very small DNA binding proteins with little potential to form folded structures, which is necessary for the thermal denaturation assay. An alternative approach was evaluated using Micro Scale Thermophoresis (MST), a biophysical technique that measures the strength of binding between two molecules by fluorescence. MST was used in an attempt to evaluate binding between commercially synthesized C2 protein and DNA from the promoter region previously demonstrated to bind C2 by gel retardation assays. Attempts to use MST failed, indicating that the C2 protein was unstable in the buffer conditions needed for MST. A high throughput fluorimetric thermal denaturation screen was first used to identify chemicals that bound to the (large) Las peroxiredoxin target. A total of 320 phytochemicals were screened, resulting in the identification of fourteen (14) lead candidates for phytochemical control of HLB. Several of the lead candidates are generally recognized as safe (GRAS) and are not pharmaceutical drugs. The larger library of 1,600 chemicals, including drugs, was then further screened using a direct enzymatic activity inhibition assay to independently verify the results of the fluorimetric assay and also potentially identify additional inhibitors that directly affect the secreted Las peroxiredoxin. Peroxiredoxins react with hydrogen peroxide and both aliphatic and aromatic hydroperoxide substrates. Of 1,600 chemicals screened, 28 exhibited a strong inhibitory effect on the Las peroxiredoxin. Based on possible commercial value as being both GRAS and relatively inexpensive, 7 chemicals were selected for further study. Three of the 7 chemicals were confirmed repeatedly as having a strong inhibitory effect. One of these was confirmed inhibitory by both fluorimetry and direct enzyme inhibition assays. Field trials in commercially grown, Las infected citrus failed to demonstrate a practical level of reduction of Las titer in heavily infected citrus. We speculate that Las peroxiredoxin is required for initial colonization and early establishment of infections, but once an infection is established, reduction in peroxiredoxin activity did not lead to clearing of existing infections. This compound will be further evaluated for efficacy in preventing new Las infections in commercial citrus replants.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
