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.
In the first three months of this project, we started evaluating the performance of a ground penetrating radar (GPR) to map root architecture of HLB-infected citrus trees. The GPR can be used to map tree roots because it is relatively easy to use in the field and it is non-destructive to trees, roots, and the root-soil environment. Numerous tree root scans can be performed and replicated in a short time without interfering with root growth.Although the GPR has been used to detect roots for different plant species, most studies were conducted in controlled environments or in simulations to evaluate signal processing algorithms. Evaluation of the GPR for root detection in an agricultural field setting such as a citrus grove has not yet been conducted. Additionally, many questions remain to regarding the suitability of the technology for studying the impact of diseases on tree root structure and distribution.One objective of this study is to evaluate the performance of a ground penetrating radar to accurately detect citrus tree roots and generate 3D morphology root maps of citrus trees grown in a complex field environment in Florida. To achieve this goal, we are investigating the influence of several limiting factors on the performance of a GPR to accurately detect citrus roots and determine their main structural characteristics. First, single-factor experiments were conducted to evaluate GPR performance. Factors evaluated were: (i) GPR frequency (900 and1,600 MHz); (ii) root diameter; (iii) root moisture level; (iv) root depth; (v) root spacing; (vi) survey angle; and (vii) soil moisture level. Second, two multi-factor field experiments were conducted to evaluate the performance of the GPR in complex citrus grove environments in southwest Florida and to develop 3D morphology root maps. Experiments were conducted at the citrus research grove of the University of Florida Southwest Florida Research and Education Center (SWFREC) in Immokalee, FL, USA. A ground penetrating radar (GPR) (TRU Model, Tree Radar, Inc., USA) mounted to a mobile scanning cart and equipped with: (i) a 1,600 MHz antenna, and (ii) a 900 MHz antenna was utilized to generate a 3D map of the root system. The transmitter of the GPR antenna transmits electromagnetic waves (pulses), and the receiver collects the reflectance when an object is detected beneath the soil surface. The relative distance from the starting point was measured with a wheel recorder. A commercial software (TreeWin Roots and TRU Tree Radar Unit) was used to generate root morphology and root density maps. To present the root layout by location and depth, 3D images were created.Our initial experiments showed that the 1,600 MHz GPR was more accurate in detecting citrus roots and their location than the 900 MHz GPR. Upon target (root) detection, the GPR generated a hyperbola in the radar profile; from the width of the hyperbola the diameter of the root was successfully determined when roots were larger than 0.5 cm in diameter. The GPR also distinguished live from dead roots, which is indispensable for studying the effects of soil-borne and other diseases on the citrus tree root system.
This project produces transgenics, cis/intragenics & subgenics, in agronomically acceptable cultivars, for field testing & potential commercialization.The original proposal was for a 3 year funding period, but the project was only funded for 1 year because the CRDF wanted transgenics made by a company in Brazil. In 2016, after the CRDF realized that logististically, transgenics could not be easily made in Brazil, the mature citrus facility (MCF) was funded 2 more years. In total, 3 proposals were written for this project. Because of instability in funding, similar to what is presently occurring with short-term contracts, it has been difficult to keep good employees & maintain productivity. The significant objectives for the 1st & 2nd funding periods were: Mature plant production as a service using with Agrobacterium harboring vectors with disease resistance genes & molecular analyses to show copy number of the transgenes & gene expression; Plant propagation to form replicates for field testing; Increase micrografting efficiencies, bypass it altogether, or root mature scion; Test different selectable markers & reporters; Develop a biolistics protocol for immature/mature citrus; Introduce new, high yielding cultivars for tests in transformation; Apply for external funding. At the beginning of this funding cycle, new customers were charged a nominal fee for transgenics because previously our services were free. All of the abovementioned objectives have been addressed. Plant production for customers, for technology development, & for increasing efficiency produced ~437 transgenics for this 3 year period (~558 in total since 2014). An additional 400 transgenics were propagated for customers, either through budding or rooting. Mature scion cannot be rooted, micrografting cannot be bypassed, but micrografting efficiencies are stable at 77% by 1 operator. Molecular analyses were conducted for customers (~120 qPCR assays for 1 customer) & for publications, & thousands of endpoint PCRs for the gene of interest conducted. Several grants proposals were submitted & 2 small proposals were funded. Biolistic transformation was developed for immature & mature citrus, & an new selectable marker significantly increased efficiency. This was the first report of biolistic transformation of citrus with plant regeneration. This objective will become increasingly important considering new 2018 USDA APHIS guidelines in which APHIS will not oversee field tests for cis/intragenics/subgenics that do not carry plant pest or vector sequences, & these trees can be fast-tracked to growers at reduced expense, essentially similar to cultivars produced with traditional breeding. High yielding cultivars from the Plant Improvement Team were introduced & Agrobacterium & biolistics transformation efficiencies determined. There is one new scion cultivar that has an extermely high Agrobacterium transformation efficiency & another scion cultivar has reproduciby high efficiency. Most cultivars can be transformed using biolistics, although none have exceedingly high transformation efficiency yet. A cisgenic selectable marker, constructed by Drs. Zale & Dutt, is being tested in mature citrus to increase transformation efficiency. An intragenic citrus reporter from Dr. Dutt works well to replace GUS. A UF CREC Initiative during this timeframe was to stack two genes in transgenics to prevent the bacteria from overcoming resistance of one gene. An unfunded UF scientist, complied with the request to make the stacked gene constructs, & the MCF produced ~150 transgenics to meet this UF CREC Initiative. Biolistic-mediated gene editing of the PDS gene was achieved in immature Carrizo & Valencia. One customer’s Agrobacterium vector had a tendency to rearrange, in Agrobacterium prior to transformation. A total of 33 trees were produced for this scientist, but the transgenes rearranged in all but 2 events. This mutation was documented by restriction digests of vector DNA grown in E. coli vs Agrobacterium. A consecutive double budding method was devised so that mature citrus is reinvigorated well prior to experimentation. New services were added that offer biolistic transformation of minimal cis/intragenic expression cassettes. This service, if utilized by scientists, will provide significantly more monetary revenue for the mature citrus facility. However the CRDF should encourage scientists to use bioistics for cis/intragenics for faster & cheaper deregulation. Growth room maintenance is expensive & it is expensive to staff the MCF. A number of publications were generated by this project.
We requested a NCE for this grant to allow proper completion of a promising greenhouse citrus root growth study that was repeated with Carrizo rootstock. This is part of objective 2 (Determine soil conditions that favor root hair and VAM proliferation) and does not affect the subawards and efforts of Vashisth, Wright and Morgan. We don’t anticipate any travel or field work. Objective 1: Leaf nutrient thresholds We implemented the full Diagnosis and Recommendation Integrated System (DRIS) method for leaf nutrient analysis that provides protection from cross-correlation of variables and environmental effects. Reference nutrient data for DRIS was obtained from high-yielding ‘Hamlin’ trees growing in the Ft. Meade area prior to HLB (>700 boxes/acre average). The DRIS method has proved very valuable for indicating ranges of critical deficiency for K, Mn, Zn, Fe. Mg, B, and Cu. Some nutrients, including S, N, Ca and P did not correlate well enough using DRIS to pinpoint critical thresholds, but general trends were still useful. These results look promising for publication and ultimately will be used to revise existing IFAS thresholds for citrus. We continue to analyze the large survey dataset from every angle and with new analytical tools since it contains an abundance of important information. Objective 2: Determine soil conditions that favor root hair and VAM proliferation The results so far with Carrizo look much better than the original root study which was using rooted scion (Murcott) material. In order to get the required information from the trial, we captured more SEM images to quantify root hair growth (Dec 2018). Visually the root hair growth was dramatically increased by tricalcium phosphate in equilibrium with the growing solution. We will quantify root hairs from the images in early 2019. When psyllid adults become available in February, we plan to inoculate the hydroponic citrus trees with CLas so that the impacts of infection and nutrient solution treatment can be measured in root hair growth (until about May 2019). The final report will be completed in May/June.
During this reporting period, we have been tested the effectiveness of the gene that can reduce chimeras of mutant and wild-type plants of citrus using mature and juvenal tissues as explants. The gene can be useful when Agrobacterium-mediated transient expression of the Cas9 and sgRNA are used to produce transgene free edited plants. The results from the first set of testing experiment will be available in two weeks. We continue to repeat some previously observed results using the proposed genes and also chemicals we have identified. We have observed some shoots 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. We have also started in-planta transformation of mature citrus shoots. We have worked with Mr. Phillip Rucks and Ms. Beth Lamb ( Phillip Rucks Citrus Nursery in Frostproof, FL) who have kindly provided us significant amounts of mature citrus shoots for our experiments so that we can work on development of an in-planta transformation method for mature citrus tissues. We have shown that we can use in-planta transform method to transform juvenile tissues of citrus.
The goal of this project is to generate green fluorescence protein (GFP) labeled Ca. Liberibacter asiaticus (Las), test its application in study of Las movement and distribution in planta, and investigate the control effect of different measurements including heat treatment and antimicrobial treatment. Las and other HLB-associating Liberibacters have not been cultured outside of their hosts in cell-free artificial culture media; therefore, traditional molecular and genetic analyses cannot be applied. This has greatly hampered our efforts to understand the virulence mechanisms of Las. We have been looking for alternative approaches to genetically manipulate Las in vivo. This has been made possible by the large population of Las in psyllid and availability of molecular tools to perform genetic manipulation in vivo. Alternatively, Las can survive for a short time in the media after acquired from psyllid gut and we aim to genetically modify Las with GFP immediately after Las being acquired from psyllids. To achieve the goal of this study, we will pursue the following specific objectives:1) GFP labeling of Candidatus Liberibacter asiaticus. 2) Elucidation of plant-Las interaction through real-time monitoring of Las movement and multiplication in planta using GFP labeled Las. 3) Investigate the effect of different control approaches on the dynamic population of Las in planta using GFP labeled Las. Previously, the reporter plasmid, pBAM1::R-PgyrA-GFP, composed of Tn5 and narrow host-range origin was constructed and therefore the GFP gene can be inserted into the genome of bacteria. However, it was only successfully transferred into a genome of Pseudomonas fluorescence with low transformation efficiency and failed with other bacteria including Escherichia coli DH5a, Sinorhizobium meliloti Rm1021, and Liberibacter crescens BT-1. Recently, pDH3::PgyrA-GFP was constructed which has a wide bacterial host range replicon, repW, but cannot be inserted into a genome. Transformation of E. coli by PEG mediated method with pDH3::PgyrA-GFP showed high transformation efficiency (~2 x 104 CFU/ g of DNA) than with previous reporter plasmid (failed). Following application with L. crescens BT-1 by electroporation was also successful (1.9 x 103 CFU/ g of DNA). Transformants and the GFP expression in L. crescens BT-1 were confirmed by PCR and fluorescent microscopic analysis, respectively. As L. crescens is a phylogenetically closest species to Ca. L. asiaticus, there is a possibility that pDH3::PgyrA-GFP would be useful for GFP labeling of Ca. L. asiaticus. We have further confirmed the Lcr-GFP using western blot. The GFP plasmid is being used to transform Las. To facilitate Las transformation, we have tested multiple novel methods of culturing. Las population was observed to decrease at the beginning, and increase slowly. Repeated experiments show similar pattern which suggest we might be able to acquire enough Las cells for transformation after further optimization. We are testing new methods for culturing Las. Especially, we are testing co-culturing Las with citrus tissue culture and psyllid tissue culture. Currently, we are in the process of establishing a pure psyllid cell culture. We have used two approaches to label L. crescens. Preliminary data showed one approach works for Las in vitro. We are testing whether we can label Las in vivo and observe its movement. 2) We have conducted Las movement and multiplication in planta based on qPCR method. We have tested approaches to prevent Las movement in planta. One manuscript has been submitted. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. 3) We have been testing the effect of different control approaches including application with bactericides. One manuscript entitled: “Control of Citrus Huanglongbing via Trunk Injection of Plant Defense Activators and Antibiotics” has been published by Phytopathology. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. This manuscript has been accepted for publication with revision by Phytopathology.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. The following work has been conducted in this quarter: (1) We have started to treat the three independent transgenic lines ( Duncan 57-28, Hamlin 13-3, and Hamlin 13-29), which have gone through the long-term HLB test and exhibited robust tolerance to HLB disease. The first batch of plants, including two replicates of the transgenic line 57-28, three replicates of the line 13-3, and one replicate of the line 13-29, have been treated under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours) for two months. These plants have generated some new shoots during the treatment. We have tested if heat treatment is able to remove CTV and the CLas bacteria. So far the new shoots are negative for both CTV and CLas, indicating that the treatment is effective. The new shoots will be used for generating citrus trees for field trials. (2) We have screened 28 new transgenic lines against HLB-infected psyllids. These lines were generated by the mature transformation laboratory. The following lines still look great and haven’t shown any HLB symptoms: #82-6 Hamlin, #70-4 Hamlin, #26 Hamlin, #65 Hamlin, #82 Hamlin, #73-5 Hamlin, #11 Pineapple, #33 Pineapple, #73-5 Pineapple, #78 Pineapple. Based on the nymph production phenotype, these plants should have been infected by HLB. We have tested bacterial titers in these plants by qPCR, and indeed, the majority of these plants are CLas positive. The bacterium-free plants (with low CLas titers) will be inoculated again. (3) The eight new transgenic lines (A99, A100, A102, A101, A72, A73, A97, and A98) were irrigated and fertilized regularly. After they reach appropriate size, they will be screened against HLB-infected psyllids. (4) The manuscript titled Overexpression of the Arabidopsis NPR1 protein in citrus confers tolerance to Huanglongbing has been revised and published in the Journal of Citrus Pathology in this quarter.
During this reporting period, we have constructed a gene vector that may reduce chimeras of mutant and wild-type plants of citrus using mature and juvenal tissues as explants. The vector contains a Cas9 gene and a sgRNA gene to target on a citrus gene that may lead to an endogenous chemical resistance. The gene may be useful to reduce wt and mutant chimaera when Agrobacterium-mediated transient expression of the Cas9 and sgRNA are used to produce transgene-free edited plants. We will start testing its efficiency in mature tissues of Valencia and juvenile tissues of Carrizo. We have been continuing to repeat some previously observed results using the proposed genes such as AGO and NPRD genes that showed some positive effects of transformation efficiencies. We have also repeated the experiments for the effects of PAT and SMZ we have previously observed some positive effects on transformation of both juvenile and mature tissues of Valencia and Washington navel. Our goals are to generate sufficient and repeatable for the genes and chemicals we have used results for scientific publications and patent applications. We have been working on production of fresh young shoots from mature citrus tissues and using them directly for regeneration and transformation studies. We have observed that reasonable amount of shoots can be produced from callus tissues derived from mature tissues of Valencia. We observed more shoots produced from these callus tissues if chemicals such as NPA or 5-Aza were used. When using the young shoot tissues derived from nature tissues of Valencia as explants for the transformation experiments, in addition to the genes originally described in the proposal, we will test the effects of the Wus and BBM genes. Over-expression of both genes has been shown very effective in enhancing transformation efficiencies of a large number of plants that are difficult to be transformed. We are designing and constructing Wus and BBM genes for citrus transformation and regeneration.
The project has three objectives: (1) Obtain mature tissues of the best transgenic lines. (2) Determine whether transgenics prevent psyllids from being infected. (3) Continue testing generations of vegetative propagation from the best transgenic lines. Major accomplishments per objective (1) Obtain mature tissues of the best transgenic lines: successfully achieved. The citrus flower-promoting gene FT3 was previously cloned into the CTV vector by the Dawson lab. The CTV-FT3 construct was introduced into Agrobacterium. Tobacco leaves were infiltrated with the resulting Agrobacterium. CTV-FT3 recombinant virions were purified from systemically infected tobacco leaves and bark flap inoculated into C. macrophylla seedlings, which began blooming in about five months. Buds from the matured C. macrophylla were grafted onto the original plants of all transgenic lines (EDS5-Dun-205-9, ELP3-Dun-207-8, ZMSN-Ham-73-1, ZMSN-Dun-137-2, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25). All plants began blooming in 6-18 months. We have successfully achieved this objective and also demonstrated that CTV-FT3 is efficient for converting juvenile tissues to mature tissues. The CTV-FT3-infected C. macrophylla plants that have bloomed can be used as bud source for promoting maturation. The three independent transgenic lines (NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25) that have shown robust tolerance to HLB have been treated under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours) to remove CTV and the CLas bacteria. The resulting clean germplasms will be used to generate trees for field trials. (2) Determine whether transgenics prevent psyllids from being infected: accomplished with negative results. CLas-infected psyllids can transfer the CLas bacteria to the next generation of psyllids by inoculating the flush area in which the nymphs develop, which allows the next generation of psyllids to continue spreading HLB without the need for another source plant. In our experiments, we noticed that several of the transgenic lines exhibit delayed or reduced levels of CLas after infection. We started to test if the delayed or reduced production of CLas is sufficient to prevent or reduce the infection of the progeny psyllids. CLas bacteria-carrying transgenic plants were placed in cages, and clean psyllids (not infected by CLas) were moved into the cages. The progenies of the psyllids were collected and tested for CLas titers. A total of six rounds of cage experiments with vegetatively propagated plants from the transgenic lines ELP3-Dun-207-8, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25 were conducted. Results showed that none of the transgenes was able to pre-vent psyllids from being infected by CLas. (3) Continue testing generations of vegetative propagation from the best transgenic lines: successfully achieved. Three independent transgenic lines, NPR1-Ham-13-3, NPR1-Ham-13-29, and NPR1-Dun-57-25, have gone through at least six rounds of HLB inoculation. Three generations of progenies (18 replicates for NPR1-Ham-13-3, 31 replicates for NPR1-Ham-13-29, and 25 replicates for NPR1-Dun-57-25) were inoculated with CLas-infected psyllids. The inoculation was repeated until all plants were CLas positive based on qPCR. All progeny plants have shown no or minor HLB symptoms. The three transgenic lines are thus highly tolerant to HLB and will be put into the field for field trials.
Objective 1: Leaf nutrient thresholds The complete data from the quarterly field survey over three years in three citrus growing regions have been collated and checked. Final analyses are underway, including the Diagnosis and Recommendation Integrated System (DRIS) method for leaf nutrient analysis that provides protection from cross-correlation of variables and environmental effects. Objective 2: Determine soil conditions that favor root hair and VAM proliferation Starting in July, new seedling Carrizo plants were established for use. Six weeks after germination, 27 uniform seedlings were selected and divided into 3 hydroponic tanks (9 seedlings each). Tank 1, is a control fertilizer mix, including all nutrients and pH adjusted to approximately 7.0. Tank 2, is a reduced Phosphorus fertilizer, similar to Tank 1, except it has no soluble phosphorus and Triple Calcium Phosphate added as the sole source of phosphate. The pH of Tank 2 is approximately 7.2 (unadjusted). Tank 3, is the same as Tank 2, except we have added CaCO3 to the mixture to increase the pH (approximately 7.8) of the solution and reduce the amount of available phosphorus. Tank 3 is a replicate from the first series tanks using Murcott seedlings (successful root hair development). All 3 tanks receive an extra dose, 100 ppm, of calcium (calcium chloride). The added calcium and the pH at approximately 7, should drive the chemical reactions in solution towards the removal of available phosphorus, keeping the available phosphorus in the solution at the concentration that favors root hair development, while still providing enough phosphorus to keep the plants nutrition balanced. The Carrizo seedlings from all 3 tanks are healthy, showing no visual signs of nutrient deficiency, and with visible differences in root hair development. Observing with light microscopy, Tanks 1 and 2 show significant development of root hairs; while Tank 3 has some root hairs, they are in much fewer numbers. We are currently assessing the numbers of root hairs using Scanning Electron Microscopy (SEM). Several roots from each tank, along with roots from untreated Carrizo seedlings, have been selected, and prepared for viewing with the SEM. Currently we are taking images and counting root hairs to asses whether there are significant differences. The density of root hairs on several of the roots is so high, SEM has been difficult. Tanks 1 and 2 had sample prep issues relating to the sputter coating stage and needed to be resampled. Those results should be available soon.
For the Third Quarter of 2018, Project #16-007 conducted or completed the following activities: 1) Revised Plot Plans and “Ground-Truthed” all entries for the three grapefruit plantings (July,2017; Nov. 2017; and May 2018), of the “Scott Seedling Variants” and will submit field maps to both the CRDF Project Manager, and Grower Cooperator (Daniel Scott) after cooperator “Walk-Through” 2) Summarized Breeding Line Entry List for each of the above plantings and prepared those lists/maps for both CRDF Project Manger and Grower Cooperator, after cooperator “Walk-Through”. 3) The USHRL farm crew continues to maintain the grapefruit seedling planted previously on the Picos Farm. The three relevant plantings include: a) Pre-CRDF/NIFA Proposal Planting: Block 2, Rows 48 (planted Feb., 2015), and Rows 50-54 (planted July, 2017); b) “Planting 1A: Block 2, Rows 9-15 planted in Nov., 2017; and c) “Planting 1B: Block 4, Rows 27-32 planted in May, 2018. 4) Inventoried greenhouse materials that belong to Grower Cooperator including the number of rootstocks on which they were budded. As of October 1, 696 of the 750 trees have been budded or grafted onto 3 rootstocks; 5) More rootstock trees have been acquired to complete the budding and grafting operation for the planting on the Scott Grove; 6) Revised budget of Project #16-007, and submitted it to CRDF; 7) Project advisor meet with both RMC and CRDF BOD to answer any questions relative to project 16-007, and provide a verbal report of progress.
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