The experimental design of the Polk County field trial is completed. Christopher Vincent has designed this trial carefully to include sufficient replication. He also planned the trial to reduce the effect of disease severity variablity on the results of the experiment. There will be ten replications for each of the four treatments and three trees per experimental unit. Only the middle tree will be sampled for CLas titer. The canopy denisty of each tree will also be measured.
The trees in the Poly County trial will be sampled next Tuesday prior to the first phosphate applications. This will give us baseline numbers on disease severity in each tree. The first phosphate spray applications will occur later that same week. Trees will be spray with one of four phosphate concentrations, including one treatment with no phosphate.
Another trial is being planned with Southern Gardens in Hendry County. With the two sites, we will have field trials in the two major soil types for citrus in Florida. We are delayed in the establishment of ths site as Southern Gardens continues its search for plots that would be suitable I do not expect this to take very long. Southern Gardens is committed to doing this.
The current plan is for Nian Wang’s lab at the CREC to determine CLas titer in samples from the Polk County trial. The Hendry County samples will be processed in Eric Triplett’s lab in Gainesville.
Meanwhile, the Triplett and Killiny labs have greenhouse experiments underway. In the Triplett lab, the optimal level of phosphate comcentration for foliar fertilization has been determined. Also, a new experiment is underway to compare calcium phosphate fertilizaiton on roots versus foliar phosphate fertilization. The former is expected to mimic field conditions where the plant is forced to reduce the pH to solubilize phosphate. The leaf sprays will mimic the intervention we have planned for the control of HLB.
In the Killiny lab, similar experiments are underway in the greenhouse as described in the paragraph above. In all of these greenhouse experiments, Killiny will measure citrate levels in leaf midribs from all treatments.
We will sample eight leaves per tree, four times each year for CLas titer. Canopy density will be used to measure disease severity. We plan to spray the plants four times a year.
The experimental design of the Polk County field trial is completed. Christopher Vincent has designed this trial carefully to include sufficient replication. He also planned the trial to reduce the effect of disease severity variablity on the results of the experiment. There will be ten replications for each of the four treatments and three trees per experimental unit. Only the middle tree will be sampled for CLas titer. The canopy denisty of each tree will also be measured.
The trees in the Poly County trial will be sampled next Tuesday prior to the first phosphate applications. This will give us baseline numbers on disease severity in each tree. The first phosphate spray applications will occur later that same week. Trees will be spray with one of four phosphate concentrations, including one treatment with no phosphate.
Another trial is being planned with Southern Gardens in Hendry County. With the two sites, we will have field trials in the two major soil types for citrus in Florida. We are delayed in the establishment of ths site as Southern Gardens continues its search for plots that would be suitable I do not expect this to take very long. Southern Gardens is committed to doing this.
The current plan is for Nian Wang’s lab at the CREC to determine CLas titer in samples from the Polk County trial. The Hendry County samples will be processed in Eric Triplett’s lab in Gainesville.
Meanwhile, the Triplett and Killiny labs have greenhouse experiments underway. In the Triplett lab, the optimal level of phosphate comcentration for foliar fertilization has been determined. Also, a new experiment is underway to compare calcium phosphate fertilizaiton on roots versus foliar phosphate fertilization. The former is expected to mimic field conditions where the plant is forced to reduce the pH to solubilize phosphate. The leaf sprays will mimic the intervention we have planned for the control of HLB.
In the Killiny lab, similar experiments are underway in the greenhouse as described in the paragraph above. In all of these greenhouse experiments, Killiny will measure citrate levels in leaf midribs from all treatments.
We will sample eight leaves per tree, four times each year for CLas titer. Canopy density will be used to measure disease severity. We plan to spray the plants four times a year.
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, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infectd plants. 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 at the Picos Research Farm. Second group of the propagates on three different root stocks mentioned above (Ca. 750 plants) have been budded and grown in our greenhouse, and are expected to be planted in Scott grove this month. All the propagates have been tested for the presence of Las. The propagates that carry a low titer of Las will be palnted separately and serve as the experiment for “Cross-protection”.
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, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infectd plants. 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 at the Picos Research Farm. Second group of the propagates on three different root stocks mentioned above (Ca. 750 plants) have been budded and grown in our greenhouse, and are expected to be planted in Scott grove this month. All the propagates have been tested for the presence of Las. The propagates that carry a low titer of Las will be palnted separately and serve as the experiment for “Cross-protection”.
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, which was also highly correlated with the titers of Ca. Liberibacter asiaticus (Las) in infectd plants. 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 at the Picos Research Farm. Second group of the propagates on three different root stocks mentioned above (Ca. 750 plants) have been budded and grown in our greenhouse, and are expected to be planted in Scott grove this month. All the propagates have been tested for the presence of Las. The propagates that carry a low titer of Las will be palnted separately and serve as the experiment for “Cross-protection”.
In the first six months of this project, we investigated the influence of several limiting factors on the performance of a ground penetrating radar (GPR) to accurately detect HLB-affected citrus roots and determine their main structural characteristics. First, single-factor experiments were conducted to evaluate GPR performance. Factors evaluated were: (i) root diameter; (ii) root moisture level; (iii) root depth; (iv) root spacing; (v) survey angle; and (vi) soil moisture level. Second, two multi-factor field experiments were conducted to evaluate the performance of the GPR in complex orchard environments. Experiments were conducted at the citrus research grove of the University of Florida Southwest Florida Research and Education Center (SWFREC) in Immokalee, FL, USA.According to the GPR work principle, three subsystems must be investigated at the outset for accurate root detection: (I) the tree root system; (II) the soil system; and (III) the GPR system. In this study, three types of limiting factors were studied: (1) root property (root diameter and root water content); (2) root position (root depth, horizontal and vertical distance between roots) and scan direction; (3) soil property (soil type and moisture). Four output parameters were evaluated: (i) the shape of the hyperbola; (ii) the signal strength; (iii) the signal overlap and interference, and (iv) the signal noise.Upon 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 6 mm 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. If two or more roots are located in close proximity, the GPR can distinguish the roots only if their horizontal distance is larger than 10 cm and their vertical distance is larger than 5 cm. The results demonstrated that GPR is useful for studying citrus tree root systems under southwest Florida growing conditions.Furthermore, we started developing an automated (remote-controlled) platform for the GPR technology. The current GPR technology requires an operator to manually complete 360-degree peripheral scans around the tree trunk (per tree) to cover the entire rooting system. A wheel encoder measures and records the distance covered by the GPR. This manual procedure generates many errors ( noise ), mainly because it is difficult for the operator to follow a perfect circular path (360-degree peripheral scan) around the tree (for more information please check our preliminary work at Derival et al., 2018). This automated system will increase data collection accuracy and decrease data collection time. The first prototype is ready for field trials. In the next quarters, we will:1) A GPR evaluation manuscript is under preparation. We will submit it to a high-quality peer-reviewed journal. 2) Evaluate the remote-controlled prototype in the field. Based on the evaluation metrics, we will develop further and improve the first prototype.In this activity, we will compare the automated GPR collected data and the developed 3D maps with the actual root systems of citrus trees. First, we will generate the 3D maps using the automated GPR; then, we will excavate the trees to compare the developed maps with the actual root system. This activity will be limited to non-commercial experimental trials in place at SWFREC.
During this reporting period, we have conducted the following studies. We have continued to work on method development to produce gene-edited and transgene-free plants derived from mature citrus tissues. For the approach to create a chemical resistance to reduce chimeras, we have not observed resistance. We are trying to determine what are possible reasons. We also started a different strategy to create chemical resistance. For that purpose, we have made some gene constructs and will test their efficiencies.
We have completed experiments to repeat some previously conducted experiments in which we observed inconsistent results using the proposed genes regarding their effects when used in mature citrus tissues. We have also completed to test the effects of several chemicals on transient and stable expression activities and also regeneration efficiencies in mature citrus tissues. Our results suggest that there are some improvement of shoot regeneration from calli derived from mature tissues of citrus. We will write a manuscript to report these results.
We have observed that fresh young shoot tissues propagated from mature citrus shoots through vegetative propagation can be regenerated but the efficiencies are somewhat low. The Agrobacterium-mediated transformation efficiency of young fresh tissues generated from mature tissues is very low. To circumvent these problems, we have constructed additional genes. We are starting to test their effectivities using both juvenile and mature citrus tissues.
To develop an in-planta transformation method for mature citrus tissues, we have done additional experiments from the last report. We observed low efficiencies for the Agrobacterium infection based on our transient or stable transformation assays. We have also observed low de novo regeneration efficiencies using mature citrus plants in our in-planta transformation experiments. Thus, we believe that new tools have to be incorporated into the in-planta transformation method in order to successfully use it in mature citrus plants. We are preparing tissues for test whether the new genes that can enhance shoot regeneration we have recently constructed can enhance regeneration efficiencies from mature plants in our in-planta transformation experiments.
The project has five objectives:(1) Remove the flowering-promoting CTV and the HLB bacterial pathogen in the transgenic plants(2) Graft CTV- and HLB-free buds onto rootstocks(3) Generate a large number of vigorous and healthy citrus trees(4) Plant the citrus trees in the site secured for testing transgenic citrus for HLB responses(5) Collect the field trial data In this quarter, we have focused on the following two activities:(1) Continue treating HLB-tolerant transgenic plants under the alternating temperature conditions (25 C for 4 hours and 42 C for 4 hours). New shoots that have emerged from the previously treated trees have been tested by ELISA for CTV and by qPCR for CLas. Results have shown that both CTV and CLas do not exist in the new shoots. We have started to graft the budwoods from the new shoots onto rootstocks to generate CTV and CLas free plants, but the number is small. We are waiting for more budwoods. (2) About 50 transgenic progeny plants that have been tested for HLB tolernace were tested by ELISA for CTV and by qPCR for CLas. Results showed that all of these plants are CTV negative and CLas postive. The majority of these plants have no HLB symptoms and three plants show mild HLB symptoms. We will plant these trees into the field in May. The field has been prepared with the help from Dr. Ed Stover in Ft Pierce USDA ARS.
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. 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. A manuscript entitled Targeted Early Detection of Citrus Huanglongbing Causal Agent ‘Candidatus Liberibacter asiaticus’ Before Symptom Expression has been published by Phytopathology. We determined the in planta minimum inhibitory concentration of oxytetracycline against Candidatus Liberibacter asiaticus effective for control of citrus Huanglongbing which is being submitted to Phytopathology.
Objective 1: Leaf nutrient thresholds for HLB+ treesWe 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). Critical nutrient thresholds from HLB+ trees in this survey study were higher than published values for K, Cu, B; slightly higher for Mn, Zn {Note: these are not yet recommendations}. We obtained additional leaf data from collaborating grower’s commercial grove databases in order to expand our new critical nutrient thresholds to include N, P, S, and Ca. The data that we collected during this project survey did not include enough values in the deficient range for those nutrients, which prevented us from establishing critical nutrient thresholds.The results of this research were presented at grower events: Citrus Nutrition Day, Bartow; Citrus School, Arcadia; Citrus Institute, Avon Park, and published in the April 2019 Citrus Industry magazine. Objective 2: Determine soil conditions that favor root hair and VAM proliferationBased on good results with healthy Carrizo rootstock trees grown hydroponically, we set up a final experiment where infected psyllids were allowed to inoculate some replications of the experiment with CLas. This treatment serves to monitor the impacts and interaction of HLB and nutrient solution treatment, as measured in root hair growth (until about May 2019). The final report will be completed in June. A paper on this research will be presented at the FSHS conference in June.
This project is an continuation of an objective of existing CRDF funded project (# 00124558 ; ending in March 2019) with some added treatments to be evaluated. The added treatments are:1. CRF + Tiger Micronutrients+ Mn 50%2. CRF + Tiger Micronutrients+ Zn 50%3. CRF + Tiger Micronutrients+ Fe 50%4. CRF + Tiger Micronutrients+ B 50%5. CRF + Tiger Micronutrients+ Mn +Zn 20%6. CRF + Tiger Micronutrients+ Mn +Fe 20%7. CRF + Tiger Micronutrients+ Zn +Fe 20%8. CRF + Tiger Micronutrients+ Zn +B 20%9. CRF + Tiger Micronutrients+ Fe + B 20%10. CRF + Tiger Micronutrients+ Mn +Zn 50%11. CRF + Tiger Micronutrients+ Mn +Fe 50%12. CRF + Tiger Micronutrients+ Zn +Fe 50%13. CRF + Tiger Micronutrients+ Zn +B 50%14. CRF + Tiger Micronutrients+ Fe + B 50%The treatment for objective 3: 1.CRF + Foliar Micronutrients + Tiger 90; 2.CRF + Tiger Micronutrients These treatments will be initiated one the block that was being used for hybrid fertilizer trial (comparison of different rate of controlled nitrogen). This treatment willend with harvest in spring 2019. In time duration, December-February, we have surveyed the site, ‘valencia’ on swingle between 11-15 year, set up an experimental layout for the newly added treatments, collected pre-treatment data on tree growth, leaf and soil nutrient analysis. The fertiizer application will be made in spring 2019. after harvest of the existing trial. We have also solicited fertilizer donation from Harrell’s and TIger Sul.
A number of trials are underway at the Picos Test Site funded through the CRDF. A detailed current status is outlined below this paragraph. In the last quarter, the most signficant advances have been: 1) A replicated planting of 32 transgenic trees and controls produced by Dr. Jeff Jones at UF. These trees include two very different constructs, each quite specific in attacking the citrus canker pathogen. 2) Initiation of an experiment on pollen flow from transgenic trees. Data on flowering overlap was collected on transgenic US-802 and non-transgenic FF-5-51-2. The FF-5-51-2 trees are slightly more than 1000 ft from the US-802, and are self-incompatible and mono-embryonic. When seed mature in the FF-5-51-2, thousands will be collected and tested for nptII, a transgene in the US-802. Controlled crosses were also made of US-802 pollen onto 36 flowers of FF-5-51-2. This will demonstrate cross-compatibility and provide seed to validate testing protocols. If pollen from transgenic trees is not detected from open-pollination, it should reduce isolation distances required by BRS. 3) Annual BRS inspection of the planting site was conducted. Previously established at the site: 1) The UF Grosser, Dutt and Gmitter transgenic effort has a substantial planting of diverse transgenics. These are on an independent permit, while all other transgenics on the site are under the Stover permit. 2) A broad cross-section of Poncirus derived material is being tested by USDA-ARS-Riverside and UCRiverside, and led by Chandrika Ramadugu. These are seedlings of 82 seed source trees from the Riverside genebank and include pure trifoliate accessions, hybrids of Poncirus with diverse parents, and more advanced accessions with Poncirus in the pedigree. Plants are replicated and each accession includes both graft-inoculated trees and trees uninfected at planting. Likely 2019 will be the last year for data collection. 3) 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. Manuscripts have been published reporting HLB tolerance associated QTLs and differences in ACP colonization. Trees continue to be useful for documenting tolerance in a new NIFA project. 4) A replicated 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 planting also includes a number of related hybrids (including our easy peeling remarkably HLB-tolerant 5-51-2) and released cultivars. Genotyping, HLB phenotyping and growth data have been collected and will continue to be conducted under a new NIFA grant. 5) 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.). 6) In a project led by Fred Gmitter there is a planting of 1132 hybrids of C. reticulata x C. latipes. C. latipes is among the few members of genus Citrus reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 7) Seedlings with a range of pedigree contributions from Microcitrus are planted in a replicated trial, in a collaboration between Malcolm Smith (Queensland Dept. of Agriculture and Fisheries) and Ed Stover. Microcitrus is reported to have HLB resistance, and it is expected that there will be segregation for such resistance. The resulting plants may be used in further breeding and may permit mapping for resistance genes. 8) Conventional scions on Mthionin-producing transgenic Carrizo are planted from the Stover team and are displaying superior growth to trees on control Carrizo. 9) 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 Zhonglin Mou of UF, Tim McNellis of PSU will be planted in the next quarter. 10) Availability of the test site for planting continues to be announced to researchers.
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.
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